JP5217546B2 - Process for producing amino acid-N-carboxyanhydride - Google Patents

Description

  The present invention relates to an efficient method for producing an amino acid-N-carboxyanhydride useful as an intermediate raw material for polypeptide synthesis.

  Amino acid-N-carboxyanhydrides are useful as intermediates in the synthesis of polypeptides from amino acids. As a method for synthesizing the amino acid-N-carboxyanhydride, a method in which phosgene is reacted with an amino acid has a high yield, and thus has become mainstream.

  However, since phosgene is an extremely toxic gas, handling thereof requires strict attention from the viewpoint of environmental problems and safety. Therefore, the use of phosgene is severely limited, limiting the industrial use of amino acid-N-carboxyanhydrides. From this point of view, various methods that do not use phosgene have been studied (Patent Documents 1 to 4 and Non-Patent Documents 1 to 3), but none of them are industrially applicable or use phosgene as a raw material. There was a problem.

Therefore, the present inventor reacted an amino acid with bis (substituted phenyl) carbonate, or reacted an amino acid ester with bis (substituted phenyl) carbonate to form a carbamate form of the amino acid ester, and after removing the ester, the amino acid was heated. It discovered that -N-carboxyanhydride was obtained efficiently and applied for a patent (patent document 5).
US Pat. No. 5,359,086 JP-A-11-29560 JP 2000-327666 A JP 2002-322160 A JP 2007-22932 A Tetrahedron Letters, 1996, 37, 9043. Chemistry Letters, 2003, 32, 830. Macromolecules 2004, 37, 251.

The method described in Patent Document 5 is a method that can be applied industrially without using phosgene, but the substituent on the phenyl group of bis (substituted phenyl) carbonate is 2,4-dinitro group, pentafluoro group. In the case of a strong electron-withdrawing group such as, the reaction proceeds at a high yield, but in the case where the substituent on the phenyl group is a group having a weak electron-withdrawing group such as a p-nitro group. The rate was low.
Accordingly, an object of the present invention is to provide a new method for producing an amino acid-N-carboxy anhydride in a high yield without using phosgene.

  The present inventor examined the reason why the yield of amino acid-N-carboxyanhydride is low when the substituent on the phenyl group of bis (substituted phenyl) carbonate is a group with weak electron-withdrawing property such as p-nitro group. As a result, it was found that the produced amino acid-N-carboxyanhydride was decomposed. Then, when the means for suppressing the decomposition of amino acid-N-carboxyanhydride and proceeding the reaction to amino acid-N-carboxyanhydride was studied, an amino acid obtained by the reaction of bis (substituted phenyl) carbonate with an amino acid. It has been found that by reacting an ester carbamate in the presence of a protonic acid, decomposition of the amino acid-N-carboxyanhydride is prevented, and the amino acid-N-carboxyanhydride can be obtained quantitatively. The amino acid ester carbamate obtained by the reaction of a bis (phenyl) carbonate having no substituent with an amino acid is used not only in the case where the substituent is a group having a weak electron-withdrawing property such as a p-nitro group. By reacting in the presence, amino acid-N-carboxyanhydride is obtained quantitatively Heading the door, and have completed the present invention.

  That is, the present invention relates to the general formula (1)

(Wherein a ¹ R ^{1 s} are the same or different and each represents an electron-withdrawing substituent selected from a nitro group, a halogen atom, a cyano group, an acyl group, an alkyloxycarbonyl group, a perfluoroalkyl group, and a perchloroalkyl group. A represents an integer of 0 to 5; R ² and R ³ are each independently a hydrogen atom, an optionally substituted alkyl group, an optionally substituted cycloalkyl group, An aryl group which may be substituted or a heterocyclic ring which may be substituted, or R ² and R ³ may combine to form a cycloalkyl group, and the cycloalkyl group may be an aromatic ring or a hetero ring as a condensed ring. (It may have a ring.)
The amino acid carbamate represented by the general formula (2) is reacted in the presence of a protonic acid.

(In the formula, R ² and R ³ are the same as above.)
The manufacturing method of the amino acid-N-carboxy anhydride represented by these is provided.

  According to the production method of the present invention, amino acid-N-carboxyanhydride can be obtained in a high yield by a simple method without using phosgene.

