WO2012108408A1 - Method for producing dipeptide and tripeptide - Google Patents
Method for producing dipeptide and tripeptide Download PDFInfo
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- WO2012108408A1 WO2012108408A1 PCT/JP2012/052684 JP2012052684W WO2012108408A1 WO 2012108408 A1 WO2012108408 A1 WO 2012108408A1 JP 2012052684 W JP2012052684 W JP 2012052684W WO 2012108408 A1 WO2012108408 A1 WO 2012108408A1
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- amino acid
- dipeptide
- glutamylvalylglycine
- tripeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
Definitions
- the present invention relates to a method for efficiently producing a dipeptide such as Val-Gly and a tripeptide such as ⁇ -Glu-X-Gly (X is an amino acid or amino acid derivative) obtained therefrom.
- Patent Document 1 discloses a synthesis method based on a complete protection method. Improvement of the synthesis method is demanded because it is difficult to improve.
- N-phthaloylglutamic anhydride when N-phthaloylglutamic anhydride is reacted with an amino acid, a ⁇ -peptide is selectively obtained.
- hydrazine having mutagenicity is essential for deprotection and is unsuitable for commercial production.
- a method for producing an N-protected glutamic acid ⁇ -derivative a method is known in which an N-protected glutamic anhydride is reacted with an amino acid or a derivative thereof in the presence of N-hydroxysuccinimide (Patent Document 2).
- this method can also be suitably applied to the production of ⁇ -Glu-X-Gly, that is, can be suitably applied to the reaction of N-protected glutamic anhydride and X-Gly type peptide. It is not clear whether or not. Therefore, a method for producing ⁇ -Glu-X-Gly efficiently and economically is required.
- the present inventors reacted N-protected neutral amino acid with N-hydroxysuccinimide to form N-hydroxysuccinimide ester of N-protected amino acid which is an active ester, and further reacted with neutral amino acid.
- N-protected neutral amino acid with N-hydroxysuccinimide
- N-hydroxysuccinimide ester of N-protected amino acid which is an active ester
- neutral amino acid is an active ester
- a dipeptide in which one of the terminal amino groups is protected can be efficiently produced, particularly when the reaction is carried out under conditions of pH 8.5 to 11.5.
- this dipeptide is used as a raw material, the terminal amino group is deprotected (N deprotection) and then reacted with N-protected glutamic anhydride in the presence of N-hydroxysuccinimide for efficient and high efficiency.
- the present invention provides a method for producing a dipeptide having a terminal amino group protected, which comprises reacting a neutral amino acid with an N-hydroxysuccinimide ester of an N-protected neutral amino acid.
- the present invention also provides a method for producing a dipeptide which performs N deprotection of the dipeptide obtained by the above method.
- the present invention also provides an N-protected glutamic acid ⁇ , wherein the dipeptide obtained by N deprotection by the above method is reacted with an N-protected glutamic anhydride in the presence of N-hydroxysuccinimide.
- the present invention also provides Z- ⁇ -glutamylvalylglycine, or a salt thereof. Furthermore, crystals of Z- ⁇ -glutamyl valylglycine are provided.
- the present invention also provides a method for producing glutamic acid ⁇ -tripeptide, which performs N deprotection of the tripeptide obtained by the above method.
- the present invention also provides a method for producing Z- ⁇ -glutamylvalylglycine, characterized by reacting valylglycine with Z-glutamic anhydride in the presence of N-hydroxysuccinimide,
- the present invention also provides a method for producing ⁇ -glutamylvalylglycine, which N-deprotects the obtained Z- ⁇ -glutamylvalylglycine.
- a dipeptide with a short reaction time and few by-products can be produced.
- N-protected glutamic acid ⁇ -tripeptide can be produced efficiently and highly selectively, although the raw materials used are inexpensive and the reaction operation is simple.
- 3 shows a powder X-ray crystal diffraction pattern of Z- ⁇ -glutamylvalylglycine A crystal.
- 3 shows a powder X-ray crystal diffraction pattern of Z- ⁇ -glutamylvalylglycine B crystal.
- 2 shows a powder X-ray crystal diffraction pattern of Z- ⁇ -glutamylvalylglycine C crystal.
- 3 shows a powder X-ray crystal diffraction pattern of Z- ⁇ -glutamylvalylglycine D crystal.
- 3 shows a powder X-ray crystal diffraction pattern of Z- ⁇ -glutamylvalylglycine E crystal.
- 3 shows a powder X-ray crystal diffraction pattern of Z- ⁇ -glutamylvalylglycine F crystal.
- 2 shows a powder X-ray crystal diffraction pattern of ⁇ -glutamylvalylglycine crystal.
- the neutral amino acid targeted in the present invention refers to an amino acid having no amino group or carboxyl group in the side chain, and includes glycine (Gly), alanine (Ala), ⁇ -alanine, valine (Val), and leucine (Leu). , Isoleucine (Ile), norvaline (Nva), 2-aminobutanoic acid (Abu), proline (Pro), methionine (Met), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr) and the like. can give.
- glycine, alanine, ⁇ -alanine, valine, leucine, isoleucine, norvaline and 2-aminobutanoic acid are preferred.
- the amino group of the neutral amino acid is protected with a protecting group such as benzyloxycarbonyl group (Z), t-butyloxycarbonyl group (Boc), formyl group, etc. Can be given.
- a protecting group such as benzyloxycarbonyl group (Z), t-butyloxycarbonyl group (Boc), formyl group, etc.
- Z benzyloxycarbonyl group
- Boc t-butyloxycarbonyl group
- formyl group etc.
- the same or different amino acid as the N-protected one can be used as the neutral amino acid.
- N-hydroxysuccinimide ester of N-protected neutral amino acid is 0.5 to 2 moles, preferably 1.0 to 1 mole of N-protected neutral amino acid in an organic solvent such as ethyl acetate, acetonitrile or tetrahydrofuran.
