JP7063408B1 - Liquid phase peptide production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid phase peptide production method capable of removing an amino acid active ester by liquid phase treatment in a peptide production method using a carrier for liquid phase peptide synthesis. A liquid phase peptide production method comprising the following steps a to d. a. A step of condensing an amino acid, peptide or amino acid amide to which a carrier for liquid phase peptide synthesis is bound and an amino acid or peptide whose amino group is protected by an amino-protecting group in a solvent containing an organic solvent, b. A step of adding an amino acid active ester scavenger selected from aminosulfonic acids, aminosulfates, aminophosphonic acids, aminophosphates and aminoalcohols to the reaction solution after the condensation reaction, c. The step of removing the amino-protecting group of the compound in which the amino-protecting group is protected by the amino-protecting group in the reaction solution, d. After adding the aqueous solution to the reaction solution, the solution is separated to contain a condensate of the amino acid, peptide or amino acid amide to which the carrier for liquid phase peptide synthesis is bound and the amino acid or peptide from which the amino-protecting group has been removed. A step of obtaining a solvent layer. [Selection diagram] None

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Published in "Molecules 2021, 26 (12), 3497" (https://doi.org/10.339/molecules26123497) on June 8, 3rd year of Reiwa. ..

The present invention relates to a method for producing a peptide in a liquid phase.

Peptide production techniques include solid phase peptide synthesis methods and liquid phase peptide synthesis methods, but liquid phase peptide synthesis methods suitable for mass production are widely adopted for producing peptides used as pharmaceuticals and the like. ing. Recently, carriers (Tags) for synthesizing liquid phase peptides, which are compounds that greatly improve the solubility of protected amino acids and protected peptides in organic solvents, have been reported (Patent Documents 1 to 14).

In liquid phase peptide synthesis, an excess of amino acids and a condensing agent are used to prevent the loss of amino acid residues. Therefore, in the peptide extension reaction, an amino acid active ester remains during the amino acid condensation reaction, and the remaining amino acid active ester is the next amino acid. The reaction also occurs during the elongation reaction, and an unwanted "double hit" is generated, which causes a problem that the yield and purity of the target product are lowered.

As a means for removing the amino acid active ester, a method of hydrolyzing the amino acid active ester with alkaline water (Patent Document 15), an alkylamine having 1 to 14 carbon atoms, an aromatic amine, and a hydroquilamine were added and quenched during the condensation reaction. Later, a method for solid-liquid separation of by-products (Patent Document 16) has been reported. In addition, amino acid activity is achieved by adding a divalent water-soluble amine to the reaction solution after the condensation reaction, adding an acid to the reaction solution after the deprotection step to neutralize it, and further adding an acidic aqueous solution for washing. A method for removing both ester and dibenzofulven (DBF) has been reported (Patent Document 17). DBF is a by-product produced when deprotecting the Fmoc group, which is a protecting group for amino groups.

Japanese Patent No. 5113118 Japanese Patent No. 5929756 Japanese Patent No. 6092513 Japanese Patent No. 5768712 Japanese Patent No. 5803674 Japanese Patent No. 6116782 Japanese Patent No. 620176 Japanese Patent No. 6283774 Japanese Patent No. 6283775 Japanese Patent No. 6322350 Japanese Patent No. 6393857 Japanese Patent No. 6531235 International Publication No. 2020/175472 International Publication No. 2020/175473 International Publication No. 2007/099656 International Publication No. 2016/140232 Japanese Patent No. 67036668

However, the methods for removing amino acid active esters described in Patent Documents 15 and 16 require solid-liquid separation means and cannot be applied to one-pot synthesis. Further, in the method described in Patent Document 17, since liquid separation under acidic conditions is required, there is a problem that liquid separation failure between the amino acid active ester and the peptide as a product is likely to occur.
An object of the present invention is to provide a liquid phase peptide production method capable of removing an amino acid active ester by a liquid phase treatment in a peptide production method using a carrier for liquid phase peptide synthesis.

Therefore, the present inventor condenses an amino acid, a peptide or an amino acid amide to which a carrier for liquid phase peptide synthesis is bound and an amino acid or a peptide whose amino group is protected by an amino-protecting group in a solvent containing an organic solvent. By using a compound selected from aminosulfonic acids, aminosulfates, aminophosphonic acids, aminophosphates and aminoalcohols as a scavenger for the amino acid active ester that remains without condensation, the system is not subject to acidic conditions. , It has been found that the amino acid active ester can be removed, and the problems of solid-liquid separation and poor liquid separation in the conventional method have been solved, and the present invention has been completed.

That is, the present invention provides the following inventions [1] to [6].
[1] A method for producing a liquid phase peptide, which comprises the following steps a to d.
a. A step of condensing an amino acid, peptide or amino acid amide to which a carrier for liquid phase peptide synthesis is bound and an amino acid or peptide whose amino group is protected by an amino protecting group in a solvent containing an organic solvent.
b. A step of adding an amino acid active ester scavenger selected from aminosulfonic acids, aminosulfates, aminophosphonic acids, aminophosphates and aminoalcohols to the reaction solution after the condensation reaction.
c. The step of removing the amino-protecting group of the compound whose amino-protecting group is protected by the amino-protecting group in the reaction solution.
d. After adding the aqueous solution to the reaction solution, the solution is separated to contain a condensate of the amino acid, peptide or amino acid amide to which the carrier for liquid phase peptide synthesis is bound and the amino acid or peptide from which the amino protecting group has been removed. A step of obtaining a solvent layer.
[2] The amino acid active ester scavenger is an aminosulfonic acid or aminosulfuric acid represented by the following general formula (1).

(R ¹ indicates a divalent organic group having 1 to 10 carbon atoms, and X ¹ indicates a single bond or an oxygen atom)
Aminophosphonic acids and aminophosphates represented by the general formula (2), and

(R ² indicates a divalent organic group having 1 to 10 carbon atoms, and X ² indicates a single bond or an oxygen atom)
The liquid phase peptide production method according to [1], which is a compound selected from amino alcohols represented by the general formula (3).

(N indicates an integer from 0 to 20, and R ³ and R ⁴ independently indicate a hydrogen atom, a methyl group, an ethyl group, or a hydroxymethyl group).
[3] Amino sulfonic acids represented by the following general formula (1a), wherein the amino acid active ester scavenger is represented by the following general formula (1a).

(R ¹ indicates a divalent organic group having 1 to 10 carbon atoms)
Aminophosphonic acids and aminophosphates represented by the general formula (2), and

(R ² indicates a divalent organic group having 1 to 10 carbon atoms, and X ² indicates a single bond or an oxygen atom)
The liquid phase peptide production method according to [1], which is a compound selected from amino alcohols represented by the general formula (3).

(N indicates an integer from 0 to 20, and R ³ and R ⁴ independently indicate a hydrogen atom, a methyl group, an ethyl group, or a hydroxymethyl group).
[4] The liquid phase peptide production method according to [1] to [3], wherein the amino protecting group is a protecting group selected from an Fmоc group, a Bоc group, a Cbz group and an Ac group.
[5] The liquid phase peptide synthesis carrier is a compound that binds to an amino acid, peptide or amino acid amide directly or via a linker to make them soluble in an organic solvent and insoluble in water [1] to [ 4] The liquid phase peptide production method according to any one of.
[6] The carrier for liquid phase peptide synthesis has the following formula (I):

[During the ceremony,
Ring A shows an aromatic ring of C4-18 which may contain a heteroatom and may be polycyclic;
When R ¹¹ is a hydrogen atom or ring A is a benzene ring and Rb is a group represented by the following formula (a), it shows a single bond together with R ¹⁴ and shows ring A. And a fluorene ring may be formed with ring B, or a xanthene ring may be formed with rings A and B via an oxygen atom;
The p Xs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^17- (R ¹⁷ is a hydrogen atom, an alkyl group or Indicates an aralkyl group.) Indicates;
The p R ^12s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
The q R ^13s represent organic groups each independently having an aliphatic hydrocarbon group, which may be a hydrogen atom independently or may be substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
p and q are integers of 0 to 3 and p + q is 1 or more and 4 or less, respectively;
Ring A is a group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom, in addition to p XR ¹² . May have substituents selected from;
Ra represents an aromatic ring that may be substituted with a hydrogen atom or a halogen atom;
Rb is a hydrogen atom, or formula (a) :.

(In the formula, * indicates the bond position;
r and s are integers of 0 to 3 and r + s is 4 or less, respectively;
The r Zs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^18- (R ¹⁸ is a hydrogen atom, an alkyl group or Indicates an aralkyl group.) Indicates;
The r R ^15s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
The s R ^16s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
R ¹⁴ either exhibits a hydrogen atom or exhibits a single bond with R ¹¹ to form a fluorene ring with rings A and B, or a xanthene ring with rings A and B via an oxygen atom. May form;
Ring B is a group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom, in addition to r ZR ¹⁵ . It may have a substituent selected from. ) Indicates a group;
Y represents a hydroxy group, NHR ¹⁹ (R ¹⁹ indicates a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom. ]
The liquid phase peptide production method according to any one of [1] to [4], which is a compound represented by.

According to the present invention, after condensing an amino acid, a peptide or an amino acid amide to which a carrier for liquid phase peptide synthesis is bound and an amino acid or a peptide whose amino group is protected by an amino-protecting group in a solvent containing an organic solvent. If a compound selected from aminosulfonic acids, aminosulfates, aminophosphonic acids, aminophosphates and aminoalcohols is used as a scavenger for the remaining amino acid active ester, the amino acid active ester can be used without making the inside of the system acidic. We found that it could be removed, and solved the problems of solid-liquid separation and poor liquid separation in the conventional method.
This has made it possible to efficiently produce the liquid phase of the peptide in one pot.

In the peptide production method of the present invention, an amino acid, a peptide or an amino acid amide to which a carrier for liquid phase peptide synthesis is bound is condensed with an amino acid or a peptide whose amino group is protected by an amino-protecting group in a solvent containing an organic solvent. , A liquid phase peptide production method.

The carrier for liquid phase peptide synthesis used is a carrier that protects an amino acid, peptide or amino acid amide and solubilizes the protected amino acid, peptide or amino acid amide in an organic solvent.
Examples of such a carrier for liquid phase peptide synthesis include the compounds described in Patent Document 1-14. Preferred liquid phase peptide synthesis carriers include compounds represented by the following formula (I).

[During the ceremony,
Ring A shows an aromatic ring of C4-18 which may contain a heteroatom and may be polycyclic;
When R ¹¹ is a hydrogen atom or ring A is a benzene ring and Rb is a group represented by the following formula (a), it shows a single bond together with R ¹⁴ and shows ring A. And a fluorene ring may be formed with ring B, or a xanthene ring may be formed with rings A and B via an oxygen atom;
The p Xs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^17- (R ¹⁷ is a hydrogen atom, an alkyl group or Indicates an aralkyl group.) Indicates;
The p R ^12s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
The q R ^13s represent organic groups each independently having an aliphatic hydrocarbon group, which may be a hydrogen atom independently or may be substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
p and q are integers of 0 to 3 and p + q is 1 or more and 4 or less, respectively;
Ring A is a group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom, in addition to p XR ¹² . May have substituents selected from;
Ra represents an aromatic ring that may be substituted with a hydrogen atom or a halogen atom;
Rb is a hydrogen atom, or formula (a) :.

(In the formula, * indicates the bond position;
r and s are integers of 0 to 3 and r + s is 4 or less, respectively;
The r Zs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^18- (R ¹⁸ is a hydrogen atom, an alkyl group or Indicates an aralkyl group.) Indicates;
The r R ^15s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
The s R ^16s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
R ¹⁴ either exhibits a hydrogen atom or exhibits a single bond with R ¹¹ to form a fluorene ring with rings A and B, or a xanthene ring with rings A and B via an oxygen atom. May form;
Ring B is a group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom, in addition to r ZR ¹⁵ . It may have a substituent selected from. ) Indicates a group;
Y represents a hydroxy group, NHR ¹⁹ (R ¹⁹ indicates a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom. ]

Ring A in formula (I) may contain a heteroatom and represents an aromatic ring of C4-18 which may be monocyclic or polycyclic. Examples of the aromatic ring include an aromatic hydrocarbon ring of C6 to 18 and an aromatic heterocycle of C4 to 10.
Specific examples of the C6-18 aromatic hydrocarbon ring include a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a triphenylene ring, a tetracene ring, an indane ring, an indene ring, a fluorene ring, and a biphenyl ring. Of these, a benzene ring, a naphthalene ring, a phenanthrene ring, and a fluorene ring are more preferable.
As the aromatic heterocycle of C4 to 10, a 5-membered to 10-membered aromatic heterocycle containing 1 to 3 selected from a nitrogen atom, an oxygen atom and a sulfur atom as a heteroatom is preferable, and specifically, a 5-membered ring to a 10-membered aromatic ring is preferable. , Pyrol ring, furan ring, thiophene ring, indole ring, benzofuran ring, benzothiophene ring, carbazole ring, pyrazole ring, indazole ring, imidazole ring, pyridine ring, quinoline ring, isoquinoline ring and the like. Of these, a 5-membered to 8-membered aromatic heterocycle containing 1 to 3 selected from a nitrogen atom, an oxygen atom and a sulfur atom as the heteroatom is preferable, and a pyrazole ring, a furan ring, a thiophene ring, an indole ring, etc. A benzofuran ring, a benzothiophene ring, a carbazole ring, a pyrazole ring, and an indazole ring are more preferable.