A R ¹ in the general formula (1) is an electron-withdrawing substituent selected from a nitro group, a halogen atom, a cyano group, an acyl group, an alkyloxycarbonyl group, a perfluoroalkyl group, and a perchloroalkyl group; is there. Among these, a nitro group, a halogen atom, and a cyano group are more preferable from the viewpoint of availability of raw materials and stability. R ¹ is preferably substituted at the p-position of the carbamate group.
As an integer of 0-5 represented by a, 0-3 are preferable, and 0 or 1 is more preferable.
Therefore, as an ester residue including a phenyl group and R ¹ , a p-nitrophenyl group, a p-chlorophenyl group, a p-cyanophenyl group, and a phenyl group are particularly preferable.

R ² and R ^{3 in} general formulas (1) and (2) are each independently a hydrogen atom, an optionally substituted alkyl group, an optionally substituted cycloalkyl group, or an optionally substituted aryl. Or R ² and R ³ may be combined to form a cycloalkyl group, and the cycloalkyl group has an aromatic ring or a heterocyclic ring as a condensed ring. You may do it. Here, as an alkyl group, a C1-C12 linear or branched alkyl group is mentioned, A C1-C6 linear or branched alkyl group is preferable. Examples of the cycloalkyl group include cycloalkyl groups having 3 to 6 carbon atoms. Examples of the aryl group include an aryl group having 6 to 10 carbon atoms, such as a phenyl group. Examples of the heterocyclic ring include indole, pyrrolidine, imidazole, pyrrole, piperidine, dihydroquinoline and the like. Examples of the cycloalkyl group formed by combining R ² and R ³ include cycloalkyl groups having 3 to 6 carbon atoms. Examples of the group that may be substituted with these groups include a phenyl group, a hydroxy group, a mercapto group, an amino group, a carboxyl group, a cyano group, and an alkoxy group. Here, as an alkoxy group, a C1-C6 alkoxy group is preferable.

  The compound of general formula (1), which is a raw material compound of the production method of the present invention, can be produced, for example, according to the following reaction formula.

(Wherein R ⁴ represents an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocycle or a commonly used ester protecting group; R ¹ , a, R ² and R ³ are the same as above.)

  That is, compound (1) can be obtained by reacting bis ((substituted) phenyl) carbonate (3) with amino acid ester (4) and then removing the ester protecting group.

  Here, bis ((substituted) phenyl) carbonate (3) can be produced safely and inexpensively by the reaction of dimethyl carbonate and (substituted) phenol. Specific examples of bis ((substituted) phenyl) carbonate (3) include bis (4-nitrophenyl) carbonate, bis (2-nitrophenyl) carbonate, bis (4-chlorophenyl) carbonate, bis (2,4-dichlorophenyl). ) Carbonate, bis (pentafluorophenyl) carbonate, bis (phenyl) carbonate, and the like.

  Examples of amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, histidine, methionine, cysteine, cystine, arginine, lysine, serine, threonine, glutamic acid, glutamine, aspartic acid, asparagine and the like. Examples include α-amino acids that are the main constituents of proteins, ortinin, norleucine, selenocysteine, cysteine sulfonic acid, and the like. Further, β-amino acids, γ-amino acids and the like can be used according to the purpose of use. Although the amino acid ester represented by Formula (4) is not specifically limited, What is obtained by making a basic compound, such as an amine, act on amino acid ester-acid salt like amino acid ester-hydrochloride can be used. Examples of amino acid ester-acid salts include amino acid ester hydrochloride, amino acid ester sulfate, and amino acid ester p-toluenesulfonate. As the amino acid ester represented by the formula (4), t-butyl ester is preferably used because deprotection can be easily performed. Examples of the amine include triethylamine, pyridine, and imidazole.

  The reaction between bis ((substituted) phenyl) carbonate (3) and amino acid ester (4) is, for example, 0.1 to 10 mol of bis ((substituted) phenyl) carbonate (3) per 1 mol of amino acid ester (4). ) And stirring with heating. The reaction is preferably performed in a solvent such as tetrahydrofuran at a temperature of 60 ° C. for 24 hours.

  The deprotection reaction of the obtained amino acid ester carbamate (5) is preferably carried out by reacting an acid or base such as trifluoroacetic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide or the like.

  By reacting the obtained amino acid carbamates (1) in the presence of a protonic acid, the target amino acid-N-carboxy anhydride can be quantitatively obtained. Examples of the protonic acid to be used include phenols, phosphoric acids, sulfonic acids, compounds represented by the general formula (6), and the like.