- the reaction of N-hydroxysuccinimide ester of N-protected neutral amino acid with neutral amino acid is, for example, 0.5 to 2 moles compared to N-hydroxysuccinimide ester of N-protected neutral amino acid, Preferably, 0.8 to 1.2 moles of neutral amino acid is carried out in a solvent at ⁇ 20 to 80 ° C., preferably 0 to 50 ° C., for 30 minutes to 24 hours. At this time, the reaction is preferably carried out under conditions of pH 8.5 to 10.5, more preferably 8.5 to less than 9.5. Further, when the reaction is carried out at a low temperature, it is also preferably carried out at a pH of 9.5 to 10.5.
- solvent used in the reaction examples include organic solvents such as ethyl acetate, acetonitrile, tetrahydrofuran, dioxane, chloroform and N, N-dimethylformamide. These solvents may be used as a mixture, or a mixed solvent with water may be used.
- N deprotection of the dipeptide obtained by the above method is then performed by a conventional method.
- the dipeptide reaction solution obtained by the above method is preferably substituted with argon, palladium carbon is added, and hydrogen is substituted to deprotect N.
- the dipeptide obtained by N-deprotection by the above method is then reacted with N-protected glutamic anhydride in the presence of N-hydroxysuccinimide to give N-protected glutamic acid ⁇ -tri A peptide is produced.
- the N-protected glutamic acid ⁇ -tripeptide means a tripeptide in which the ⁇ -position of N-protected glutamic acid is bound to a dipeptide.
- Z- ⁇ -glutamylvalylglycine is a novel compound and is extremely useful as an intermediate for production of ⁇ -glutamylvalylglycine.
- crystals of Z- ⁇ -glutamyl valyl glycine may be obtained. In this case, purification becomes extremely easy, and high purity ⁇ -glutamyl valyl glycine can be produced. .
- crystals of Z- ⁇ -glutamylvalylglycine have diffraction angles (2 ⁇ ) of 10.9, 16.3, 19.2, 19.7, 20.3 in the powder X-ray diffraction pattern. It is characterized by showing a peak.
- N-protected glutamic anhydride can be produced by a conventional method such as treating N-protected glutamic acid with a dehydrating agent such as dicyclohexylcarbodiimide (DDC) or acetic anhydride.
- a dehydrating agent such as dicyclohexylcarbodiimide (DDC) or acetic anhydride.
- the reaction of the above dipeptide with N-protected glutamic anhydride is, for example, 0.5 to 2-fold mol, preferably 0.8 to 1.2-fold mol of dipeptide with respect to N-protected glutamic anhydride.
- N-hydroxysuccinimide (HOSu) in a solvent at ⁇ 20 to 80 ° C., preferably 0 to 50 ° C. for 30 minutes.
- the reaction is preferably carried out in the presence of an alkali metal carbonate.
- Alkali metal carbonates include normal salts (sodium carbonate, potassium carbonate, etc.) or bicarbonates (sodium bicarbonate, potassium bicarbonate, etc.). Preferred are sodium hydrogen carbonate and potassium hydrogen carbonate.
- the alkali metal carbonate is preferably used in an amount of 1.0 to 1.2 times the amount of N-protected glutamic acid.
- the solvent used in the reaction include organic solvents such as ethyl acetate, acetonitrile, tetrahydrofuran, dioxane, chloroform and N, N-dimethylformamide. These solvents may be used as a mixture, or a mixed solvent with water may be used.
- N deprotection of the tripeptide obtained by the above method is performed by a conventional method.
- the tripeptide reaction solution obtained by the above method is replaced with argon, palladium carbon is added, and hydrogen is replaced with N. It should be deprotected.
- the by-product ⁇ -form may be separated from the ⁇ -form by a conventional method such as crystallization or chromatography before or after removal of the protecting group.
- a conventional method such as crystallization or chromatography before or after removal of the protecting group.
- the salt of Z- ⁇ -glutamylvalylglycine produced by the method of the present invention include sodium salt and potassium salt.
- Run 6 was performed as follows. To 1.57 g (1.05 eq) of glycine was dissolved by adding 25 ml of water, and 5.1 g (3.0 eq) of sodium bicarbonate was added and stirred. To this solution, an ethyl acetate solution (19.9 mmol) of hydroxysuccinimide ester of Z-valine was added and stirred at 40 ° C. for 18 hours. 6M hydrochloric acid was added and stirred to adjust the pH to 8. The reaction solution was purged with argon, 0.05 g of 50% wet 10% palladium on carbon was added, purged with hydrogen, and stirred overnight at room temperature. After replacing the reaction solution with argon, palladium carbon was removed by separation. The reaction solution was separated to obtain an aqueous solution of valylglycine. Yield 87.4%
- the precipitated dicyclohexylurea was separated by filtration and washed with 37 ml of ethyl acetate. An ethyl acetate solution of Z-glutamic anhydride was obtained. 7.36 g (1.0 eq vs. Z-Glu) of sodium hydrogen carbonate was added to and dissolved in the valylglycine aqueous solution (including HOSu) produced in Run 3 of Example 1, and an ethyl acetate solution of Z-glutamic anhydride was added thereto. It was dripped at room temperature over 1.5 hours. After completion of the dropping, the mixture was stirred for 1 hour to obtain a layer separation and an aqueous solution of Z- ⁇ -glutamylvalylglycine.
- Z- ⁇ -glutamylvalylglycine has the following crystal polymorphs of A crystal to F crystal. Of these, the crystal E is preferred as the crystal.
- the main peak [diffraction angle (2 ⁇ )] and production examples are shown below.
- the powder X-ray crystal diffraction patterns of these A crystals to F crystals are shown in FIGS.
- Crystal A Crystals crystallized acidic (10.9, 16.3, 19.2, 19.7, 20.3)
- Crystal B Crystal obtained by slurry washing with methanol (6.0, 7.3, 8.3, 15.0, 20.0) 1.5 ml of methanol was added to the crystal A (0.3 g) of the crude Z- ⁇ -glutamylvalylglycine obtained in (3) and stirred at 20 ° C.