R ¹¹ represents a hydrogen atom, or when ring A is a benzene ring and Rb is a group represented by the above formula (a), it shows a single bond together with R ¹⁴ and shows ring A. And a fluorene ring may be formed with the ring B, or a xanthene ring may be formed with the ring A and the ring B via an oxygen atom. As the ring in which R ¹¹ and R ¹⁴ may be formed together, a fluorene ring or a xanthene ring is preferable.

The p Xs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^17- (R ¹⁷ is a hydrogen atom, an alkyl group or It indicates an aralkyl group.).
Here, as R ¹⁷ , a hydrogen atom, an alkyl group of C1 to 10 or an aralkyl group of C7 to 20 is preferable. Alkyl groups include linear or branched C1 to such as methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group and hexyl group. Examples include 10 alkyl groups.
Examples of the aralkyl group include a C7 to 16 aralkyl group, for example, a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-phenylpropyl group, a naphthylmethyl group, a 1-naphthylethyl group and the like.

The p R ^12s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom.
The q R ^13s represent organic groups each independently having an aliphatic hydrocarbon group which may be a hydrogen atom or may be substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom.
p and q are integers of 0 to 3 and p + q is 1 or more and 4 or less, respectively.

In the present specification, the organic group having an aliphatic hydrocarbon group is a monovalent organic group having an aliphatic hydrocarbon group in its molecular structure. The site of the aliphatic hydrocarbon group in the organic group having the aliphatic hydrocarbon group is not particularly limited, and may be present at the terminal or at any other site.
The aliphatic hydrocarbon group present in the organic group is a linear, branched or cyclic saturated or unsaturated aliphatic hydrocarbon group, and is C5 or higher aliphatic hydrocarbon from the viewpoint of organic solvent solubility. Hydrogen groups are preferred, C5-30 aliphatic hydrocarbon groups are more preferred, and C8-30 aliphatic hydrocarbon groups are even more preferred. Specific examples of the aliphatic hydrocarbon group include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group and the like, but an alkyl group, a cycloalkyl group and an alkenyl group are particularly preferable, and an alkyl group is more preferable. Further, a linear or branched alkyl group of C5 to 30, a cycloalkyl group of C3 to 8, a linear or branched alkenyl group of C5 to 30, is preferable, and a linear or branched alkyl group of C5 to 30 is preferable. , C3-8 cycloalkyl groups are more preferred, C5-30 linear or branched alkyl groups are even more preferred, and C8-30 linear or branched alkyl groups are even more preferred.

Specific examples of the alkyl group include an alkyl group having 1 to 30 carbon atoms, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, and a pentyl group. , Hexyl group, octyl group, decyl group, lauryl group, tridecyl group, myristyl group, cetyl group, stearyl group, araquil group, behenyl group, tetracosanyl group, hexacosanyl group, isostearyl group and other monovalent groups, they Examples thereof include a divalent group derived from, and a group obtained by excluding hydroxyl groups from various steroid groups.
Examples of the alkenyl group include a monovalent group such as a vinyl group, a 1-propenyl group, an allyl group, an isopropenyl group, a butenyl group, an isobutenyl group and an oleyl group, and a divalent group derived from them.
Examples of the alkynyl group include an ethynyl group, a propargyl group, a 1-propynyl group and the like.

The above aliphatic hydrocarbon group may be substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom.
The silyl group capable of substituting the aliphatic hydrocarbon group via the oxygen atom is three selected from a linear or branched alkyl group having 1 to 6 carbon atoms and an aryl group which may have a substituent. A silyl group substituted with is preferable. Therefore, the aliphatic hydrocarbon group is substituted with a silyloxy group substituted with three selected from a linear or branched alkyl group having 1 to 6 carbon atoms and an aryl group which may have a substituent. May be. Here, examples of the aryl group which may have a substituent include a phenyl group and a naphthyl group.
The silyl group substituted via a preferable oxygen atom is a silyloxy group substituted with three linear or branched alkyl groups having 1 to 6 carbon atoms, and more preferably a linear or branched silyl group having 1 to 4 carbon atoms. It is a silyloxy group in which three alkyl groups in the chain are substituted. The three alkyl or aryl groups substituted with the silyloxy group may be the same or different. The silyloxy group is preferably substituted with 1 to 3 aliphatic hydrocarbon groups.

Examples of the aliphatic hydrocarbon group capable of substituting the aliphatic hydrocarbon group via an oxygen atom include a linear or branched alkoxy group having 1 to 6 carbon atoms, an alkenyloxy group having 2 to 6 carbon atoms, and 3 carbon atoms. Examples thereof include monovalent groups such as cycloalkyloxy groups of up to 6 and divalent groups derived from them.

p and q are integers of 0 to 3 and p + q is 1 or more and 4 or less, respectively. Here, p is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2.

Ring A is a group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom, in addition to p XR ¹² . It may have a substituent selected from.
Examples of the halogen atom include a chlorine atom, a fluorine atom, a bromine atom and an iodine atom. Examples of the C1-6 alkyl group that may be substituted with a halogen atom include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group and a hexyl group. , Dichloromethyl group, trichloromethyl group, trifluoromethyl group and the like. Examples of the C1-6 alkoxy group which may be substituted with a halogen atom include a methoxy group, an ethoxy group, a propyloxy group, an isopropyloxy group, a butyloxy group, an isobutyloxy group, a sec-butyloxy group, a tert-butyloxy group and a trichloromethoxy. Examples include a group and a trifluoromethoxy group.

Ra represents an aromatic ring that may be substituted with a hydrogen atom or a halogen atom.
Here, examples of the aromatic ring include an aromatic hydrocarbon ring of C6 to 18 and an aromatic heterocycle of C4 to 10.
Specific examples of the C6-18 aromatic hydrocarbon ring include a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a triphenylene ring, a tetracene ring, an indane ring, an indene ring, a fluorene ring, and a biphenyl ring. Of these, a benzene ring, a naphthalene ring, a phenanthrene ring, and a fluorene ring are more preferable.
The aromatic heterocycle of C4 to 10 is preferably a 5-membered to 10-membered heterocycle containing 1 to 3 selected from a nitrogen atom, an oxygen atom and a sulfur atom as the heteroatom, and specifically, pyrrole. Examples thereof include a ring, a furan ring, a thiophene ring, an indole ring, a benzofuran ring, a benzothiophene ring, a carbazole ring, a pyrazole ring, an indazole ring, an imidazole ring, a pyridine ring, a quinoline ring, and an isoquinoline ring. Of these, a 5-membered to 8-membered heterocycle containing 1 to 3 selected from a nitrogen atom, an oxygen atom and a sulfur atom is preferable as the heteroatom, and a pyrrole ring, a furan ring, a thiophene ring, an indole ring and a benzofuran ring are preferable. , Benzothiophene ring, carbazole ring, pyrazole ring, indole ring are more preferable.
The aromatic ring of Ra may be substituted with 1 to 3 halogen atoms.

Rb represents a hydrogen atom or a group represented by the above formula (a).
R and s in the formula (a) are integers of 0 to 3 and r + s is 0 to 4, respectively.
r is preferably 0 to 4, more preferably 1 to 3, and even more preferably 1 to 2.

The r Zs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^18- (R ¹⁸ is a hydrogen atom, an alkyl group or It indicates an aralkyl group.).
Here, as R ¹⁸ , a hydrogen atom, an alkyl group of C1 to 10 or an aralkyl group of C7 to 20 is preferable. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group and a hexyl group.
Examples of the aralkyl group include a C7 to 16 aralkyl group, for example, a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-phenylpropyl group, a naphthylmethyl group, a 1-naphthylethyl group and the like.

The r R ^15s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
The s R ^16s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom.
The organic group having an aliphatic hydrocarbon group which may be substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom represented by R ¹⁵ and R ¹⁶ is the same as that of R ¹² and R ¹³ described above. The same as R ¹² and R ¹³ described above is preferable.

R ¹⁴ either exhibits a hydrogen atom or exhibits a single bond with R ¹¹ to form a fluorene ring with rings A and B, or a xanthene ring with rings A and B via an oxygen atom. May be formed.

Ring B is a group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom, in addition to r ZR ¹⁵ . It may have a substituent selected from.
Examples of the halogen atom include a chlorine atom, a fluorine atom, a bromine atom and an iodine atom. Examples of the C1-6 alkyl group that may be substituted with a halogen atom include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group and a hexyl group. , Dichloromethyl group, trichloromethyl group, trifluoromethyl group and the like. Examples of the C1-6 alkoxy group which may be substituted with a halogen atom include a methoxy group, an ethoxy group, a propyloxy group, an isopropyloxy group, a butyloxy group, an isobutyloxy group, a sec-butyloxy group, a tert-butyloxy group and a trichloromethoxy. Examples include a group and a trifluoromethoxy group.

Y represents a hydroxy group, NHR ¹⁹ (R ¹⁹ indicates a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom.
Here, as R ¹⁹ , a hydrogen atom, an alkyl group of C1 to 10 or an aralkyl group of C7 to 20 is preferable. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group and a hexyl group.
Examples of the aralkyl group include a C7 to 16 aralkyl group, for example, a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-phenylpropyl group, a naphthylmethyl group, a 1-naphthylethyl group and the like.

An amino acid to which a carrier for synthesizing a liquid phase peptide or a peptide to which a carrier for synthesizing a liquid phase peptide is bound, which is one of the raw materials used in the production method of the present invention, is one of the reactive groups of the amino acid or the peptide. An amino acid or peptide that is bound to a carrier for synthesizing a liquid phase peptide and has at least one amino group in a reactive state. Preferably, the carboxyl group of the amino acid or peptide binds to the carrier for liquid phase peptide synthesis, while the amino group is unprotected and reactive. The amino acid amide to which the carrier for synthesizing the liquid phase peptide is bound is that at least one amide group of the amino acid amide is bound to the carrier for synthesizing the liquid phase peptide, and at least one amino group is unprotected and reactive. Amino acid amide.
When the amino acid, peptide or amino acid amide to which the carrier for liquid phase peptide synthesis is bound has a highly reactive functional group such as a hydroxyl group, an amino group, a guanidyl group, a carboxyl group, a thiol group, an indol group and an imidazole group. A general protective group used in peptide synthesis may be introduced into these functional groups, and the target compound can be obtained by removing and removing the protective group as necessary after the reaction is completed. Examples of the hydroxyl group protective group include a tBu group, a Trt group, a Bz group, an acetyl group, a silyl group and the like, and examples of the amino group protective group include a Boc group, an Fmoc group, a Cbz group, a Trt group and an Mmt group. , IvDde group and the like, examples of the guanidyl group protecting group include Pbf group, Pmc group, nitro group and the like, and examples of the carboxyl group protecting group include tBu group, methyl group, ethyl group, Bz group and the like. Examples of the thiol group protective group include a Trt group, an Acm group, a tBu group, an S—tBu group and the like, and examples of the indol group protection group include a Boc group and the like, and examples of the imidazole group protection group include a Boc group and the like. , Boc group, Bom group, Bum group, Trt group and the like.

The amino acid, peptide or amino acid amide to which the liquid phase peptide synthesis carrier is bound dissolves the liquid phase peptide synthesis carrier in an organic solvent such as THF, and for example, a Boc-protected amino acid, peptide or amino acid amide and a condensing agent, for example. N, N'-diisopropylcarbodiimide (DIPCI) is added to perform condensation, and an N-Boc-liquid phase synthesis carrier, which is an intermediate in which a liquid phase peptide synthesis carrier is bound to the carboxyl group of an amino acid, peptide or amino acid amide. Protected amino acids, peptides or amino acid amides can be produced.

Further, the carrier for liquid phase peptide synthesis can also be bound to the carboxyl group of the amino acid or peptide via a linker.
The term "linker" as used herein is an organic group having two reactive groups, one of which binds to the carboxyl group of the amino acid or peptide and the other of which binds to a carrier for liquid phase peptide synthesis. A preferred linker is an organic group having a molecular weight of about 2000 or less (preferably about 1500 or less, more preferably about 1000 or less), and the reactive groups may be the same or different, as well as an amino group, a carboxyl group, and a halomethyl group. It is a compound having at least two groups in the molecule selected from the group consisting of. Examples of the linker include the following compounds.

（式中、Ｙは１～６、好ましくは１～４の整数である）。

(In the formula, Y is an integer of 1 to 6, preferably 1 to 4).

（式中、Ｘはハロゲン原子、好ましくは塩素又は臭素である）。

(In the formula, X is a halogen atom, preferably chlorine or bromine).