R ⁵ (C (R ⁶ ) (R ⁷ )) _n COOH (6)
(In the formula, R ⁵ , R ⁶ and R ⁷ are each independently a hydrogen atom, an alkyl group which may have a substituent, an aryl group which may have a substituent, a halogen atom, a hydroxyl group or an alkoxy; A group, an aryloxy group, a ketone group, an ester group or a carboxyl group; n represents a number from 0 to 10. However, when n = 0, R ⁵ may have a hydrogen atom or a substituent. (It is an aryl group which may have an alkyl group or a substituent.)

Of these, phenols and compounds represented by the above general formula (6) are preferable.
Examples of phenols include electron-withdrawing group-substituted phenols such as 2,4-dinitrophenol, pentafluorophenol, and cyanophenol.

In General formula (6), a C1-C12 linear or branched alkyl group is mentioned as an alkyl group shown by R < ⁵ >, R < ⁶ >, R < ⁷ >. Examples of the aryl group include an aryl group having 6 to 14 carbon atoms, and a phenyl group, a naphthyl group, and the like are preferable. Examples of the halogen atom include fluorine, chlorine, bromine and iodine. Examples of the alkoxy group include linear or branched alkoxy groups having 1 to 12 carbon atoms. Examples of the aryloxy group include aryloxy groups having 6 to 14 carbon atoms, and a phenoxy group is preferable. Examples of the group that can be substituted on the alkyl group or aryl group include 1 to 5 groups selected from a halogen atom, a nitro group, a hydroxyl group, a mercapto group, a cyano group, an alkoxy group, and the like.

In the compound represented by the general formula (6), preferably R ⁵ , R ⁶ and R ⁷ are each independently selected from a hydrogen atom and an aryl group which may have a substituent, and n is 0 or 1 It is. Among these, when n = 0, R ⁵ is preferably a methyl group, a phenyl group or a naphthyl group which may have a substituent, and when n = 1, R ⁵ , R ⁶ and R ⁷ are each It is preferably a hydrogen atom or a phenyl group which may have a substituent. Preferable specific examples of the compound represented by the general formula (6) include benzoic acid, p-chlorobenzoic acid, p-nitrobenzoic acid, pentafluorobenzoic acid, 2,4-dinitrobenzoic acid, acetic acid, phenylacetic acid. , Naphthoic acid such as diphenylacetic acid and 1-naphthoic acid.
Examples of phosphoric acids include phosphoric acid, phosphorous acid, and hypophosphorous acid.
Examples of the sulfonic acids include aliphatic sulfonic acid, aromatic sulfonic acid, sulfuric acid, and the like. Specifically, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and benzenesulfonic acid are preferably used.

  The amount of the protonic acid used is preferably 0.1 to 10 mol, particularly 0.3 to 5 mol, per 1 mol of the amino acid carbamates (1).

  The reaction is preferably carried out in an organic solvent. Specific examples of the organic solvent that can be used in the present invention include ethers such as tetrahydrofuran, 1,4-dioxane, diethyl ether and ethylene glycol dimethyl ether; halogenated aliphatic hydrocarbons such as chloroform and dichloromethane; ethyl acetate and acetic acid. Esters such as butyl; Ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; Aromatic hydrocarbons such as benzene, toluene, and xylene; Nitriles such as acetonitrile and propionitrile; Carbonates such as dimethyl carbonate; Hexane, Aliphatic hydrocarbons such as petroleum ether; halogenated aromatic hydrocarbons such as chlorobenzene and dichlorobenzene. The use of an organic solvent is not essential and there is no particular limitation on the amount used. These solvents may be used alone or in combination of two or more.

  The reaction conditions in the production method of the present invention are not particularly limited. The reaction can usually be carried out in the atmosphere, but it is desirable to carry out the reaction under an inert gas atmosphere such as argon or nitrogen because the compounds and products used are decomposed by moisture. This reaction can be carried out under normal pressure, reduced pressure, or increased pressure. The reaction temperature is usually selected from the range of −78 to 120 ° C., preferably −10 to 100 ° C. The reaction time usually requires 0.1 to 100 hours.

By carrying out the reaction under such reaction conditions, amino acid-N-carboxyanhydride is quantitatively generated.
The produced amino acid-N-carboxyanhydride is purified by a commonly used method such as recrystallization or column chromatography.