- Crystals Crystal obtained by recrystallizing Crystal A from ethanol (6.1, 15.1, 17.7, 24.6, 26.9) Ethanol (10 ml) was added to the crude Z- ⁇ -glutamylvalylglycine crystal A (1.0 g) obtained in (3), and the mixture was stirred at 30 ° C. for 3 hours and further at 50 ° C. for 1 hour. Thereafter, the mixture was cooled to 20 ° C. over 6 hours and stirred at 20 ° C. overnight. The crystals were separated and dried under reduced pressure overnight at 40 ° C. to obtain 0.85 g of Z- ⁇ -glutamylvalylglycine crystals (C crystals).
- Crystal D Crystal obtained by recrystallizing Crystal B from ethanol (6.6, 6.7, 19.0, 19.5, 31.9) 108 B of ethanol was added to B crystal (6.0 g) of crude Z- ⁇ -glutamylvalylglycine, and the mixture was stirred at 65 ° C. for 1 hour. Thereafter, the mixture was cooled to 10 ° C. over 10 hours and stirred at 10 ° C. overnight. The crystals were separated and dried under reduced pressure at 40 ° C. overnight to obtain 3.8 g of Z- ⁇ -glutamylvalylglycine crystals (crystal D).
- Crystal E Crystal obtained by recrystallizing Crystal A from methanol (8.5, 14.1, 18.0, 21.6, 22.9) 73.3 ml of methanol was added to the crude Z- ⁇ -glutamylvalylglycine obtained in (3) above and stirred at 40 ° C. for 3 hours. Thereafter, the mixture was cooled to 10 ° C. over 3 hours and stirred at 10 ° C. overnight. The crystals were separated, washed with 12 ml of methanol, and dried under reduced pressure at 40 ° C. overnight to obtain 8.0 g of Z- ⁇ -glutamylvalylglycine crystals.
- the precipitated dicyclohexylurea was removed by filtration and washed with 40 ml of ethyl acetate to obtain a solution of Z-glutamic anhydride in ethyl acetate.
- 7.36 g (1.0 eq vs. Z-Glu) of sodium bicarbonate was added to an aqueous valylglycine solution (including HOSu) and stirred, and the ethyl acetate solution of Z-glutamic anhydride prepared above was added dropwise over 1.5 hours. did. After the dropwise addition, the mixture was stirred at room temperature for 1 hour, 70 ml of THF was added, and the mixture was heated to 40 ° C.
- Concentrated hydrochloric acid (22 ml) was added to adjust to pH 3.0 and extraction was performed. The layers were separated, and the ethyl acetate layer was washed with 100 ml of water. To the ethyl acetate layer, 120 ml of water and 0.75 g of 50% wet 10% palladium carbon were added to replace with hydrogen, followed by deprotection with stirring at 40 ° C. After completion of the reaction, palladium carbon was filtered and separated. The aqueous layer was concentrated to 100 ml under reduced pressure, insoluble material was removed by Millipore filtration (0.45 ⁇ m), and the mixture was again concentrated to 50 g under reduced pressure.
- FIG. 7 shows a powder X-ray crystal diffraction pattern of the crystals of ⁇ -glutamylvalylglycine.
- Example 3 Synthesis of norvalylglycine and ⁇ -glutamylnorvalylglycine (1) Synthesis of hydroxysuccinimide ester of Z (benzyloxycarbonyl) -norvaline Z-norvaline 10 g (39.8 mmol) was dissolved in 60 ml of ethyl acetate, and 4.59 g (1 eq) of N-hydroxysuccinimide (HOSu) was dissolved. ) And cooled to 5 ° C. 8.21 g (1 eq) of N, N-dicyclohexylcarbodiimide was dissolved in 25 ml of ethyl acetate and slowly added at 10 ° C. or lower.
- the precipitated dicyclohexylurea was removed by filtration and washed with 20 ml of ethyl acetate to obtain a solution of Z-glutamic anhydride in ethyl acetate.
- 3.68 g (1.0 eq vs. Z-Glu) of sodium hydrogen carbonate was added to an aqueous solution of norvalylglycine (including HOSu) and stirred, and the ethyl acetate solution of Z-glutamic anhydride prepared above was added over 1.5 hours. It was dripped. After dropping, the mixture was stirred at room temperature for 1 hour and heated to 40 ° C. Concentrated hydrochloric acid (11 ml) was added to adjust to pH 3.0 and extraction was performed.
- the layers were separated, and the ethyl acetate layer was washed with 50 ml of water.
- the ethyl acetate layer was purged with hydrogen by adding 50 ml of water, 25 ml of methanol, and 0.37 g of 50% wet 10% palladium on carbon, and deprotected by stirring at 20 to 25 ° C.
- palladium carbon was removed by Millipore filtration (0.45 ⁇ m), and the mixture was concentrated under reduced pressure to 40 g.
- the mixture was heated to 50 ° C. and 50 ml of methanol was added dropwise.
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Abstract
A glutamic acid γ-tripeptide can be efficiently produced by a method for producing an N-protected glutamic acid γ-tripeptide, said method comprising a process wherein an N-protected neutral amino acid is formed into an N-hydroxysuccinimide ester and then made to react with a neutral amino acid so as to synthesize a dipeptide having a protected terminal amino group, and then after performing N-deprotection, the thus-obtained dipeptide is caused to react with an N-protected glutamic acid anhydride in the presence of N-hydroxysuccinimide.
Description
本発明は、Ｖａｌ-Ｇｌｙなどのジペプチド、及びこれから得られるγ－Ｇｌｕ－Ｘ－Ｇｌｙ（Ｘはアミノ酸またはアミノ酸誘導体）などのトリペプチドを効率的に製造する方法に関する。
The present invention relates to a method for efficiently producing a dipeptide such as Val-Gly and a tripeptide such as γ-Glu-X-Gly (X is an amino acid or amino acid derivative) obtained therefrom.