（式中、Ｚは２～４０、好ましくは２～３５、より好ましくは、２～２８の整数である）。
（上記リンカーの構造式は、側鎖官能基等に結合する前の状態かつ液相ペプチド合成用担体と結合する前の状態を示す）。

(In the formula, Z is an integer of 2 to 40, preferably 2 to 35, more preferably 2 to 28).
(The structural formula of the linker indicates a state before binding to a side chain functional group or the like and a state before binding to a carrier for liquid phase peptide synthesis).

The other raw material, an amino acid or peptide whose amino group is protected by an amino-protecting group, is a reaction in which the amino group of the amino acid or peptide is protected by an amino-protecting group, while the carboxyl group is not protected. Means an amino acid or peptide that is of sex. When an amino acid or peptide has one or more amino groups, it is sufficient that at least one amino group is protected by an amino protecting group.
Examples of the amino-protecting group include an Fmоc group, a Bоc group, a Cbz group, an Ac group and the like, and among these, the Fmоc group that can be deprotected under basic conditions is more preferable.
When the amino acid or peptide whose amino group is protected by the amino-protecting group has a highly reactive functional group such as a hydroxyl group, an amino group, a guanidyl group, a carboxyl group, a thiol group, an indol group and an imidazole group, these A general protective group used in peptide synthesis may be introduced into the functional group, and the target compound can be obtained by removing the protective group as necessary at any time after the reaction is completed.
Examples of the protecting group of the hydroxyl group include a tBu group, a Trt group, a Bz group, an acetyl group and a silyl group, and examples of the protecting group of the amino group include a Boc group, an Fmoc group, a Cbz group, a Trt group, an Mmt group and an ivDde group. Examples of the guanidyl group protective group include a Pbf group, a Pmc group, a nitro group and the like, and examples of the carboxyl group protective group include a tBu group, a methyl group, an ethyl group, a Bz group and the like, and thiol. Examples of the protective group of the group include a Trt group, an Acm group, a tBu group, an S—tBu group and the like, examples of the protective group of the indol group include a Boc group and the like, and examples of the protective group of the imidazole group include a Boc group. , Bom group, Bum group, Trt group and the like.

The amino acid or peptide whose amino group is protected by an amino-protecting group is, for example, an amino acid or peptide whose amino group is to be protected by an amino-protecting group, and a condensing agent of chlorogenic acid 9-fluorenylmethyl ester in a solvent such as THF. It can be produced by reacting in the presence of.

The step a of the present invention is a step of condensing the raw materials, and the reaction solvent used in the step a is a solvent containing an organic solvent. If the amino acid, peptide or amino acid amide is protected by the above-mentioned liquid phase peptide synthesis carrier used in the present invention, the amino acid, peptide or amino acid amide bound to the liquid phase peptide synthesis carrier will be dissolved in an organic solvent. Therefore, liquid phase peptide synthesis becomes possible.
Such organic solvents include, for example, tetrahydrofuran (THF), dimethylformamide (DMF), cyclohexane, cyclopentyl methyl ether (CPME), methyl-tert-butyl ether (MTBE), 2-methylTHF, 4-methyltetrahydropyran ( 4-Methyl THP), isopropyl acetate, chloroform, dichloromethane, N-methylpyrrolidone can be mentioned, preferably THF, DMF, cyclohexane, CPME, MTBE, 2-methyl THF, 4-methylTHP, isopropylacetate, N. -Methylpyrrolidone. Further, a mixed solvent of two or more kinds of the above solvents may be used.

The condensation reaction involves an amino acid, a peptide or an amino acid amide (hereinafter abbreviated as a carrier-binding peptide for liquid phase peptide synthesis) to which the carrier for liquid phase peptide synthesis is bound in a solvent containing the organic solvent, and the amino protective group. It can be carried out by mixing an amino acid or peptide having an amino group protected in (hereinafter, abbreviated as amino group protected amino acid) and a condensing agent.

The amount of the amino group-protected amino acid used for the carrier-binding peptide for liquid phase peptide synthesis is usually 1.01 to 4 equivalents, preferably 1.03 to 3 equivalents, more preferably with respect to the carrier-bound peptide for liquid phase peptide synthesis. Is 1.05 to 2 equivalents, more preferably 1.1 to 1.5 equivalents. In the peptide production method of the present invention, the active ester of an unreacted amino acid can be captured and inactivated by a scavenger added thereafter. Therefore, even if an excess of amino group-protected amino acids is used, the problem of residual does not occur.

As the condensing agent, a condensing agent generally used in peptide synthesis can also be used in the present invention, for example, 4- (4,6-dimethoxy-1,3,5-triazine-2-yl)-. 4-Methylmorphonium chloride (DMT-MM), O- (benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O- (7-azabenzo) Triazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O- (6-chlorobenzotriazole-1-yl) -1,1,3,3-tetramethyl Uronium hexafluorophosphate (HBTU (6-Cl)), O- (benzotriazole-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O- (6-chloro Benzotriazole-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylamino-morpholino-carbodinium Hexafluorophosphate (COMU), diisopropylcarbodiimide (DIPCI), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC) can be mentioned. Preferably, it is DMT-MM, HBTU, HATU, or COMU. The amount of the condensing agent used is preferably 1 to 4 equivalents, more preferably 1 to 2 equivalents, still more preferably 1.05 to 1.3 equivalents, relative to the carrier-binding peptide for liquid phase peptide synthesis.

In the condensation step, an activator is preferably added in order to promote the reaction and suppress side reactions such as racemization. Here, the activator is a reagent that, when coexistent with a condensing agent, leads an amino acid to a corresponding active ester, symmetric acid anhydride, or the like to facilitate the formation of a peptide bond (amide bond). As the activator, an activator generally used in peptide synthesis can be used. For example, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-1H-1,2,3-triazole ethyl carboxylate (HOCt), 1-hydroxy-7-azabenzotriazole (HOAt), 3-hydroxy-4- Ketobenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornen-2,3-dicarboxyimide (HONb), pentafluorophenol, cyano ( Hydroxyimino) ethyl acetate (Oxyma) and the like can be mentioned. Preferred are HOBt, HOOBt, HOCt, HOAt, HONb, HOSu, and Oxyma. The amount of the activator used is preferably 1 to 4 equivalents, more preferably 1 to 2 equivalents, still more preferably 1.05 to 1.3 equivalents, relative to the carrier-binding peptide for liquid phase peptide synthesis.

The amount of the solvent used is such that the concentration in which the carrier-binding peptide for liquid phase peptide synthesis is dissolved is preferably 0.1 mM to 1 M, and more preferably 1 mM to 0.5 M.
The reaction temperature is preferably a temperature generally used in peptide synthesis, for example, −20 to 40 ° C., more preferably 0 to 30 ° C. The reaction time (time for one cycle) is usually 0.5 to 30 hours.

Step b is a step of adding an amino acid active ester scavenger selected from aminosulfonic acids, aminosulfates, aminophosphonic acids, aminophosphates and aminoalcohols to the reaction solution after the condensation reaction.
This step is a step of scavenging the amino acid active ester produced as a by-product in the condensation reaction, and is characterized by using a compound selected from aminosulfonic acids, aminosulfates, aminophosphonic acids, aminophosphates and aminoalcohols as the scavenger. And. By using these compounds as scavengers, amino acid active esters can be captured and removed without making the system acidic.

Examples of the amino acid active ester scavenger include aminosulfonic acids and aminosulfuric acids represented by the following general formula (1);

(R ¹ indicates a divalent organic group having 1 to 10 carbon atoms, and X ¹ indicates a single bond or an oxygen atom)
Aminophosphonic acids and aminophosphates represented by the general formula (2);

(R ² indicates a divalent organic group having 1 to 10 carbon atoms, and X ² indicates a single bond or an oxygen atom)
Amino alcohols represented by the general formula (3) are preferable.

(N indicates an integer from 0 to 20, and R ³ and R ⁴ independently indicate a hydrogen atom, a methyl group, an ethyl group, or a hydroxymethyl group).

R ¹ in the general formula (1) and R ² in the general formula (2) are independently divalent organic groups having 1 to 10 carbon atoms, preferably linear or linear groups having 1 to 10 carbon atoms. Examples thereof include an alkylene group of a branched chain and an arylene group having 6 to 10 carbon atoms. Specific examples thereof include a methylene group, an ethylene group, a trimethylene group, a propylene group, a tetramethylene group, a butylene group, a pentamethylene group, a phenylene group and a naphthylene group.
Of these, from the viewpoint of solubility of these compounds, a linear or branched alkylene group having 1 to 6 carbon atoms and an arylene group having 6 to 8 carbon atoms are more preferable, and a linear or branched group having 1 to 6 carbon atoms is more preferable. The alkylene group is more preferable, the linear or branched alkylene group having 1 to 5 carbon atoms is more preferable, the linear alkylene group having 1 to 3 carbon atoms is further preferable, and the linear alkylene group having 1 or 2 carbon atoms is more preferable. The group is most preferred.
In the general formula (1), when X ¹ is a single bond, it is an aminosulfonic acid, and when X ¹ is an oxygen atom, it is an aminosulfate.
In the general formula (2), when X ² is a single bond, it is an aminophosphonic acid, and when X ² is an oxygen atom, it is an aminophosphoric acid.

N in the general formula (3) represents an integer of 0 to 20. Of these, n is preferably 0 or 2 to 20, more preferably 0 or 2 to 6, and even more preferably 0 or 2 to 4. As R ³ and R ⁴ in the general formula (3), a hydrogen atom or a hydroxylmethyl group is preferable.

The amount of the scavenger of the present invention added in step b is preferably 1 to 10 equivalents, more preferably 1 to 6 equivalents, still more preferably 1 to 4 equivalents, relative to 1 equivalent of the theoretically remaining active amino acid ester. If the amount of the scavenger added in the present invention is too small, the scavenging (capture) of the amino acid active ester becomes insufficient, and a double hit occurs in which the remaining amino acid active ester reacts with the amino group generated in step d, resulting in purity and yield. To reduce. On the other hand, if the amount is too large, the deaminoprotecting group reaction proceeds at the same time, and the remaining amino acid active ester reacts with the regenerated amino group due to the desorption of the aminoprotecting group, resulting in a double hit, which lowers the purity and yield. Let me.

Further, as another aspect of the step b, the following can be mentioned.
Another aspect of step b is a step of adding an amino acid active ester scavenger selected from aminosulfonic acids, aminophosphonic acids, aminophosphates and aminoalcohols to the reaction solution after the condensation reaction.
This step is a step of scavenging the amino acid active ester produced as a by-product in the condensation reaction, and is characterized by using a compound selected from aminosulfonic acids, aminophosphonic acids, aminophosphates and aminoalcohols as the scavenger. By using these compounds as scavengers, amino acid active esters could be removed without making the system acidic, and solid-liquid separation and liquid separation defects were eliminated.

The amino acid active ester scavenger includes aminosulfonic acids represented by the following general formula (1a);

(R ¹ indicates a divalent organic group having 1 to 10 carbon atoms)
Aminophosphonic acids and aminophosphates represented by the general formula (2);

(R ² indicates a divalent organic group having 1 to 10 carbon atoms, and X ² indicates a single bond or an oxygen atom)
Amino alcohols represented by the general formula (3) are preferable.

(N indicates an integer from 0 to 20, and R ³ and R ⁴ independently indicate a hydrogen atom, a methyl group, an ethyl group, or a hydroxymethyl group).

R ¹ in the general formula (1a) and R ² in the general formula (2) are independently divalent organic groups having 1 to 10 carbon atoms, preferably linear or linear groups having 1 to 10 carbon atoms. Examples thereof include an alkylene group of a branched chain and an arylene group having 6 to 10 carbon atoms. Specific examples thereof include a methylene group, an ethylene group, a trimethylene group, a propylene group, a tetramethylene group, a butylene group, a pentamethylene group, a phenylene group and a naphthylene group.
Of these, from the viewpoint of solubility of these compounds, a linear or branched alkylene group having 1 to 6 carbon atoms and an arylene group having 6 to 8 carbon atoms are more preferable, and a linear or branched group having 1 to 6 carbon atoms is more preferable. The alkylene group is more preferable, the linear or branched alkylene group having 1 to 5 carbon atoms is more preferable, the linear alkylene group having 1 to 3 carbon atoms is further preferable, and the linear alkylene group having 1 or 2 carbon atoms is more preferable. The group is most preferred.
The compound of the general formula (1a) is an aminosulfonic acid.
In the general formula (2), when X ² is a single bond, it is an aminophosphonic acid, and when X ² is an oxygen atom, it is an aminophosphoric acid.

N in the general formula (3) represents an integer of 0 to 20. Of these, n is preferably 0 or 2 to 20, more preferably 0 or 2 to 6, and even more preferably 0 or 2 to 4. As R ³ and R ⁴ in the general formula (3), a hydrogen atom or a hydroxylmethyl group is preferable.