  EXAMPLES Hereinafter, although an Example is hung up and this invention is demonstrated, this invention is not restrict | limited to these Examples.

Example 1
In a 100 mL two-necked round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 804 mg (2 mmol) of (4-nitrophenoxy) carbonyl-γ-benzyl-L-glutamic acid and 441 mg (2.4 mmol) of 2,4-dinitrophenol were added. ), 2-butanone (20 mL) was added, and the mixture was stirred at 60 ° C. for 24 hours. The reaction mixture was subjected to NMR quantification using dioxane as an internal standard to confirm that γ-benzyl-N-carboxy-L-glutamic anhydride was obtained in a yield of 98%.

Example 2
In a 100 mL two-necked round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 804 mg (2 mmol) of (4-nitrophenoxy) carbonyl-γ-benzyl-L-glutamic acid, 244 mg (2 mmol) of benzoic acid, 20 mL of 2-butanone And stirred at 60 ° C. for 24 hours. The reaction mixture was subjected to NMR quantification using dioxane as an internal standard to confirm that γ-benzyl-N-carboxy-L-glutamic anhydride was obtained in a yield of 98%.

Example 3
In a 100 mL two-necked round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 804 mg (2 mmol) of (4-nitrophenoxy) carbonyl-γ-benzyl-L-glutamic acid and 334 mg (2 mmol) of p-chlorobenzoic acid, 2 -20 mL of butanone was added and stirred at 60 ° C for 17 hours. The reaction mixture was subjected to NMR quantification using dioxane as an internal standard to confirm that γ-benzyl-N-carboxy-L-glutamic anhydride was obtained in a yield of 98%.

Example 4
In a 100 mL two-neck round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 804 mg (2 mmol) of (4-nitrophenoxy) carbonyl-γ-benzyl-L-glutamic acid and 334 mg (2 mmol) of p-nitrobenzoic acid, 2 -20 mL of butanone was added and stirred at 60 ° C for 27 hours. The reaction mixture was subjected to NMR quantification using dioxane as an internal standard to confirm that γ-benzyl-N-carboxy-L-glutamic anhydride was obtained in a yield of 98%.

Example 5
In a 100 mL two-necked round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 804 mg (2 mmol) of (4-nitrophenoxy) carbonyl-γ-benzyl-L-glutamic acid and 424 mg (2 mmol) of pentafluorobenzoic acid, 2- 20 mL of butanone was added and stirred at 60 ° C. for 72 hours. The reaction mixture was subjected to NMR quantification using dioxane as an internal standard to confirm that γ-benzyl-N-carboxy-L-glutamic anhydride was obtained in a yield of 96%.

Example 6
In a 100 mL two-necked round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 804 mg (2 mmol) of (4-nitrophenoxy) carbonyl-γ-benzyl-L-glutamic acid and 424 mg (2 mmol) of 2,4-dinitrobenzoic acid were added. 2-butanone 20 mL was added and stirred at 60 ° C. for 72 hours. The reaction mixture was subjected to NMR quantification using dioxane as an internal standard to confirm that γ-benzyl-N-carboxy-L-glutamic anhydride was obtained in a yield of 97%.

Example 7
In a 100 mL two-neck round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 593 mg (2 mmol) of (4-nitrophenoxy) carbonyl-L-leucine, 424 mg (2 mmol) of 2,4-dinitrobenzoic acid, 2-butanone 20 mL was added and stirred at 60 ° C. for 72 hours. NMR quantification of the reaction mixture using dioxane as an internal standard confirmed that N-carboxy-L-leucine anhydride was obtained in a yield of 97%.

Example 8
In a 100 mL two-necked round bottom flask equipped with a Dimroth condenser under a nitrogen atmosphere, 804 mg (2 mmol) of (4-nitrophenoxy) carbonyl-γ-benzyl-L-glutamic acid, 424 mg (2 mmol) of diphenylacetic acid, 20 mL of 2-butanone And stirred at 60 ° C. for 8 hours. The reaction mixture was subjected to NMR quantification using dioxane as an internal standard to confirm that γ-benzyl-N-carboxy-L-glutamic anhydride was obtained in a yield of 97%.