γ－Ｇｌｕ－Ｘ－Ｇｌｙ（Ｘは上記の通りである）などペプチド類は、コク味を有するペプチドとして発見され、食品分野における応用が期待されている（特許文献１）。
γ－Ｇｌｕ－Ｘ－Ｇｌｙ型のペプチドのうち、γ－Ｇｌｕ－Ｖａｌ－Ｇｌｙの製造方法として、特許文献１に完全保護法による合成方法が開示されているが、保護アミノ酸が高価であり、スケールアップが困難なため、その合成方法の改善が要望されている。
一方、γ－Ｇｌｕ－Ｘ－Ｇｌｙのように、N末アミノ酸であるグルタミン酸のγ位と縮合したペプチド（以下、「γ－グルタミルペプチド」という。）の合成に関して、グルタミン酸が縮合可能なカルボン酸基を２つ有するため、N－保護グルタミン酸からγ－グルタミルペプチドを選択的に製造することには課題があった。
すなわち、Ｎ－保護グルタミン酸をＮ－保護グルタミン酸無水物とし、アミノ酸と反応させると、α／γ＝２．０／１．０～３．０／１．０とα－ペプチドが優位的に得られることが報告されている。またＮ－フタロイルグルタミン酸無水物はアミノ酸と反応させるとγ－ペプチドが選択的に得られるが、脱保護に変異原性を有するヒドラジンが必須であり、商業生産には不適であった。
また、Ｎ－保護グルタミン酸γ－誘導体の製法に関し、Ｎ－ヒドロキシコハク酸イミドの存在下にＮ－保護グルタミン酸無水物とアミノ酸またはその誘導体とを反応させる方法が知られている（特許文献２）。しかしながら、この方法が、γ－Ｇｌｕ－Ｘ－Ｇｌｙの製法においても好適に適用可能であるか否か、すなわち、Ｎ－保護グルタミン酸無水物とＸ－Ｇｌｙ型のペプチドとの反応に好適に適応可能であるか否かは明確ではない。
したがって、効率的かつ経済的に、γ－Ｇｌｕ－Ｘ－Ｇｌｙを製造する方法が求められている。 Peptides such as γ-Glu-X-Gly (X is as described above) have been discovered as peptides having a rich taste and are expected to be applied in the food field (Patent Document 1).
Among the γ-Glu-X-Gly type peptides, as a method for producing γ-Glu-Val-Gly, Patent Document 1 discloses a synthesis method based on a complete protection method. Improvement of the synthesis method is demanded because it is difficult to improve.
On the other hand, as with γ-Glu-X-Gly, regarding the synthesis of a peptide condensed with the γ-position of N-terminal amino acid glutamic acid (hereinafter referred to as “γ-glutamyl peptide”), a carboxylic acid group to which glutamic acid can be condensed Therefore, there was a problem in selectively producing γ-glutamyl peptide from N-protected glutamic acid.
That is, when N-protected glutamic acid is converted to N-protected glutamic anhydride and reacted with an amino acid, α / γ = 2.0 / 1.0 to 3.0 / 1.0 and α-peptide can be obtained predominantly. It has been reported. Further, when N-phthaloylglutamic anhydride is reacted with an amino acid, a γ-peptide is selectively obtained. However, hydrazine having mutagenicity is essential for deprotection and is unsuitable for commercial production.
Further, regarding a method for producing an N-protected glutamic acid γ-derivative, a method is known in which an N-protected glutamic anhydride is reacted with an amino acid or a derivative thereof in the presence of N-hydroxysuccinimide (Patent Document 2). However, this method can also be suitably applied to the production of γ-Glu-X-Gly, that is, can be suitably applied to the reaction of N-protected glutamic anhydride and X-Gly type peptide. It is not clear whether or not.
Therefore, a method for producing γ-Glu-X-Gly efficiently and economically is required.
γ－Ｇｌｕ－Ｘ－Ｇｌｙ型のペプチドのうち、γ－Ｇｌｕ－Ｖａｌ－Ｇｌｙの製造方法として、特許文献１に完全保護法による合成方法が開示されているが、保護アミノ酸が高価であり、スケールアップが困難なため、その合成方法の改善が要望されている。
一方、γ－Ｇｌｕ－Ｘ－Ｇｌｙのように、N末アミノ酸であるグルタミン酸のγ位と縮合したペプチド（以下、「γ－グルタミルペプチド」という。）の合成に関して、グルタミン酸が縮合可能なカルボン酸基を２つ有するため、N－保護グルタミン酸からγ－グルタミルペプチドを選択的に製造することには課題があった。
すなわち、Ｎ－保護グルタミン酸をＮ－保護グルタミン酸無水物とし、アミノ酸と反応させると、α／γ＝２．０／１．０～３．０／１．０とα－ペプチドが優位的に得られることが報告されている。またＮ－フタロイルグルタミン酸無水物はアミノ酸と反応させるとγ－ペプチドが選択的に得られるが、脱保護に変異原性を有するヒドラジンが必須であり、商業生産には不適であった。
また、Ｎ－保護グルタミン酸γ－誘導体の製法に関し、Ｎ－ヒドロキシコハク酸イミドの存在下にＮ－保護グルタミン酸無水物とアミノ酸またはその誘導体とを反応させる方法が知られている（特許文献２）。しかしながら、この方法が、γ－Ｇｌｕ－Ｘ－Ｇｌｙの製法においても好適に適用可能であるか否か、すなわち、Ｎ－保護グルタミン酸無水物とＸ－Ｇｌｙ型のペプチドとの反応に好適に適応可能であるか否かは明確ではない。
したがって、効率的かつ経済的に、γ－Ｇｌｕ－Ｘ－Ｇｌｙを製造する方法が求められている。 Peptides such as γ-Glu-X-Gly (X is as described above) have been discovered as peptides having a rich taste and are expected to be applied in the food field (Patent Document 1).
Among the γ-Glu-X-Gly type peptides, as a method for producing γ-Glu-Val-Gly, Patent Document 1 discloses a synthesis method based on a complete protection method. Improvement of the synthesis method is demanded because it is difficult to improve.
On the other hand, as with γ-Glu-X-Gly, regarding the synthesis of a peptide condensed with the γ-position of N-terminal amino acid glutamic acid (hereinafter referred to as “γ-glutamyl peptide”), a carboxylic acid group to which glutamic acid can be condensed Therefore, there was a problem in selectively producing γ-glutamyl peptide from N-protected glutamic acid.