The amount of the scavenger of the present invention added in step b is preferably 1 to 10 equivalents, more preferably 1 to 6 equivalents, still more preferably 1 to 4 equivalents, relative to 1 equivalent of the theoretically remaining active amino acid ester. If the amount of the scavenger added in the present invention is too small, the scavenging (capture) of the amino acid active ester becomes insufficient, and a double hit occurs in which the remaining amino acid active ester reacts with the amino group generated in step c, resulting in purity and yield. To reduce. On the other hand, if the amount is too large, the deaminoprotecting group reaction proceeds at the same time, and the remaining amino acid active ester reacts with the regenerated amino group due to the desorption of the aminoprotecting group, resulting in a double hit, which lowers the purity and yield. Let me.

Step c is a step of removing the amino-protecting group of the compound whose amino-protecting group is protected by the amino-protecting group in the reaction solution.
The step of removing the amino-protecting group differs depending on the type of the amino-protecting group. For example, when the amino protecting group is an Fmoc group, the reaction solution may be used as a basic condition. When the amino-protecting group is a Boc group, the reaction solution may be subjected to acidic conditions. When the amino protecting group is a Cbz group, catalytic reduction may be performed. When the amino-protecting group is an Ac group, it may be deprotected under strong acid or strong base conditions. Of these, for one-pot liquid phase synthesis, it is more preferable to use an amino protecting group as an Fmoc group.

The step of removing the amino-protecting group when the amino-protecting group is an Fmoc group will be described.
The Fmoc desorption step may be such that the reaction solution can be basic, but amine compounds such as 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), 1,5-diazabicyclo [4.3]. .0] -5-nonen (DBN), 1,4-diazabicyclo [2.2.2] -octane (DABCO), triethylamine, tributylamine and other tertiary amines; 1-methylpiperazine, 4-aminopiperidine, Use divalent or higher water-soluble amines having at least one primary or secondary amino group such as diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N, N-dimethylethylenediamine, ethylenediamine, piperidine, piperazine. be able to. DBU, piperidine, 1-methylpiperazine, 4-aminopiperidine and diethylenetriamine are preferable, and DBU, piperidine and 1-methylpiperazine are more preferable. More preferably, it is DBU.
The equivalent of the amine compound added in step c is 1 to 30 equivalents, preferably 4 to 20 equivalents, more preferably 4 to 10 equivalents, relative to the amount of Fmoc groups present in the system.

Further, in addition to the amine compound, it is preferable to add a trapping agent of DBF (dibenzofulvene) generated by the de-Fmoc reaction. Examples of the DBF trapping agent used here include mercapto compounds. The mercapto compound that can be used is not particularly limited as long as it has a mercapto group and the compound that has reacted with DBF exhibits water solubility, but for example, a mercapto fatty acid or an alkali metal salt thereof, the following general formula (4). Or (5)

(In the formula, L1 and L2 each represent a divalent organic group, and M represents a hydrogen atom or an alkali metal).
Examples thereof include compounds represented by.

As the mercapto fatty acid or the alkali metal salt thereof, mercapto C1-20 fatty acid or the alkali metal salt thereof is preferable, and mercapto C1-6 fatty acid or the alkali metal salt thereof is more preferable.
L1 and L2 in the general formula (4) or (5) represent divalent organic groups, respectively. As the divalent organic group, a divalent organic group having 1 to 10 carbon atoms is preferable, and more preferably, a linear or branched alkylene group having 1 to 10 carbon atoms which may have a mercapto group. Examples thereof include an arylene group having 6 to 10 carbon atoms which may have a mercapto group and a heteroarylene group having 4 to 9 carbon atoms which may have a mercapto group. Specifically, methylene group, ethylene group, trimethylene group, propylene group, mercaptotrimethylene group, mercaptopropylene group, tetramethylene group, butylene group, pentamethylene group, phenylene group, naphthylene group, indol group, benzimidazole group, Examples thereof include a quinolyl group and an isoquinolin group.
M represents a hydrogen atom or an alkali metal. Specific examples include a hydrogen atom, sodium and potassium.
Specifically, sodium mercaptomethane sulfonate, sodium 2-mercaptoethanesulfonate, 2-mercaptoethanesulfonic acid, sodium 3-mercaptopropanesulfonic acid, 1,3-dimercaptopropanesulfonic acid, 2-mercaptobenzimidazole- Examples thereof include sodium 5-sulfonic acid, sodium mercaptomethanephosphonate, mercaptoethanephosphonic acid, sodium 3-mercaptopropanephosphonate, and sodium 1,3-dimercaptopropanephosphonate.

The amount of the mercapto compound added is theoretically preferably 1 to 30 equivalents, more preferably 1 to 10 equivalents, still more preferably 1 to 5 equivalents, relative to the amount of DBF produced as a by-product.
The amine compound and the mercapto compound may be added at the same time, or may be added in the order of the mercapto compound and then the amine compound, or the mercapto compound may be added after adding a base to remove the Fmoc group.
The Fmoc desorption step may be carried out at a temperature of −20 to 40 ° C. for 5 minutes to 5 hours.

In step d, after adding an aqueous solution to the reaction solution, the solution is separated to form a condensate of an amino acid, peptide or amino acid amide to which a carrier for liquid phase peptide synthesis is bound and an amino acid or peptide from which the amino protecting group has been removed. This is a step of obtaining an organic solvent layer containing.
After adding the aqueous solution to the reaction solution in step c, the aqueous layer and the organic solvent layer are separated.
The aqueous layer contains a condensate of an amino acid or peptide from which the amino-protecting group has been eliminated and an active ester scavenger, and a DBF-trapping agent adduct. That is, the condensate of the amino acid or peptide from which the amino-protecting group has been removed and the active ester scavenger can be easily extracted into the aqueous layer only by adding the aqueous solution in step d.
On the other hand, the organic solvent layer contains a condensate of an amino acid, a peptide or an amino acid amide to which a carrier for liquid phase peptide synthesis is bound, and an amino acid or a peptide from which the amino protecting group has been removed.
Here, examples of the aqueous solution used include water and an aqueous solution having a pH near neutrality. Specifically, water, sodium chloride aqueous solution, sodium carbonate aqueous solution, potassium carbonate aqueous solution, disodium hydrogen phosphate aqueous solution, trisodium phosphate aqueous solution, sodium hydrogen carbonate aqueous solution, potassium hydrogen carbonate aqueous solution, dipotassium hydrogen phosphate aqueous solution, phosphorus. Examples thereof include a tripotassium acid aqueous solution.

As described above, according to the steps a to d of the present invention, it is not necessary to use an acidic aqueous solution by simply adding an aqueous solution and separating the liquids. Therefore, the liquid separation between the amino acid active ester and the peptide as a product is required. No defects occur. In addition, since solid-liquid separation is not required, one-pot synthesis is possible because the peptide extension reaction in the next step can be performed without performing an isolation operation in the liquid phase production of the peptide. The series of steps described above may be carried out using microflow techniques. The peptide synthesis technique using the microflow technique is described in, for example, Nature Communications 7, Article number: 13491 (2016). Further, the obtained organic solvent layer can be further used for a condensation reaction with any amino acid.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
The following compounds were used as the carrier for liquid phase peptide synthesis.
-TIPS2-OH (C11) type benzyl compound (manufactured by Sekisui Medical Co., Ltd.) (hereinafter, may be referred to as B-Stag). However, TIPS represents a triisopropylsilyl group.

-TIPS2-OH (C11) type diphenylmethane compound (manufactured by Sekisui Medical Co., Ltd.) (hereinafter, may be referred to as D-Stag). However, TIPS represents a triisopropylsilyl group.

Fmoc-NH- (D-Stag) is a compound in which the amino group of D-Stag is protected by an Fmoc group.
-TIPS2-OH (C11) type xanthene compound (manufactured by Sekisui Medical Co., Ltd.) (hereinafter, may be referred to as X-Stag). However, TIPS indicates a triisopropylsilyl group.

Fmoc-NH- (X-Stag) is a compound in which the amino group of X-Stag is protected by an Fmoc group.
Further, in the following examples, the compound in which B-Stag is bound to Fmoc-protected tBu-protected glutamic acid (Fmoc-Glu (OtBu) -OH) is referred to as Fmoc-Glu (OtBu) -O- (B-Stag). , The following structure shall be shown. Not limited to Fmoc-Glu (OtBu) -OH, the notation conforms to this when bound to other amino acids.

Further, a compound in which D-STAg is bound to Fmoc-protected tBu-protected threonine (Fmoc-Thr (tBu) -OH) is referred to as Fmoc-Thr (tBu) -NH- (D-Stag), which has the following structure. Shall indicate. Not limited to threonine, when combined with other amino acids, the notation is based on this.

Further, a compound in which X-Stag is bound to Fmoc-protected phenylalanine (Fmoc-Phe-OH) is referred to as Fmoc-Phe-NH- (X-Stag), which has the following structure. Not limited to phenylalanine, when combined with other amino acids, the notation is based on this.

Example 1
Examination of removal rate of condensate of amino acid active ester and scavenger by washing with water Taurine (Example 1-1), 2-AEHS (2-aminoethyl hydrogensulfate: 2-aminoethyl sulfate) (Example 1) as scavengers. The following studies were carried out using either -2) or AEAE (2- (2-aminoethylamino) ethanol) (Comparative Example 1). Taurine was added as a 0.6 M aqueous solution, and 2-AEHS was added as a 0.25 M DMSO solution.
In a mixed solution of CPME 7 mL and DMF 3 mL, Fmoc-Cys (Trt) -OH 0.88 g (1.5 mmol), COMU 0.64 g (1.5 mmol), Oxyma 0.64 g (4.5 mmol), DIPEA 1. 55 g (12.0 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. After adding 1.8 mmol of scavenger and stirring at room temperature for 15 minutes, this solution was analyzed by HPLC to verify the amount of condensate of amino acid active ester and scavenger. Subsequently, 0.69 g (4.5 mmol) of DBU was added, and the mixture was stirred for 30 minutes. After analyzing this solution by HPLC (analysis (1)), 4.5 mL of 1N hydrochloric acid was added and the solution was separated. The obtained organic layer was analyzed by HPLC (analysis (2)), and the removal rate of the condensate of the amino acid active ester and the scavenger into the aqueous layer, that is, the detergency was verified. The results are shown in Table 1.

HPLC analysis condition column: MonoBis column 3.2 × 150mm low pressure type, mesopore diameter 11mm, ODS / end cap (Kyoto Monotech Co., Ltd., product number 32150L11ODS)
Mobile phase A: 0.1% formic acid-containing 5% isopropanol-5% isopropyl ether aqueous solution Mobile phase B: 0.1% formic acid-containing 85% isopropanol-5% isopropyl ether aqueous solution Flow velocity: 1.0 mL / min
Column temperature: 60 ° C
Detection wavelength: 215 nm
Radiant conditions: 5% B (0 minutes) → 5% B (1 minute) → 100% B (11 minutes) → 100% B (16 minutes) → 5% B (17 minutes) → 5% B (20 minutes)

The removal rate of the amino acid active ester and scavenger condensate is determined by the HPLC peak area A of the amino acid active ester and scavenger condensate in analysis (1) and the HPLC peak area of the amino acid active ester and scavenger condensate in analysis (2). Calculated from B.
Removal rate = {(AB) / A} x 100 (%)
The higher the removal rate, the easier it is for the condensate of the active ester of Fmoc-Cys (Trt) -OH and the scavenger to be removed in the aqueous layer, and it can be said that the scavenger is useful.

From Table 1, when taurine of Example 1-1 and 2-AEHS of Example 1-2 were used as the scavenger, the condensed product of the active ester of Fmoc-Cys (Trt) -OH and the scavenger was all put into the aqueous layer. I was able to remove it. This was an extremely good result as compared with 34% when AEAE of Comparative Example 1 was used. AEAE is a divalent water-soluble amine, and the divalent water-soluble amine is a compound which is useful as a scavenger in Patent Document 17.

Example 2
When 3.0 equivalents of Fmoc-protected phenylalanine (Fmoc-Phe-OH) was added to X-Stag and no scavenger was used in the reaction of extending the peptide chain in three steps (Comparative Example 2). , The synthesis results of H-Phe-Phe-Phe-NH ₂ when taurine was used as a scavenger (Example 2) were compared. Taurine was added as a 0.6 M aqueous solution.