Example 9
Under a nitrogen atmosphere, a phenoxycarbonyl-γ-benzyl-L-glutamic acid 714 mg (2 mmol), 1-naphthoic acid 344 mg (2 mmol), 2-butanone 20 mL were placed in a 100 mL two-necked round bottom flask equipped with a Dimroth condenser. The mixture was stirred at 80 ° C. for 96 hours. NMR quantification of the reaction mixture using dioxane as an internal standard confirmed that N-carboxy-α-glutamic acid-γ-benzyl anhydride was obtained in a yield of 55%.

Example 10
Under a nitrogen atmosphere, 714 mg (2 mmol) of phenoxycarbonyl-γ-benzyl-L-glutamic acid, 212 mg (1 mmol) of diphenylacetic acid, and 20 mL of 2-butanone were placed in a 100 mL two-necked round bottom flask equipped with a Dimroth condenser. For 40 hours. NMR quantification of this reaction mixture using dioxane as an internal standard confirmed that N-carboxy-α-glutamic acid-γ-benzyl anhydride was obtained in a yield of 97%.

Example 11
Under a nitrogen atmosphere, a phenoxycarbonyl-γ-benzyl-L-glutamic acid 714 mg (2 mmol), p-nitrobenzoic acid 334 mg (2 mmol), and 2-butanone 20 mL were placed in a 100 mL two-necked round bottom flask equipped with a Dimroth condenser. , And stirred at 80 ° C. for 120 hours. NMR quantification of this reaction mixture using dioxane as an internal standard confirmed that N-carboxy-α-glutamic acid-γ-benzyl anhydride was obtained in a yield of 54%.

Example 12
Under a nitrogen atmosphere, put 714 mg (2 mmol) of phenoxycarbonyl-γ-benzyl-L-glutamic acid, 0.115 mL (2 mmol) of acetic acid, and 20 mL of 2-butanone in a 100 mL two-necked round bottom flask equipped with a Dimroth condenser. Stir at 23 ° C. for 23 hours. NMR quantification of the reaction mixture using dioxane as an internal standard confirmed that N-carboxy-α-glutamic acid-γ-benzyl anhydride was obtained in a yield of 96%.

Example 13
Under a nitrogen atmosphere, put 714 mg (2 mmol) of phenoxycarbonyl-γ-benzyl-L-glutamic acid, 0.345 mL (6 mmol) of acetic acid, 20 mL of 2-butanone in a 100 mL two-necked round bottom flask equipped with a Dimroth condenser. Stir at 30 ° C. for 30 hours. NMR quantification of the reaction mixture using dioxane as an internal standard confirmed that N-carboxy-α-glutamic acid-γ-benzyl anhydride was obtained in a yield of 94%.

Reference example 1
The reaction was carried out for 7 hours in the same manner as in Example 1 except that 2,4-dinitrophenol was not added. The yield of N-carboxy-α-glutamic acid-γ-benzyl anhydride in the reaction mixture was 19%. It was.

Claims (1)

General formula (1)

(Wherein a ¹ R ^{1 s} are the same or different and each represents an electron-withdrawing substituent selected from a nitro group, a halogen atom, a cyano group, an acyl group, an alkyloxycarbonyl group, a perfluoroalkyl group, and a perchloroalkyl group. A represents an integer of 0 to 5; R ² and R ³ are each independently a hydrogen atom, an optionally substituted alkyl group, an optionally substituted cycloalkyl group, An aryl group which may be substituted or a heterocyclic ring which may be substituted, or R ² and R ³ may combine to form a cycloalkyl group, and the cycloalkyl group may be an aromatic ring or a hetero ring as a condensed ring. (It may have a ring.)
Amino acid carbamates represented by the formula: phosphoric acids, sulfonic acids and general formula (6)
R ⁵ (C (R ⁶ ) (R ⁷ )) _n COOH (6)
(In the formula, R ⁵ , R ⁶ and R ⁷ are each independently a hydrogen atom, an alkyl group which may have a substituent, an aryl group which may have a substituent, a halogen atom, a hydroxyl group or an alkoxy; A group, an aryloxy group, a ketone group, an ester group or a carboxyl group; n represents a number from 0 to 10. However, when n = 0, R ⁵ may have a hydrogen atom or a substituent. (It is an aryl group which may have an alkyl group or a substituent.)
The reaction is carried out in the presence of a protonic acid selected from the compounds represented by formula (2)

(In the formula, R ² and R ³ are the same as above.)
The manufacturing method of the amino acid-N-carboxyanhydride represented by these.