That is, when N-protected glutamic acid is converted to N-protected glutamic anhydride and reacted with an amino acid, α / γ = 2.0 / 1.0 to 3.0 / 1.0 and α-peptide can be obtained predominantly. It has been reported. Further, when N-phthaloylglutamic anhydride is reacted with an amino acid, a γ-peptide is selectively obtained. However, hydrazine having mutagenicity is essential for deprotection and is unsuitable for commercial production.
Further, regarding a method for producing an N-protected glutamic acid γ-derivative, a method is known in which an N-protected glutamic anhydride is reacted with an amino acid or a derivative thereof in the presence of N-hydroxysuccinimide (Patent Document 2). However, this method can also be suitably applied to the production of γ-Glu-X-Gly, that is, can be suitably applied to the reaction of N-protected glutamic anhydride and X-Gly type peptide. It is not clear whether or not.
Therefore, a method for producing γ-Glu-X-Gly efficiently and economically is required.
本発明は、中性アミノ酸を２分子結合させてなるジペプチドを効率的に製造できる方法を提供することを目的とする。
本発明は、又、グルタミン酸γ－トリペプチドを効率的に製造できる方法を提供することを目的とする。 An object of the present invention is to provide a method capable of efficiently producing a dipeptide obtained by bonding two molecules of neutral amino acids.
Another object of the present invention is to provide a method capable of efficiently producing a glutamic acid γ-tripeptide.
本発明は、又、グルタミン酸γ－トリペプチドを効率的に製造できる方法を提供することを目的とする。 An object of the present invention is to provide a method capable of efficiently producing a dipeptide obtained by bonding two molecules of neutral amino acids.
Another object of the present invention is to provide a method capable of efficiently producing a glutamic acid γ-tripeptide.
本発明者らは、Ｎ－保護中性アミノ酸をＮ－ヒドロキシコハク酸イミドと反応させて活性エステルであるＮ－保護アミノ酸のＮ－ヒドロキシコハク酸イミドエステルを形成せしめ、さらに、中性アミノ酸と反応させると、特に、ｐＨ８.５～１１.５の条件下で反応させると、効率的に末端アミノ基の１つが保護されたジペプチドを製造できることを見出した。さらに、このジペプチドを原料として、末端アミノ基の脱保護（Ｎ脱保護）を行った後に、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｎ－保護グルタミン酸無水物と反応させると、効率的かつ高選択的にＮ－保護グルタミン酸γ－トリペプチドを製造できることを見いだし、この知見に基づいて本発明を完成したのである。
すなわち、本発明は、Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルに、中性アミノ酸を反応させることを特徴とする末端アミノ基が保護されたジペプチドの製造方法を提供する。 The present inventors reacted N-protected neutral amino acid with N-hydroxysuccinimide to form N-hydroxysuccinimide ester of N-protected amino acid which is an active ester, and further reacted with neutral amino acid. In particular, it has been found that a dipeptide in which one of the terminal amino groups is protected can be efficiently produced, particularly when the reaction is carried out under conditions of pH 8.5 to 11.5. Furthermore, when this dipeptide is used as a raw material, the terminal amino group is deprotected (N deprotection) and then reacted with N-protected glutamic anhydride in the presence of N-hydroxysuccinimide for efficient and high efficiency. The inventors have found that an N-protected glutamic acid γ-tripeptide can be selectively produced, and based on this finding, the present invention has been completed.
That is, the present invention provides a method for producing a dipeptide having a terminal amino group protected, which comprises reacting a neutral amino acid with an N-hydroxysuccinimide ester of an N-protected neutral amino acid.
すなわち、本発明は、Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルに、中性アミノ酸を反応させることを特徴とする末端アミノ基が保護されたジペプチドの製造方法を提供する。 The present inventors reacted N-protected neutral amino acid with N-hydroxysuccinimide to form N-hydroxysuccinimide ester of N-protected amino acid which is an active ester, and further reacted with neutral amino acid. In particular, it has been found that a dipeptide in which one of the terminal amino groups is protected can be efficiently produced, particularly when the reaction is carried out under conditions of pH 8.5 to 11.5. Furthermore, when this dipeptide is used as a raw material, the terminal amino group is deprotected (N deprotection) and then reacted with N-protected glutamic anhydride in the presence of N-hydroxysuccinimide for efficient and high efficiency. The inventors have found that an N-protected glutamic acid γ-tripeptide can be selectively produced, and based on this finding, the present invention has been completed.
That is, the present invention provides a method for producing a dipeptide having a terminal amino group protected, which comprises reacting a neutral amino acid with an N-hydroxysuccinimide ester of an N-protected neutral amino acid.
本発明は、又、上記方法で得たジペプチドのＮ脱保護を行うジペプチドの製造方法を提供する。
本発明は、又、上記方法でＮ脱保護して得られたジペプチドを、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｎ－保護グルタミン酸無水物と反応させることを特徴とするＮ－保護グルタミン酸γ－トリペプチドの製造方法を提供する。
本発明は、又、Ｚ－γ－グルタミルバリルグリシン、またはその塩を提供する。更には、Ｚ－γ－グルタミルバリルグリシンの結晶を提供する。
本発明は、又、上記方法で得たトリペプチドのＮ脱保護を行うグルタミン酸γ－トリペプチドの製造方法を提供する。
本発明は、又、バリルグリシンを、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｚ－グルタミン酸無水物と反応させることを特徴とするＺ－γ－グルタミルバリルグリシンの製造方法を提供し、更には、得られたＺ－γ－グルタミルバリルグリシンのＮ脱保護を行うγ－グルタミルバリルグリシンの製造方法を提供する。 The present invention also provides a method for producing a dipeptide which performs N deprotection of the dipeptide obtained by the above method.
The present invention also provides an N-protected glutamic acid γ, wherein the dipeptide obtained by N deprotection by the above method is reacted with an N-protected glutamic anhydride in the presence of N-hydroxysuccinimide. -Providing a method for the production of tripeptides.
The present invention also provides Z-γ-glutamylvalylglycine, or a salt thereof. Furthermore, crystals of Z-γ-glutamyl valylglycine are provided.