1) Synthesis of H-Phe-NH- (X-STAg) 0.44 g (0.5 mmol) of X-STAg was dissolved in 7 mL of CPME and 3 mL of DMF, and Fmoc-Phe-OH 0.58 g (1.5 mmol), COMU 0.64 (1.5 mmol), Oxyma 0.21 g (1.5 mmol) and DIPEA 0.26 g (2.0 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After confirming that X-Stag was 5% or less of the product Fmoc-Phe-NH- (X-Stag), 2.0 mmol was added when scavenger was added, and the mixture was stirred at room temperature for 15 minutes. .. After confirming the compound in which the active ester of Fmoc-Phe-OH was condensed with the scavenger, the mixture was cooled to 0 ° C. and 0.54 g (3.0 mmol) (1.2 mol / L) of sodium 2-mercapto-1-propanesulfonate. (Dissolved in DMSO) was added, 0.88 g (5.8 mmol) of DBU was added, and the mixture was stirred for 30 minutes. After confirming that Fmoc-Phe-NH- (X-Stag) was 5% or less of the product H-Phe-NH- (X-Stag), 3.4 mL of 1N hydrochloric acid was added dropwise to room temperature. The temperature was raised and the liquid was separated. To the obtained organic layer, 6.7 mL of a 50% aqueous dipotassium hydrogen phosphate solution, 0.18 mL of DMSO and 0.18 mL of DMF were added, and the liquids were separated. It was confirmed that there was no compound in which the active ester of Fmoc-Phe-OH was condensed with the scavenger in the obtained organic layer, and after concentrating the organic layer, 7 mL of CPME was added to the residue, and H-Phe-NH- (X-). A CPME solution containing STag) was obtained.

2) Similarly, H-Phe-Phe-NH- (X-Stag) and H-Phe-Phe-Phe-NH- (X-Stag) were synthesized. The CPME solution of the obtained H-Phe-Phe-Phe-NH- (X-Stag) was concentrated under reduced pressure, and the obtained solid was dried under reduced pressure at room temperature to obtain H-Phe-Phe-Phe-NH- (. X-Stag) was obtained.

3) Synthesis of H-Phe-Phe-Phe-NH ₂ Trifluoroacetic acid, water, triisopropylsilane, 3,6 were added to H-Phe-Phe-Phe-NH- (X-Stag) obtained under each condition. A 92.5 / 2.5 / 2.5 / 2.5 mixture of -dioxa-1,8-octanedithiol has a concentration of H-Phe-Phe-Phe-NH- (X-Stag) of 50 mM. And stirred at room temperature for 1 hour. The reaction solution was cooled to 0 ° C., 90% (v / w) of MTBE of H-Phe-Phe-Phe-NH- (X-Stag) was slowly added dropwise, and the precipitate was collected by filtration. The collected precipitate was washed 3 times with 68.4% (v / w) MTBE against H-Phe-Phe-Phe-NH- (X-Stag), and then the precipitate was washed under reduced pressure. It was dried to obtain H-Phe-Phe-Phe-NH ₂ .
The purity of the obtained H-Phe-Phe-Phe-NH ₂ was measured by HPLC. The results are shown in Table 2. The HPLC analysis conditions are the same as in Example 1.

In the case of Comparative Example 2 in which no scavenger was used, the purity of the target product, H-Phe-Phe-Phe-NH ₂ , was 62.8%, and the double-hit H-Phe-Phe-Phe-Phe. It was confirmed that -NH ₂ and H-Phe-Phe-Phe-Phe-Phe-NH ₂ which are triple hits were mixed. On the other hand, when taurine was used as a scavenger (Example 2), the purity of the target product was 88.5%, which was higher than that of Comparative Example 2. From the above results, it was shown that taurine is useful as a scavenger for amino acid active esters.

Example 3
Synthesis of H-Asp-Ala-Asn-Cys-Glu-OH 1) Synthesis of H-Glu (OtBu) -O- (B-Stag) 2.40 g (3.0 mmol) of B-Stag was dissolved in 6 mL of CPME and 9 mL of THF. Then, add 3.21 g (7.5 mmol) of Fmoc-Glu (OtBu) -OH, 1.44 g (7.5 mmol) of WSCI / HCl, and 36.8 mg (0.3 mmol) of 4-dimethylaminopyridine, and add 2 at room temperature. Stir for hours. After confirming that B-STAg was 5% or less of the product Fmoc-Glu (OtBu) -O- (B-STAg), 0.94 g (7.5 mmol) of taurine and 38 mL of DMSO were added, and room temperature was reached. Was stirred for 30 minutes. After confirming the compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with taurine used as a scavenger, the mixture was cooled to 0 ° C. and 1.42 g (8.6 mmol) of sodium 2-mercapto-1-ethanesulfonate. Was added, 5.1 mL (34 mmol) of DBU was added, and the mixture was stirred for 20 minutes. After confirming that Fmoc-Glu (OtBu) -O- (B-Stag) was 5% or less of the product H-Glu (OtBu) -O- (B-Stag), a 5% sodium carbonate aqueous solution was used. 75 mL was added dropwise, the temperature was raised to room temperature, and the liquid was separated. 17 mL of 20% saline solution and 6 mL of 5% sodium carbonate aqueous solution and 1.2 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with taurine used as a scavenger, and after concentrating the organic layer, 27 mL of CPME was added to the residue, and H-Glu ( A CPME solution containing OtBu) -O- (B-Stag) was obtained.

2) Synthesis of H-Cys (Trt) -Glu (OtBu) -O- (B-Stag) 7 mL of DMF, Fmoc-Cys ( 2.38 g (4.1 mmol) of Trt) -OH, 1.67 g (3.9 mmol) of COMU, and 2.1 mL of DIEPA were added, and the mixture was stirred at room temperature for 2 hours. After confirming that H-Glu (OtBu) -O- (B-Stag) was 5% or less of the product Fmoc-Cys (Trt) -Glu (OtBu) -O- (B-Stag), 0.55 g (3.9 mmol) of taurine and 16 mL of DMSO were added, and the mixture was stirred at room temperature for 2 hours. After confirming the compound in which the active ester of Fmoc-Cys (Trt) -OH was condensed with taurine used as a scavenger, the mixture was cooled to 0 ° C., and 1.19 g (3.9 mmol) of sodium 2-mercapto-1-ethanesulfonate was used. Was added, 2.7 mL (18 mmol) of DBU was added, and the mixture was stirred for 20 minutes. Fmoc-Cys (Trt) -Glu (OtBu) -O- (B-Stag) was 5% or less of the product H-Cys (Trt) -Glu (OtBu) -O- (B-Stag). After confirming that, 9.6 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 60 mL of a 5% sodium carbonate aqueous solution was added, the temperature was raised to room temperature, and the liquid was separated. 38 mL of 20% saline solution and 13 mL of 5% sodium carbonate aqueous solution and 2.7 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Cys (Trt) -OH was condensed with taurine used as a scavenger, and H-Cys (Trt) -Glu (OtBu) -O- (B) was confirmed. -A CPME solution containing STag) was obtained.

3) Synthesis of H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) The above H-Cys (Trt) -Glu (OtBu) -O- (B-Stag) was used. To the CPME solution containing, CPME 2 mL, DMF 7 mL, Fmoc-Asn (Trt) -OH 2.43 g (4.1 mmol), COMU 1.67 g (3.9 mmol), and DIEPA 2.1 mL were added, and the mixture was stirred at room temperature for 2 hours. H-Cys (Trt) -Glu (OtBu) -O- (B-Stag) is 5 for the product Fmoc-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag). After confirming that the content was less than%, 0.55 g (3.9 mmol) of taurine and 16 mL of DMSO were added, and the mixture was stirred at room temperature for 40 minutes. After confirming the compound in which the active ester of Fmoc-Asn (Trt) -OH was condensed with taurine used as a scavenger, the mixture was cooled to 0 ° C., and 1.19 g (3.9 mmol) of sodium 2-mercapto-1-ethanesulfonate was used. Was added, 2.7 mL (18 mmol) of DBU was added, and the mixture was stirred for 1 hour. Fmoc-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-) After confirming that the content was 5% or less with respect to STag), 60 mL of a 5% sodium carbonate aqueous solution was added dropwise, the temperature was raised to room temperature, and the liquid was separated. 38 mL of 20% saline solution and 13 mL of 5% sodium carbonate aqueous solution and 2.7 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Asn (Trt) -OH was condensed with taurine used as a scavenger, and H-Asn (Trt) -Cys (Trt) -Glu (OtBu) was confirmed. A CPME solution containing —O— (B—Stag) was obtained.

4) Synthesis of H-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) The above H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O -To a CPME solution containing (B-STAg), add 2 mL of CPME, 7 mL of DMF, 1.33 g (4.1 mmol) of Fmoc-Ala-OH monohydrate, 1.67 g (3.9 mmol) of COMU, and 2.1 mL of DIEPA. The mixture was stirred at room temperature for one and a half hours. H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product Fmoc-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- ( After confirming that the content was 5% or less based on B-Stag), 0.55 g (3.9 mmol) of taurine and 16 mL of DMSO were added, and the mixture was stirred at room temperature for 30 minutes. After confirming the compound in which the active ester of Fmoc-Ala-OH was condensed with taurine used as a scavenger, the mixture was cooled to 0 ° C., 1.19 g (3.9 mmol) of sodium 2-mercapto-1-ethanesulfonate, DMF 1 .3 mL was added, 2.7 mL (18 mmol) of DBU was added, and the mixture was stirred for 1 hour. Fmoc-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product H-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O -After confirming that the content was 5% or less with respect to (B-Stag), 9.6 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 60 mL of a 5% sodium carbonate aqueous solution was added, the temperature was raised to room temperature, and the liquid was separated. 38 mL of 20% saline solution and 13 mL of 5% sodium carbonate aqueous solution and 2.7 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Ala-OH was condensed with taurine used as a scavenger, and H-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu)-. A CPME solution containing O- (B-STAg) was obtained.

5) Synthesis of H-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) The above H-Ala-Asn (Trt) -Cys (Trt) CPME solution containing -Glu (OtBu) -O- (B-STAg) contains 2 mL of CPME, 7 mL of DMF, 1.67 g (4.1 mmol) of Fmoc-Asp (OtBu) -OH, 1.67 g (3.9 mmol) of COMU, DIEPA. 2.1 mL was added, and the mixture was stirred at room temperature for 1 hour. H-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product Fmoc-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu After confirming that the content was 5% or less based on (OtBu) -O- (B-Stag), 0.55 g (3.9 mmol) of taurine and 16 mL of DMSO were added, and the mixture was stirred at room temperature for 30 minutes. After confirming the compound in which the active ester of Fmoc-Asp (OtBu) -OH was condensed with taurine used as a scavenger, the mixture was cooled to 0 ° C., and 1.19 g (3.9 mmol) of sodium 2-mercapto-1-ethanesulfonate was used. , DMF 1.3 mL was added, DBU 2.7 mL (18 mmol) was added, and the mixture was stirred for 40 minutes. Fmoc-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product H-Asp (OtBu) -Ala-Asn (Trt) -Cys After confirming that the content was 5% or less with respect to (Trt) -Glu (OtBu) -O- (B-Stag), 9.6 mL of a 1 M sulfuric acid aqueous solution was added dropwise, and then 60 mL of a 5% sodium carbonate aqueous solution was added at room temperature. The temperature was raised to the maximum and the liquid was separated. 38 mL of 20% saline solution and 13 mL of 5% sodium carbonate aqueous solution and 2.7 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Ala-OH was condensed with taurine used as a scavenger, and H-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt)-. A CPME solution containing Glu (OtBu) -O- (B-STAg) was obtained.
The obtained CPME solution was concentrated under reduced pressure, 40 mL of MeCN was added to the residue, the precipitated solid was collected by filtration, and the obtained solid was dried under reduced pressure at 30 ° C. H-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) 4.58 g was obtained.

6) Synthesis of H-Asp-Ala-Asn-Cys-Glu-OH H-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) 962 mg ( To 0.50 mmol), 9.5 mL of trifluoroacetic acid, 0.29 mL of water, 0.29 mL of triisopropylsilane, 865 mg of dithiothreitol and 0.58 mL of anisole were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was cooled to 0 ° C., 72 mL of diisopropyl ether was slowly added dropwise, and the precipitate was collected by filtration. The precipitate collected by filtration was washed 3 times with 10 mL of diisopropyl ether, and then the precipitate was dried under reduced pressure to obtain 275 mg of H-Asp-Ala-Asn-Cys-Glu-OH. The purity of the obtained H-Asp-Ala-Asn-Cys-Glu-OH was 90.5%.