The present invention also provides a method for producing glutamic acid γ-tripeptide, which performs N deprotection of the tripeptide obtained by the above method.
The present invention also provides a method for producing Z-γ-glutamylvalylglycine, characterized by reacting valylglycine with Z-glutamic anhydride in the presence of N-hydroxysuccinimide, The present invention also provides a method for producing γ-glutamylvalylglycine, which N-deprotects the obtained Z-γ-glutamylvalylglycine.
本発明は、又、上記方法でＮ脱保護して得られたジペプチドを、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｎ－保護グルタミン酸無水物と反応させることを特徴とするＮ－保護グルタミン酸γ－トリペプチドの製造方法を提供する。
本発明は、又、Ｚ－γ－グルタミルバリルグリシン、またはその塩を提供する。更には、Ｚ－γ－グルタミルバリルグリシンの結晶を提供する。
本発明は、又、上記方法で得たトリペプチドのＮ脱保護を行うグルタミン酸γ－トリペプチドの製造方法を提供する。
本発明は、又、バリルグリシンを、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｚ－グルタミン酸無水物と反応させることを特徴とするＺ－γ－グルタミルバリルグリシンの製造方法を提供し、更には、得られたＺ－γ－グルタミルバリルグリシンのＮ脱保護を行うγ－グルタミルバリルグリシンの製造方法を提供する。 The present invention also provides a method for producing a dipeptide which performs N deprotection of the dipeptide obtained by the above method.
The present invention also provides an N-protected glutamic acid γ, wherein the dipeptide obtained by N deprotection by the above method is reacted with an N-protected glutamic anhydride in the presence of N-hydroxysuccinimide. -Providing a method for the production of tripeptides.
The present invention also provides Z-γ-glutamylvalylglycine, or a salt thereof. Furthermore, crystals of Z-γ-glutamyl valylglycine are provided.
The present invention also provides a method for producing glutamic acid γ-tripeptide, which performs N deprotection of the tripeptide obtained by the above method.
The present invention also provides a method for producing Z-γ-glutamylvalylglycine, characterized by reacting valylglycine with Z-glutamic anhydride in the presence of N-hydroxysuccinimide, The present invention also provides a method for producing γ-glutamylvalylglycine, which N-deprotects the obtained Z-γ-glutamylvalylglycine.
本発明によれば、反応時間が短く、副生物の少ないジペプチドを製造することができる。又、本発明によれば、使用原料が安価でかつ反応操作が簡便であるにも係わらず、効率的かつ高選択的にＮ－保護グルタミン酸γ－トリペプチドを製造できるという利点がある。
According to the present invention, a dipeptide with a short reaction time and few by-products can be produced. In addition, according to the present invention, there is an advantage that N-protected glutamic acid γ-tripeptide can be produced efficiently and highly selectively, although the raw materials used are inexpensive and the reaction operation is simple.
本発明で対象とする中性アミノ酸としては、側鎖にアミノ基もしくはカルボキシル基を有しないアミノ酸をいい、グリシン（Ｇｌｙ）、アラニン（Ａｌａ）、β－アラニン、バリン（Ｖａｌ）、ロイシン（Ｌｅｕ）、イソロイシン（Ｉｌｅ）、ノルバリン（Ｎｖａ）、２－アミノブタン酸（Ａｂｕ）、プロリン（Ｐｒｏ）、メチオニン（Ｍｅｔ）、セリン（Ｓｅｒ）、スレオニン（Ｔｈｒ）、フェニルアラニン（Ｐｈｅ）、チロシン（Ｔｙｒ）等があげられる。これらのうち、グリシン、アラニン、β－アラニン、バリン、ロイシン、イソロイシン、ノルバリン、２-アミノブタン酸が好ましい。
ここで、Ｎ－保護中性アミノ酸としては、上記中性アミノ酸のアミノ基を、例えばベンジルオキシカルボニル基（Ｚ）、ｔ－ブチルオキシカルボニル基（Ｂｏｃ）、ホルミル基等の保護基で保護したものがあげられる。本発明において用いるもう１つの中性アミノ酸としては、アミノ基とカルボキシル基のいずれもが保護されていないもの、すなわちフリーの中性アミノ酸を用いるのが好ましい。本発明では、中性アミノ酸として、Ｎ－保護されたものと同じ又は異なったアミノ酸を用いることができる。 The neutral amino acid targeted in the present invention refers to an amino acid having no amino group or carboxyl group in the side chain, and includes glycine (Gly), alanine (Ala), β-alanine, valine (Val), and leucine (Leu). , Isoleucine (Ile), norvaline (Nva), 2-aminobutanoic acid (Abu), proline (Pro), methionine (Met), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr) and the like. can give. Of these, glycine, alanine, β-alanine, valine, leucine, isoleucine, norvaline and 2-aminobutanoic acid are preferred.
Here, as the N-protected neutral amino acid, the amino group of the neutral amino acid is protected with a protecting group such as benzyloxycarbonyl group (Z), t-butyloxycarbonyl group (Boc), formyl group, etc. Can be given. As another neutral amino acid used in the present invention, it is preferable to use one in which neither an amino group nor a carboxyl group is protected, that is, a free neutral amino acid. In the present invention, the same or different amino acid as the N-protected one can be used as the neutral amino acid.
ここで、Ｎ－保護中性アミノ酸としては、上記中性アミノ酸のアミノ基を、例えばベンジルオキシカルボニル基（Ｚ）、ｔ－ブチルオキシカルボニル基（Ｂｏｃ）、ホルミル基等の保護基で保護したものがあげられる。本発明において用いるもう１つの中性アミノ酸としては、アミノ基とカルボキシル基のいずれもが保護されていないもの、すなわちフリーの中性アミノ酸を用いるのが好ましい。本発明では、中性アミノ酸として、Ｎ－保護されたものと同じ又は異なったアミノ酸を用いることができる。 The neutral amino acid targeted in the present invention refers to an amino acid having no amino group or carboxyl group in the side chain, and includes glycine (Gly), alanine (Ala), β-alanine, valine (Val), and leucine (Leu). , Isoleucine (Ile), norvaline (Nva), 2-aminobutanoic acid (Abu), proline (Pro), methionine (Met), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr) and the like. can give. Of these, glycine, alanine, β-alanine, valine, leucine, isoleucine, norvaline and 2-aminobutanoic acid are preferred.
Here, as the N-protected neutral amino acid, the amino group of the neutral amino acid is protected with a protecting group such as benzyloxycarbonyl group (Z), t-butyloxycarbonyl group (Boc), formyl group, etc. Can be given. As another neutral amino acid used in the present invention, it is preferable to use one in which neither an amino group nor a carboxyl group is protected, that is, a free neutral amino acid. In the present invention, the same or different amino acid as the N-protected one can be used as the neutral amino acid.
Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルは、Ｎ－保護中性アミノ酸を酢酸エチル、アセトニトリル、テトラヒドロフランなどの有機溶媒中、０．５～２倍モル、好ましくは１．０～１．２倍モルのＮ－ヒドロキシコハク酸イミドと０．５～２倍モル、好ましくは１．０～１．２倍モルのジシクロヘキシルカルボジイミド（ＤＣＣ）、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩（ＥＤＣＩ．ＨＣｌ）等の縮合剤と反応させて得ることができる。
Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルと中性アミノ酸の反応は、例えば、Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルに対して、０．５～２倍モル、好ましくは０．８～１．２倍モルの中性アミノ酸を、溶媒中、－２０～８０℃、好ましくは０～５０℃で３０分～２４時間行うのがよい。
この際、反応をｐＨ８.５～１０.５の条件下で行うのが好ましく、より好ましくは８．５～９．５未満で行うのがよい。また、反応が低温で行われる場合は、ｐＨ９．５～１０．５で行うことも好ましい。
反応に用いられる溶媒としては、酢酸エチル、アセトニトリル、テトラヒドロフラン、ジオキサン、クロロホルム、Ｎ，Ｎ－ジメチルホルムアミド等の有機溶媒があげられる。
これら溶媒は混合して用いてもよく、また水との混合溶媒を用いてもよい。 N-hydroxysuccinimide ester of N-protected neutral amino acid is 0.5 to 2 moles, preferably 1.0 to 1 mole of N-protected neutral amino acid in an organic solvent such as ethyl acetate, acetonitrile or tetrahydrofuran. 2 moles of N-hydroxysuccinimide and 0.5 to 2 moles, preferably 1.0 to 1.2 moles of dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl ) It can be obtained by reacting with a condensing agent such as carbodiimide hydrochloride (EDCI.HCl).
The reaction of N-hydroxysuccinimide ester of N-protected neutral amino acid with neutral amino acid is, for example, 0.5 to 2 moles compared to N-hydroxysuccinimide ester of N-protected neutral amino acid, Preferably, 0.8 to 1.2 moles of neutral amino acid is carried out in a solvent at −20 to 80 ° C., preferably 0 to 50 ° C., for 30 minutes to 24 hours.
At this time, the reaction is preferably carried out under conditions of pH 8.5 to 10.5, more preferably 8.5 to less than 9.5. Further, when the reaction is carried out at a low temperature, it is also preferably carried out at a pH of 9.5 to 10.5.
Examples of the solvent used in the reaction include organic solvents such as ethyl acetate, acetonitrile, tetrahydrofuran, dioxane, chloroform and N, N-dimethylformamide.
These solvents may be used as a mixture, or a mixed solvent with water may be used.
Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルと中性アミノ酸の反応は、例えば、Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルに対して、０．５～２倍モル、好ましくは０．８～１．２倍モルの中性アミノ酸を、溶媒中、－２０～８０℃、好ましくは０～５０℃で３０分～２４時間行うのがよい。
この際、反応をｐＨ８.５～１０.５の条件下で行うのが好ましく、より好ましくは８．５～９．５未満で行うのがよい。また、反応が低温で行われる場合は、ｐＨ９．５～１０．５で行うことも好ましい。
反応に用いられる溶媒としては、酢酸エチル、アセトニトリル、テトラヒドロフラン、ジオキサン、クロロホルム、Ｎ，Ｎ－ジメチルホルムアミド等の有機溶媒があげられる。
これら溶媒は混合して用いてもよく、また水との混合溶媒を用いてもよい。 N-hydroxysuccinimide ester of N-protected neutral amino acid is 0.5 to 2 moles, preferably 1.0 to 1 mole of N-protected neutral amino acid in an organic solvent such as ethyl acetate, acetonitrile or tetrahydrofuran. 2 moles of N-hydroxysuccinimide and 0.5 to 2 moles, preferably 1.0 to 1.2 moles of dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl ) It can be obtained by reacting with a condensing agent such as carbodiimide hydrochloride (EDCI.HCl).
The reaction of N-hydroxysuccinimide ester of N-protected neutral amino acid with neutral amino acid is, for example, 0.5 to 2 moles compared to N-hydroxysuccinimide ester of N-protected neutral amino acid, Preferably, 0.8 to 1.2 moles of neutral amino acid is carried out in a solvent at −20 to 80 ° C., preferably 0 to 50 ° C., for 30 minutes to 24 hours.
At this time, the reaction is preferably carried out under conditions of pH 8.5 to 10.5, more preferably 8.5 to less than 9.5. Further, when the reaction is carried out at a low temperature, it is also preferably carried out at a pH of 9.5 to 10.5.
Examples of the solvent used in the reaction include organic solvents such as ethyl acetate, acetonitrile, tetrahydrofuran, dioxane, chloroform and N, N-dimethylformamide.
These solvents may be used as a mixture, or a mixed solvent with water may be used.