Example 4
Synthesis of H-Gln-Trp-Glu-Arg-Thr-NH ₂ 1) Synthesis of H-Thr (tBu) -NH- (D-Stag) Fmoc-NH- (D-Stag) 1.09 g (1.0 mmol) ) Was dissolved in 9 mL of CPME and 2 mL of DMF, cooled to 0 ° C., 0.32 mL (1.9 mmol) of DIEPA and 0.30 g (1.8 mmol) of sodium 2-mercapto-1-ethanesulfonate were added, and DBU0. 67 mL (4.5 mmol) was added dropwise. After stirring at 0 ° C. for 1 hour and confirming that Fmoc-NH- (D-Stag) was 5% or less of the product NH _2- (D-Stag), 2.4 mL of 1 M sulfuric acid aqueous solution was added dropwise. Then, 10 mL of a 5% aqueous sodium carbonate solution was added, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 25 mL of 20% saline solution and 8 mL of 5% sodium carbonate aqueous solution and 1.8 mL of DMF were added and separated to obtain a CPME solution containing NH _2- (D-Stag).
In this CPME solution containing NH _2- (D-Stag), CPME 0.6 mL, DMF 2 mL, Fmoc-Thr (tBu) -OH 0.55 g (1.4 mmol), COMU 0.56 g (1.3 mmol), DIEPA 0 .71 mL (4.1 mmol) was added, and the mixture was stirred at room temperature for 70 minutes. After confirming that NH _2- (D-Stag) was 5% or less of the product Fmoc-Thr (tBu) -NH- (D-Stag), 2- [2- (2-aminoethoxy) Ethoxy] Ethanol (AEEE) 62 μL (0.5 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. After confirming the compound in which the active ester of Fmoc-Thr (tBu) -OH was condensed with AEEE used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2.4 mmol) of sodium 2-mercapto-1-ethanesulfonate. , DMSO 2.4 mL was added, DBU 0.91 mL (6.1 mmol) was added, and the mixture was stirred for 70 minutes. After confirming that Fmoc-Thr (tBu) -NH- (D-Stag) was 5% or less of the product H-Thr (tBu) -NH- (D-Stag), 1M aqueous sulfuric acid solution 3. 2 mL was added dropwise, 13 mL of a 5% aqueous sodium carbonate solution was added, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 25 mL of 20% saline solution and 8 mL of 5% aqueous sodium carbonate solution and 1.8 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Thr (tBu) -OH was condensed with AEEE used as a scavenger, and contained H-Thr (tBu) -NH- (D-Stag). A CPME solution was obtained.

2) Synthesis of H-Arg (Pbf) -Thr (tBu) -NH- (D-Stag) In the CPME solution containing the above H-Thr (tBu) -NH- (D-Stag), CPME 0.7 mL and DMF 2 mL , Fmoc-Arg (Pbf) -OH 0.88 g (1.4 mmol), COMU 0.56 g (1.3 mmol), DIEPA 0.71 mL (4.1 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After confirming that H-Thr (tBu) -NH- (D-Stag) was 5% or less of the product Fmoc-Arg (Pbf) -Thr (tBu) -NH- (D-Stag), 62 μL (0.5 mmol) of AEEE was added, and the mixture was stirred at room temperature for 30 minutes. After confirming the compound in which the active ester of Fmoc-Arg (Pbf) -OH was condensed with AEEE used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2.4 mmol) of sodium 2-mercapto-1-ethanesulfonate. , DMSO 2.4 mL was added, DBU 0.91 mL (6.1 mmol) was added, and the mixture was stirred for 50 minutes. Fmoc-Arg (Pbf) -Thr (tBu) -NH- (D-Stag) was 5% or less of the product H-Arg (Pbf) -Thr (tBu) -NH- (D-Stag). After confirming that, 13 mL of a 5% sodium carbonate aqueous solution was added dropwise, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Arg (Pbf) -OH was condensed with AEEE used as a scavenger, and H-Arg (Pbf) -Thr (tBu) -NH- (D) was confirmed. -A CPME solution containing Stag) was obtained.

3) Synthesis of H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) The above H-Arg (Pbf) -Thr (tBu) -NH- (D-Stag) was used. To the CPME solution containing, CPME 0.5 mL, DMF 2 mL, Fmoc-Glu (OtBu) -OH 0.58 g (1.4 mmol), COMU 0.56 g (1.3 mmol), DIEPA 0.71 mL (4.1 mmol) were added. The mixture was stirred at room temperature for 1 hour. H-Arg (Pbf) -Thr (tBu) -NH- (D-Stag) is 5 relative to the product Fmoc-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag). After confirming that the content was less than%, 62 μL (0.5 mmol) of AEEE was added, and the mixture was stirred at room temperature for 40 minutes. After confirming the compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with AEEE used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2.4 mmol) of sodium 2-mercapto-1-ethanesulfonate. , DMSO 2.4 mL was added, DBU 0.91 mL (6.1 mmol) was added, and the mixture was stirred for 65 minutes. Fmoc-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) is the product H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-) After confirming that the content was 5% or less with respect to STag), 13 mL of a 5% sodium carbonate aqueous solution was added dropwise, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with AEEE used as a scavenger, and H-Glu (OtBu) -Arg (Pbf) -Thr (tBu). A CPME solution containing -NH- (D-Stag) was obtained.

4) Synthesis of H-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) The above H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) ) -NH- (D-Stag) in CPME solution, CPME 0.5 mL, DMF 2 mL, Fmoc-Trp (Boc) -OH 0.71 g (1.4 mmol), COMU 0.56 g (1.3 mmol), DIEPA 0 .71 mL (4.1 mmol) was added, and the mixture was stirred at room temperature for 70 minutes. H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) is the product Fmoc-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu)- After confirming that the content was 5% or less with respect to NH- (D-Stag), 62 μL (0.5 mmol) of AEEE was added, and the mixture was stirred at room temperature for 40 minutes. After confirming the compound in which the active ester of Fmoc-Trp (Boc) -OH was condensed with AEEE used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2.4 mmol) of sodium 2-mercapto-1-ethanesulfonate. , DMSO 2.4 mL was added, DMF 0.4 mL and DBU 0.91 mL (6.1 mmol) were added, and the mixture was stirred for 65 minutes. Fmoc-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) is the product H-Trp (Boc) -Glu (OtBu) -Arg (Pbf)- After confirming that the ratio was 5% or less with respect to Thr (tBu) -NH- (D-Stag), 3.2 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 13 mL of a 5% sodium carbonate aqueous solution was added, and then the temperature was raised to room temperature. And separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Trp (Boc) -OH was condensed with AEEE used as a scavenger, and H-Trp (Boc) -Glu (OtBu) -Arg (Pbf). A CPME solution containing -Thr (tBu) -NH- (D-Stag) was obtained.

5) Synthesis of H-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) The above H-Trp (Boc) -Glu (OtBu) ) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) in CPME solution, CPME 0.5 mL, DMF 2 mL, Fmoc-Gln (Trt) -OH 0.82 g (1.4 mmol), COMU 0 .56 g (1.3 mmol) and 0.71 mL (4.1 mmol) of DIEPA were added, and the mixture was stirred at room temperature for 1 hour. H-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) is the product Fmoc-Gln (Trt) -Trp (Boc) -Glu (OtBu)- After confirming that the content was 5% or less with respect to Arg (Pbf) -Thr (tBu) -NH- (D-Stag), 62 μL (0.5 mmol) of AEEE was added, and the mixture was stirred at room temperature for 40 minutes. After confirming the compound in which the active ester of Fmoc-Gln (Trt) -OH was condensed with AEEE used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2.4 mmol) of sodium 2-mercapto-1-ethanesulfonate. , DMSO 3.0 mL was added, DMF 0.4 mL and DBU 0.91 mL (6.1 mmol) were added, and the mixture was stirred for 65 minutes. Fmoc-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) is the product H-Gln (Trt) -Trp (Boc)- After confirming that the content was 5% or less of Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag), 3.2 mL of 1 M sulfuric acid aqueous solution was added dropwise, and a 5% sodium carbonate aqueous solution was added. After adding 13 mL, the temperature was raised to room temperature and the solution was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Gln (Trt) -OH was condensed with AEEE used as a scavenger, and H-Gln (Trt) -Trp (Boc) -Glu (OtBu) was confirmed. A CPME solution containing -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) was obtained.
The obtained CPME solution was concentrated under reduced pressure, 24 mL of MeCN, 18 mL of IPA, 6 mL of CPME and 12 mL of water were added to the residue, the precipitated solid was collected by filtration, and the obtained solid was dried under reduced pressure at 30 ° C. H-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-Stag) 1.84 g was obtained.

6) Synthesis of H-Gln-Trp-Glu-Arg-Thr-NH ₂ H-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (D-) To 1.14 g (0.50 mmol) of Stag), 9.5 mL of trifluoroacetic acid, 0.25 mL of water and 0.25 mL of triisopropylsilane were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was cooled to 0 ° C., 70 mL of MTBE was slowly added dropwise, and the precipitate was collected by filtration. The collected precipitate was washed 3 times with 10 mL of MTBE, and then the precipitate was dried under reduced pressure to obtain 227 mg of H-Gln-Trp-Glu-Arg-Thr-NH _{2 2} . The purity of the obtained H-Gln-Trp-Glu-Arg-Thr-NH ₂ was 77.2%.

Example 5
Synthesis of H-Asp-Ala-Asn-Cys-Glu-OH 1) Synthesis of H-Glu (OtBu) -O- (B-Stag) 0.79 g (1.0 mmol) of B-Stag was dissolved in 2 mL of CPME and 3 mL of THF. Then, add 1.06 g (2.5 mmol) of Fmoc-Glu (OtBu) -OH, 0.48 g (2.5 mmol) of WSCI / HCl, and 12.2 mg (0.1 mmol) of 4-dimethylaminopyridine, and add 2 at room temperature. Stir for hours. After confirming that B-STAg was 5% or less of the product Fmoc-Glu (OtBu) -O- (B-STAg), 150 μL (2.5 mmol) of 2-aminoethanol was added, and 2 at room temperature. Stir for hours. After confirming the compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C. and 0.74 g (4) of sodium 2-mercapto-1-ethanesulfonate. .5 mmol) and 4.5 mL of DMSO were added, 1.7 mL (11.3 mmol) of DBU was added, and the mixture was stirred for 1 hour. After confirming that Fmoc-Glu (OtBu) -O- (B-Stag) was 5% or less of the product H-Glu (OtBu) -O- (B-Stag), add 6 mL of 1M sulfuric acid aqueous solution. The mixture was added dropwise, 19 mL of a 5% aqueous sodium carbonate solution was added, the temperature was raised to room temperature, and the solution was separated. 20 mL of 20% saline solution was added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with 2-aminoethanol used as a scavenger. After concentrating the organic layer, 9 mL of CPME was added to the residue, and H was added. A CPME solution containing -Glu (OtBu) -O- (B-STAg) was obtained.

2) Synthesis of H-Cys (Trt) -Glu (OtBu) -O- (B-Stag) 2 mL of DMF, Fmoc-Cys ( Trt) -OH 0.79 g (1.4 mmol), COMU 0.57 g (1.3 mmol), and DIEPA 0.71 mL were added, and the mixture was stirred at room temperature for 1 hour. After confirming that H-Glu (OtBu) -O- (B-Stag) was 5% or less of the product Fmoc-Cys (Trt) -Glu (OtBu) -O- (B-Stag), 81 μL (1.4 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 1.5 hours. After confirming the compound in which the active ester of Fmoc-Cys (Trt) -OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (1) of sodium 2-mercapto-1-ethanesulfonate. .3 mmol) and 2.4 mL of DMSO were added, 0.91 mL (6.1 mmol) of DBU was added, and the mixture was stirred for 25 minutes. Fmoc-Cys (Trt) -Glu (OtBu) -O- (B-Stag) was 5% or less of the product H-Cys (Trt) -Glu (OtBu) -O- (B-Stag). After confirming that, 3.2 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 13 mL of a 5% sodium carbonate aqueous solution was added, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Cys (Trt) -OH was condensed with 2-aminoethanol used as a scavenger, and H-Cys (Trt) -Glu (OtBu) -O was confirmed. -A CPME solution containing (B-STAg) was obtained.

3) Synthesis of H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) The above H-Cys (Trt) -Glu (OtBu) -O- (B-Stag) was used. To the CPME solution containing, CPME 0.5 mL, DMF 2 mL, Fmoc-Asn (Trt) -OH 0.81 g (1.4 mmol), COMU 0.56 g (1.3 mmol), and DIEPA 0.71 mL were added, and the mixture was stirred at room temperature for 55 minutes. .. H-Cys (Trt) -Glu (OtBu) -O- (B-Stag) is 5 for the product Fmoc-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag). After confirming that the content was less than%, 81 μL (1.4 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 25 minutes. After confirming the compound in which the active ester of Fmoc-Asn (Trt) -OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2) of sodium 2-mercapto-1-ethanesulfonate was confirmed. .4 mmol) and 2.4 mL of DMSO were added, 0.91 mL (6.1 mmol) of DBU was added, and the mixture was stirred for 75 minutes. Fmoc-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-) After confirming that the content was 5% or less with respect to STag), 3.2 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 13 mL of a 5% sodium carbonate aqueous solution was added, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Asn (Trt) -OH was condensed with 2-aminoethanol used as a scavenger, and H-Asn (Trt) -Cys (Trt) -Glu was confirmed. A CPME solution containing (OtBu) -O- (B-STAg) was obtained.

4) Synthesis of H-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) The above H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O -CPME solution containing (B-STAg) 0.5 mL CPME, 2 mL DMF, 0.47 g (1.5 mmol) Fmoc-Ala-OH monohydrate, 0.64 g (1.5 mmol) COMU, 0.78 mL DIEPA (4.5 mmol) was added, and the mixture was stirred at room temperature for 1 hour. H-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product Fmoc-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- ( After confirming that the content was 5% or less with respect to B-Stag), 89 μL (1.5 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 2 hours. After confirming the compound in which the active ester of Fmoc-Ala-OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C., and 0.40 g (2.5 mmol) of sodium 2-mercapto-1-ethanesulfonate was used. , DMSO 2.5 mL was added, DBU 0.91 mL (6.1 mmol) was added, and the mixture was stirred for 1 hour. Fmoc-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product H-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O -After confirming that the content was 5% or less with respect to (B-Stag), 3.2 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 13 mL of a 5% sodium carbonate aqueous solution was added, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that there was no compound in which the active ester of Fmoc-Ala-OH was condensed with 2-aminoethanol used as a scavenger in the obtained organic layer, and it was confirmed that H-Asn (Trt) -Cys (Trt) -Glu (OtBu). A CPME solution containing —O— (B—STAg) was obtained.

5) Synthesis of H-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) The above H-Ala-Asn (Trt) -Cys (Trt) CPME solution containing -Glu (OtBu) -O- (B-Stag) 0.5 mL CPME, 2 mL DMF, 0.56 g (1.4 mmol) Fmoc-Asp (OtBu) -OH 0.56 g (1.3 mmol) COMU , DIEPA 0.71 mL (4.1 mmol) was added, and the mixture was stirred at room temperature for 1 hour. H-Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product Fmoc-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu After confirming that the content was 5% or less with respect to (OtBu) -O- (B-Stag), 81 μL (1.4 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 70 minutes. After confirming the compound in which the active ester of Fmoc-Asp (OtBu) -OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2) of sodium 2-mercapto-1-ethanesulfonate was confirmed. .4 mmol) and 2.4 mL of DMSO were added, 0.91 mL (6.1 mmol) of DBU was added, and the mixture was stirred for 75 minutes. Fmoc-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) is the product H-Asp (OtBu) -Ala-Asn (Trt) -Cys After confirming that it was 5% or less of (Trt) -Glu (OtBu) -O- (B-Stag), add 3.2 mL of water, add 13 mL of 5% sodium carbonate aqueous solution, and raise to room temperature. It was warmed and separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Asp (OtBu) -OH was condensed with 2-aminoethanol used as a scavenger, and H-Asp (OtBu) -Ala-Asn (Trt) was confirmed. A CPME solution containing -Cys (Trt) -Glu (OtBu) -O- (B-Stag) was obtained.
The obtained CPME solution was concentrated under reduced pressure, 40 mL of MeCN was added to the residue, the precipitated solid was collected by filtration, and the obtained solid was dried under reduced pressure at 30 ° C. H-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) 1.56 g was obtained.

6) Synthesis of H-Asp-Ala-Asn-Cys-Glu-OH H-Asp (OtBu) -Ala-Asn (Trt) -Cys (Trt) -Glu (OtBu) -O- (B-Stag) 962 mg ( 7.5 mL of trifluoroacetic acid, 0.21 mL of water, 0.21 mL of triisopropylsilane, and 0.42 mL of 3,6-dioxa-1,8-octanedithiol were added to 0.50 mmol), and the mixture was stirred at room temperature for 2 hours. The reaction solution was cooled to 0 ° C., 60 mL of MTBE was slowly added dropwise, and the precipitate was collected by filtration. The collected precipitate was washed 3 times with 10 mL of MTBE, and then the precipitate was dried under reduced pressure to obtain 275 mg of H-Asp-Ala-Asn-Cys-Glu-OH. The purity of the obtained H-Asp-Ala-Asn-Cys-Glu-OH was 78.4%.

Example 6
Synthesis of H-Gln-Trp-Glu-Arg-Thr-NH ₂ 1) Synthesis of H-Thr (tBu) -NH- (X-Stag) Fmoc-NH- (X-Stag) 1.10 g (1.0 mmol) ) Was dissolved in 9 mL of CPME and 2 mL of DMF, cooled to 0 ° C., 0.33 mL (1.9 mmol) of DIEPA and 0.30 g (1.8 mmol) of sodium 2-mercapto-1-ethanesulfonate were added, and DBU0. 67 mL (4.5 mmol) was added dropwise. After stirring at 0 ° C. for 1 hour and confirming that Fmoc-NH- (X-Stag) was 5% or less of the product NH _2- (X-Stag), 4.8 mL of 1 M sulfuric acid aqueous solution was added dropwise. Then, 10 mL of a 5% aqueous sodium carbonate solution was added, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 25 mL of 20% saline solution and 8 mL of 5% sodium carbonate aqueous solution and 1.8 mL of DMF were added and separated to obtain a CPME solution containing NH _2- (X-Stag).
In this CPME solution containing NH _2- (X-Stag), CPME 0.6 mL, DMF 2 mL, Fmoc-Thr (tBu) -OH 0.54 g (1.4 mmol), COMU 0.57 g (1.3 mmol), DIEPA 0 .71 mL (4.1 mmol) was added, and the mixture was stirred at room temperature for 70 minutes. After confirming that NH _2- (X-Stag) was 5% or less of the product Fmoc-Thr (tBu) -NH- (X-Stag), 0.11 g (1.0 mmol) of aminomethanephosphonic acid. ), DMSO 2.4 mL was added, and the mixture was stirred at room temperature for 20 minutes. After confirming the compound in which the active ester of Fmoc-Thr (tBu) -OH was condensed with the aminomethanephosphonic acid used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2) of sodium 2-mercapto-1-ethanesulfonate was confirmed. .4 mmol) was added, 0.91 mL (6.1 mmol) of DBU was added, and the mixture was stirred for 70 minutes. After confirming that Fmoc-Thr (tBu) -NH- (X-Stag) was 5% or less of the product H-Thr (tBu) -NH- (X-Stag), 1M aqueous sulfuric acid solution 3. 2 mL was added dropwise, 13 mL of a 5% aqueous sodium carbonate solution was added, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 25 mL of 20% saline solution and 8 mL of 5% aqueous sodium carbonate solution and 1.8 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Thr (tBu) -OH was condensed with the aminomethanephosphonic acid used as a scavenger, and it was confirmed that there was no compound condensed with H-Thr (tBu) -NH- (X-Stag). ) Was obtained.

2) Synthesis of H-Arg (Pbf) -Thr (tBu) -NH- (X-Stag) In the CPME solution containing the above H-Thr (tBu) -NH- (X-Stag), CPME 0.7 mL, DMF 2 mL , Fmoc-Arg (Pbf) -OH 0.88 g (1.4 mmol), COMU 0.56 g (1.3 mmol), DIEPA 0.71 mL (4.1 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After confirming that H-Thr (tBu) -NH- (X-Stag) was 5% or less of the product Fmoc-Arg (Pbf) -Thr (tBu) -NH- (X-Stag), 81 μL (1.4 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 30 minutes. After confirming the compound in which the active ester of Fmoc-Arg (Pbf) -OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2) of sodium 2-mercapto-1-ethanesulfonate. .4 mmol) and 2.4 mL of DMSO were added, 0.91 mL (6.1 mmol) of DBU was added, and the mixture was stirred for 50 minutes. Fmoc-Arg (Pbf) -Thr (tBu) -NH- (X-Stag) was 5% or less of the product H-Arg (Pbf) -Thr (tBu) -NH- (X-Stag). After confirming that, 13 mL of a 5% sodium carbonate aqueous solution was added dropwise, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Arg (Pbf) -OH was condensed with 2-aminoethanol used as a scavenger, and H-Arg (Pbf) -Thr (tBu) -NH was confirmed. -A CPME solution containing (X-STAg) was obtained.

3) Synthesis of H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) The above H-Arg (Pbf) -Thr (tBu) -NH- (X-Stag) was used. To the CPME solution containing, CPME 0.5 mL, DMF 2 mL, Fmoc-Glu (OtBu) -OH 0.57 g (1.4 mmol), COMU 0.56 g (1.3 mmol), DIEPA 0.71 mL (4.1 mmol) were added. The mixture was stirred at room temperature for 1 hour. H-Arg (Pbf) -Thr (tBu) -NH- (X-Stag) is 5 relative to the product Fmoc-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag). After confirming that the content was less than%, 81 μL (1.0 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 40 minutes. After confirming the compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with AEEE used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2.4 mmol) of sodium 2-mercapto-1-ethanesulfonate. , DMSO 2.4 mL, DMF 0.4 mL was added, DBU 0.91 mL (6.1 mmol) was added, and the mixture was stirred for 65 minutes. Fmoc-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) is the product H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-) After confirming that the content was 5% or less with respect to STag), 13 mL of a 5% sodium carbonate aqueous solution was added dropwise, the temperature was raised to room temperature, and the liquid was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Glu (OtBu) -OH was condensed with 2-aminoethanol used as a scavenger, and H-Glu (OtBu) -Arg (Pbf) -Thr. A CPME solution containing (tBu) -NH- (X-STAg) was obtained.

4) Synthesis of H-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) The above H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) ) -NH- (X-Stag) in CPME solution, CPME 0.5 mL, DMF 2 mL, Fmoc-Trp (Boc) -OH 0.71 g (1.4 mmol), COMU 0.56 g (1.3 mmol), DIEPA 0 .71 mL (4.1 mmol) was added, and the mixture was stirred at room temperature for 70 minutes. H-Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) is the product Fmoc-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu)- After confirming that the content was 5% or less with respect to NH- (X-Stag), 81 μL (1.0 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 40 minutes. After confirming the compound in which the active ester of Fmoc-Trp (Boc) -OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2) of sodium 2-mercapto-1-ethanesulfonate was confirmed. .4 mmol) and 2.4 mL of DMSO were added, 0.4 mL of DMF and 0.91 mL (6.1 mmol) of DBU were added, and the mixture was stirred for 65 minutes. Fmoc-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) is the product H-Trp (Boc) -Glu (OtBu) -Arg (Pbf)- After confirming that the ratio was 5% or less with respect to Thr (tBu) -NH- (X-Stag), 3.2 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 13 mL of a 5% sodium carbonate aqueous solution was added, and then the temperature was raised to room temperature. And separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Trp (Boc) -OH was condensed with 2-aminoethanol used as a scavenger, and H-Trp (Boc) -Glu (OtBu) -Arg was confirmed. A CPME solution containing (Pbf) -Thr (tBu) -NH- (X-Stag) was obtained.

5) Synthesis of H-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) The above H-Trp (Boc) -Glu (OtBu) ) -Arg (Pbf) -Thr (tBu) -NH- (X-STAg) in CPME solution, CPME 0.5 mL, DMF 2 mL, Fmoc-Gln (Trt) -OH 0.83 g (1.4 mmol), COMU 0 .56 g (1.3 mmol) and 0.71 mL (4.1 mmol) of DIEPA were added, and the mixture was stirred at room temperature for 1 hour. H-Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) is the product Fmoc-Gln (Trt) -Trp (Boc) -Glu (OtBu)- After confirming that the content was 5% or less with respect to Arg (Pbf) -Thr (tBu) -NH- (X-Stag), 81 μL (1.0 mmol) of 2-aminoethanol was added, and the mixture was stirred at room temperature for 40 minutes. After confirming the compound in which the active ester of Fmoc-Gln (Trt) -OH was condensed with 2-aminoethanol used as a scavenger, the mixture was cooled to 0 ° C. and 0.40 g (2) of sodium 2-mercapto-1-ethanesulfonate was confirmed. .4 mmol), DMSO 3.0 mL was added, DMF 0.4 mL and DBU 0.91 mL (6.1 mmol) were added, and the mixture was stirred for 65 minutes. Fmoc-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) is the product H-Gln (Trt) -Trp (Boc)- After confirming that the content was 5% or less of Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag), 3.2 mL of 1 M sulfuric acid aqueous solution was added dropwise, and a 5% sodium carbonate aqueous solution was added. After adding 13 mL, the temperature was raised to room temperature and the solution was separated. To the obtained organic layer, 13 mL of 20% saline solution and 4 mL of 5% aqueous sodium carbonate solution and 0.9 mL of DMF were added and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Gln (Trt) -OH was condensed with 2-aminoethanol used as a scavenger, and H-Gln (Trt) -Trp (Boc) -Glu was confirmed. A CPME solution containing (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) was obtained.
The obtained CPME solution was concentrated under reduced pressure, 25 mL of MeCN and 15 mL of MeOH were added to the residue, the precipitated solid was collected by filtration, and the obtained solid was dried under reduced pressure at 30 ° C. H-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-Stag) 1.26 g was obtained.

6) Synthesis of H-Gln-Trp-Glu-Arg-Thr-NH ₂ H-Gln (Trt) -Trp (Boc) -Glu (OtBu) -Arg (Pbf) -Thr (tBu) -NH- (X-) To 1.15 g (0.50 mmol) of Stag), 9.5 mL of trifluoroacetic acid, 0.25 mL of water and 0.25 mL of triisopropylsilane were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was cooled to 0 ° C., 70 mL of MTBE was slowly added dropwise, and the precipitate was collected by filtration. The collected precipitate was washed 3 times with 10 mL of MTBE, and then the precipitate was dried under reduced pressure to obtain 358 mg of H-Gln-Trp-Glu-Arg-Thr-NH ₂ . The purity of the obtained H-Gln-Trp-Glu-Arg-Thr-NH ₂ was 78.1%.

Example 7
Synthesis of H-Gln-Asn-Cys-Arg-OH 1) Synthesis of H-Arg (Pbf) -O- (B-Stag) 0.40 g (0.5 mmol) of B-Stag was dissolved in 1 mL of CPME and 1.5 mL of THF. Then, add 0.81 g (1.3 mmol) of Fmoc-Arg (Pbf) -OH, 0.24 g (1.3 mmol) of WSCI / HCl, and 6.1 mg (0.05 mmol) of 4-dimethylaminopyridine, and add 2 at room temperature. Stirred for hours. After confirming that B-STAg was 10% or less of the product Fmoc-Arg (Pbf) -O- (B-STAg), 0.18 g (1) of 2-aminoethyl dihydrogen phosphate (AEDP). .3 mmol), 2.5 mL of DMSO was added, and the mixture was stirred at room temperature for 1 hour. After confirming the compound in which the active ester of Fmoc-Arg (Pbf) -OH was condensed with AEDP used as a scavenger, the mixture was cooled to 0 ° C. and cooled to 0 ° C., and 0.37 g (2.3 mmol) of sodium 2-mercapto-1-ethanesulfonate was used. Was added, 0.84 mL (5.6 mmol) of DBU was added, and the mixture was stirred for 65 minutes. After confirming that Fmoc-Arg (Pbf) -O- (B-STAg) was 10% or less of the product H-Arg (Pbf) -O- (B-STAg), 5% potassium hydrogen carbonate 15 mL of the aqueous solution and 3 mL of CPME were added dropwise, the temperature was raised to room temperature, and the liquid was separated. 6 mL of 20% saline solution was added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Arg (Pbf) -OH was condensed with AEDP used as a scavenger, and after concentrating the organic layer, 0.6 mL of CPME was added to the residue, and H- A CPME solution containing Arg (Pbf) -O- (B-STAg) was obtained.

2) Synthesis of H-Cys (Trt) -Arg (Pbf) -O- (B-Stag) 7 mL of DMF, Fmoc-Cys ( Trt) -OH 0.40 g (0.7 mmol), COMU 0.28 g (0.7 mmol), and DIEPA 0.24 mL were added, and the mixture was stirred at room temperature for 80 minutes. After confirming that H-Arg (Pbf) -O- (B-Stag) was 10% or less of the product Fmoc-Cys (Trt) -Arg (Pbf) -O- (B-Stag), 92 mg (0.7 mmol) of AEDP and 1.3 mL of DMSO were added, and the mixture was stirred at room temperature for 2 hours. After confirming the compound in which the active ester of Fmoc-Cys (Trt) -OH was condensed with AEDP used as a scavenger, the mixture was cooled to 0 ° C. and 0.21 g (1.2 mmol) of sodium 2-mercapto-1-ethanesulfonate. Was added, 0.45 mL (3 mmol) of DBU was added, and the mixture was stirred for 50 minutes. Fmoc-Cys (Trt) -Arg (Pbf) -O- (B-Stag) was 10% or less of the product H-Cys (Trt) -Arg (Pbf) -O- (B-Stag). After confirming that, 1.6 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 7 mL of a 5% potassium hydrogen carbonate aqueous solution was added, the temperature was raised to room temperature, and the liquid was separated. 8 mL of 20% saline solution and 0.4 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Cys (Trt) -OH was condensed with AEDP used as a scavenger, and H-Cys (Trt) -Arg (Pbf) -O- (B) was confirmed. -A CPME solution containing STag) was obtained.

3) Synthesis of H-Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-Stag) The above H-Cys (Trt) -Arg (Pbf) -O- (B-Stag) was used. To the CPME solution containing, CPME 0.2 mL, DMF 1.1 mL, Fmoc-Asn (Trt) -OH 0.40 g (0.7 mmol), COMU 0.28 g (0.7 mmol), DIEPA 0.24 mL were added, and 65 minutes at room temperature. Stirred. H-Cys (Trt) -Arg (Pbf) -O- (B-Stag) is 10 relative to the product Fmoc-Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-Stag). After confirming that the content was less than%, 92 mg (0.7 mmol) of AEDP and 1.3 mL of DMSO were added, and the mixture was stirred at room temperature for 90 minutes. After confirming the compound in which the active ester of Fmoc-Asn (Trt) -OH was condensed with AEDP used as a scavenger, the mixture was cooled to 0 ° C. and 0.21 g (1.2 mmol) of sodium 2-mercapto-1-ethanesulfonate. Was added, 0.45 mL (3 mmol) of DBU was added, and the mixture was stirred for 1 hour. Fmoc-Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-Stag) is the product H-Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-) After confirming that the content was 10% or less with respect to STag), 1.6 mL of a 1 M sulfuric acid aqueous solution was added dropwise, 7 mL of a 5% potassium hydrogen carbonate aqueous solution was added, the temperature was raised to room temperature, and the liquid was separated. 8 mL of 20% saline solution and 0.4 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Asn (Trt) -OH was condensed with AEDP used as a scavenger, and H-Asn (Trt) -Cys (Trt) -Arg (Pbf) was confirmed. A CPME solution containing —O— (B—STAg) was obtained.

4) Synthesis of H-Gln (Trt) -Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-Stag) The above H-Asn (Trt) -Cys (Trt) -Arg (Pbf) ) -O- (B-Stag) in CPME solution containing 0.2 mL of CPME, 1.1 mL of DMF, 0.48 g (0.75 mmol) of Fmoc-Gln (Trt) -OH, 0.31 g (0.73 mmol) of COMU, DIEPA 0 .39 mL was added and stirred at room temperature for 80 minutes. H-Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-Stag) is the product Fmoc-Gln (Trt) -Asn (Trt) -Cys (Trt) -Arg (Pbf)- After confirming that the content was 10% or less with respect to O- (B-STAg), 0.10 g (0.73 mmol) of AEDP and 1.5 mL of DMSO were added, and the mixture was stirred at room temperature for 45 minutes. After confirming the compound in which the active ester of Fmoc-Gln (Trt) -OH was condensed with AEDP used as a scavenger, the mixture was cooled to 0 ° C. and 0.22 g (1.4 mmol) of sodium 2-mercapto-1-ethanesulfonate. Was added, 0.50 mL (3.4 mmol) of DBU was added, and the mixture was stirred for 1 hour. Fmoc-Gln (Trt) -Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-Stag) is the product H-Gln (Trt) -Asn (Trt) -Cys (Trt)- After confirming that it was 10% or less of Arg (Pbf) -O- (B-Stag), 1.8 mL of 1 M sulfuric acid aqueous solution was added dropwise, then 7 mL of 5% potassium hydrogen carbonate aqueous solution was added, and the temperature was raised to room temperature. And separated. 8 mL of 20% saline solution and 0.4 mL of DMF were added to the obtained organic layer, and the liquids were separated. It was confirmed that the obtained organic layer had no compound in which the active ester of Fmoc-Gln (Trt) -OH was condensed with AEDP used as a scavenger, and H-Gln (Trt) -Asn (Trt) -Cys (Trt) was confirmed. A CPME solution containing -Arg (Pbf) -O- (B-STAg) was obtained.
The obtained CPME solution was concentrated under reduced pressure to obtain 1.58 g of H-Gln (Trt) -Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-STAg) as a residue. ..

5) Synthesis of H-Gln-Asn-Cys-Arg-OH The above H-Gln (Trt) -Asn (Trt) -Cys (Trt) -Arg (Pbf) -O- (B-Stag) 1.58 ( Add 10 mL of a pre-prepared trifluoroacetic acid / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol mixture (90 / 2.5 / 2.5 / 5) to (equivalent to 0.50 mmol). , Stirred at room temperature for 2 hours. The reaction solution was cooled to 0 ° C., 60 mL of MTBE was slowly added dropwise, and the precipitate was collected by filtration. The collected precipitate was washed 3 times with 10 mL of MTBE, and then the precipitate was dried under reduced pressure to obtain 275 mg of H-Gln-Asn-Cys-Arg-OH. The purity of the obtained H-Gln-Asn-Cys-Arg-OH was 82.6%.
As described above, the aminosulfonic acids, aminosulfates, aminophosphonic acids, aminophosphates and aminoalcohols found in the present invention function as scavengers for amino acid active esters, and are continuously subjected to peptide elongation reactions, especially in one pot. It has been shown to be useful for peptide elongation reactions.

Claims (5)

A method for producing a liquid phase peptide, which comprises the following steps a to d.
a. A step of condensing an amino acid, peptide or amino acid amide to which a carrier for liquid phase peptide synthesis is bound and an amino acid or peptide whose amino group is protected by an amino protecting group in a solvent containing an organic solvent.
b. Aminosulfonic acids and aminosulfuric acids represented by the following general formula (1) are added to the reaction solution after the condensation reaction.

( R ¹ indicates a divalent organic group having 1 to 10 carbon atoms, and X ¹ indicates a single bond or an oxygen atom).
Aminophosphonic acids and aminophosphates represented by the general formula (2), and

(R ² indicates a divalent organic group having 1 to 10 carbon atoms, and X ² indicates a single bond or an oxygen atom)
A step of adding an amino acid active ester scavenger selected from amino alcohols represented by the general formula (3) ,

(N indicates 0 or an integer of 2 to 20, and R ³ and R ⁴ independently indicate a hydrogen atom, a methyl group, an ethyl group, or a hydroxymethyl group).
c. The step of removing the amino-protecting group of the compound whose amino-protecting group is protected by the amino-protecting group in the reaction solution.
d. After adding the aqueous solution to the reaction solution, the solution is separated to contain a condensate of the amino acid, peptide or amino acid amide to which the carrier for liquid phase peptide synthesis is bound and the amino acid or peptide from which the amino protecting group has been removed. A step of obtaining a solvent layer. The amino acid active ester scavenger is an aminosulfonic acid represented by the following general formula (1a),

(R ¹ indicates a divalent organic group having 1 to 10 carbon atoms)
Aminophosphonic acids and aminophosphates represented by the general formula (2), and

(R ² indicates a divalent organic group having 1 to 10 carbon atoms, and X ² indicates a single bond or an oxygen atom)
The liquid phase peptide production method according to claim 1, which is a compound selected from amino alcohols represented by the general formula (3).

(N indicates 0 or an integer of 2 to 20, and R ³ and R ⁴ independently indicate a hydrogen atom, a methyl group, an ethyl group, or a hydroxymethyl group). The liquid phase peptide production method according to claim 1 or 2 , wherein the amino protecting group is a protecting group selected from an Fmоc group, a Bоc group, a Cbz group and an Ac group. Any of claims 1 to 3 , wherein the carrier for liquid phase peptide synthesis is a compound that binds to an amino acid, peptide or amino acid amide directly or via a linker to make them soluble in an organic solvent and insoluble in water. The method for producing a liquid phase peptide according to item 1. The carrier for liquid phase peptide synthesis has the following formula (I):

[During the ceremony,
Ring A represents an aromatic ring of C4-18 which may contain a hetero atom and may be polycyclic; R ¹¹ is a hydrogen atom or ring A is a benzene ring and Rb is the following formula (a). When it is a group represented by, it shows a single bond together with R ¹⁴ to form a fluorene ring with rings A and B, or xanthene with rings A and B via an oxygen atom. Rings may be formed; p Xs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^17- (R). ¹⁷ indicates a hydrogen atom, an alkyl group or an aralkyl group); p R ^12s may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom. Representing an organic group having a hydrocarbon group; q R ^13s are each independently a hydrogen atom or an aliphatic group which may be substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom. An organic group having a hydrocarbon group is shown; p and q are integers of 0 to 3 and p + q is 1 or more and 4 or less, respectively; ring A is a halogen atom and a halogen atom in addition to p XR ¹² . It may have a substituent selected from the group consisting of a C1-6 alkyl group optionally substituted with and a C1-6 alkoxy group optionally substituted with a halogen atom; Ra is a hydrogen atom. , Or an aromatic ring that may be substituted with a halogen atom; Rb is a hydrogen atom, or formula (a) :.

(In the formula, * indicates the bond position;
r and s are integers of 0 to 3 and r + s is 4 or less, respectively;
The r Zs are independently -O-, -S-, -C (= O) O-, -C (= O) NH- or -NR ^18- (R ¹⁸ is a hydrogen atom, an alkyl group or Indicates an aralkyl group.) Indicates;
The r R ^15s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
The s R ^16s represent organic groups having an aliphatic hydrocarbon group which may be independently substituted with a silyl group or an aliphatic hydrocarbon group via an oxygen atom;
R ¹⁴ either exhibits a hydrogen atom or exhibits a single bond with R ¹¹ to form a fluorene ring with rings A and B, or a xanthene ring with rings A and B via an oxygen atom. May form;
Ring B is a group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom, in addition to r ZR ¹⁵ . It may have a substituent selected from. ) Indicates a group;
Y represents a hydroxy group, NHR ¹⁹ (R ¹⁹ indicates a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom. ]
The liquid phase peptide production method according to any one of claims 1 to 4 , which is a compound represented by.