本発明では、次いで、上記の方法で得たジペプチドのＮ脱保護を常法により行う。例えば、保護基がベンジルオキシカルボニル基の場合は、上記の方法で得たジペプチド反応液をアルゴン置換し、パラジウム炭素を加えて、水素置換してＮ脱保護させるのがよい。
本発明では、次いで、上記の方法でＮ脱保護して得られたジペプチドを、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｎ－保護グルタミン酸無水物と反応させて、Ｎ－保護グルタミン酸γ－トリペプチドを製造する。尚、Ｎ－保護グルタミン酸γ－トリペプチドは、Ｎ－保護グルタミン酸のγ位がジペプチドに結合しているトリペプチドを意味する。
また、本発明の方法で得られうるＮ－保護グルタミン酸γ－トリペプチドのうち、Ｚ－γ－グルタミルバリルグリシンは新規化合物であり、γ-グルタミルバリルグリシンの製造中間体としてきわめて有用である。また、γ-グルタミルバリルグリシンの製造にあたり、Ｚ－γ-グルタミルバリルグリシンの結晶を取得してもよいが、その場合精製がきわめて容易となり、純度の高いγ-グルタミルバリルグリシンを製造することができる。
例えば、Ｚ－γ-グルタミルバリルグリシンの結晶（Ａ晶）は、粉末Ｘ線回折パターンにおいて、１０．９、１６．３、１９．２、１９．７、２０．３の回折角（２θ）にピークを示すことを特徴とする。 In the present invention, N deprotection of the dipeptide obtained by the above method is then performed by a conventional method. For example, when the protecting group is a benzyloxycarbonyl group, the dipeptide reaction solution obtained by the above method is preferably substituted with argon, palladium carbon is added, and hydrogen is substituted to deprotect N.
In the present invention, the dipeptide obtained by N-deprotection by the above method is then reacted with N-protected glutamic anhydride in the presence of N-hydroxysuccinimide to give N-protected glutamic acid γ-tri A peptide is produced. The N-protected glutamic acid γ-tripeptide means a tripeptide in which the γ-position of N-protected glutamic acid is bound to a dipeptide.
Among the N-protected glutamic acid γ-tripeptides obtainable by the method of the present invention, Z-γ-glutamylvalylglycine is a novel compound and is extremely useful as an intermediate for production of γ-glutamylvalylglycine. In production of γ-glutamyl valylglycine, crystals of Z-γ-glutamyl valyl glycine may be obtained. In this case, purification becomes extremely easy, and high purity γ-glutamyl valyl glycine can be produced. .
For example, crystals of Z-γ-glutamylvalylglycine (crystal A) have diffraction angles (2θ) of 10.9, 16.3, 19.2, 19.7, 20.3 in the powder X-ray diffraction pattern. It is characterized by showing a peak.
本発明では、次いで、上記の方法でＮ脱保護して得られたジペプチドを、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｎ－保護グルタミン酸無水物と反応させて、Ｎ－保護グルタミン酸γ－トリペプチドを製造する。尚、Ｎ－保護グルタミン酸γ－トリペプチドは、Ｎ－保護グルタミン酸のγ位がジペプチドに結合しているトリペプチドを意味する。
また、本発明の方法で得られうるＮ－保護グルタミン酸γ－トリペプチドのうち、Ｚ－γ－グルタミルバリルグリシンは新規化合物であり、γ-グルタミルバリルグリシンの製造中間体としてきわめて有用である。また、γ-グルタミルバリルグリシンの製造にあたり、Ｚ－γ-グルタミルバリルグリシンの結晶を取得してもよいが、その場合精製がきわめて容易となり、純度の高いγ-グルタミルバリルグリシンを製造することができる。
例えば、Ｚ－γ-グルタミルバリルグリシンの結晶（Ａ晶）は、粉末Ｘ線回折パターンにおいて、１０．９、１６．３、１９．２、１９．７、２０．３の回折角（２θ）にピークを示すことを特徴とする。 In the present invention, N deprotection of the dipeptide obtained by the above method is then performed by a conventional method. For example, when the protecting group is a benzyloxycarbonyl group, the dipeptide reaction solution obtained by the above method is preferably substituted with argon, palladium carbon is added, and hydrogen is substituted to deprotect N.
In the present invention, the dipeptide obtained by N-deprotection by the above method is then reacted with N-protected glutamic anhydride in the presence of N-hydroxysuccinimide to give N-protected glutamic acid γ-tri A peptide is produced. The N-protected glutamic acid γ-tripeptide means a tripeptide in which the γ-position of N-protected glutamic acid is bound to a dipeptide.
Among the N-protected glutamic acid γ-tripeptides obtainable by the method of the present invention, Z-γ-glutamylvalylglycine is a novel compound and is extremely useful as an intermediate for production of γ-glutamylvalylglycine. In production of γ-glutamyl valylglycine, crystals of Z-γ-glutamyl valyl glycine may be obtained. In this case, purification becomes extremely easy, and high purity γ-glutamyl valyl glycine can be produced. .
For example, crystals of Z-γ-glutamylvalylglycine (crystal A) have diffraction angles (2θ) of 10.9, 16.3, 19.2, 19.7, 20.3 in the powder X-ray diffraction pattern. It is characterized by showing a peak.
ここで、Ｎ－保護グルタミン酸無水物は、Ｎ－保護グルタミン酸をジシクロヘキシルカルボジイミド（ＤＤＣ）、無水酢酸等の脱水剤で処理するなどの常法により製造することができる。
上記ジペプチドとＮ－保護グルタミン酸無水物の反応は、例えば、Ｎ－保護グルタミン酸無水物に対して、０．５～２倍モル、好ましくは０．８～１．２倍モルのジペプチドを、０．２～２倍モル、好ましくは０．５～１．２倍モルのＮ－ヒドロキシコハク酸イミド（ＨＯＳｕ）の存在下に、溶媒中、－２０～８０℃、好ましくは０～５０℃で３０分～２４時間行うのがよい。
この際、反応を炭酸アルカリ金属塩の存在下で行うのが好ましい。炭酸アルカリ金属塩には、正塩（炭酸ナトリウム、炭酸カリウムなど）又は、炭酸水素塩（炭酸水素ナトリウム、炭酸水素カリウムなど）が含まれる。好ましくは炭酸水素ナトリム、炭酸水素カリウムである。炭酸アルカリ金属塩は、Ｎ－保護グルタミン酸に対して、１．０～１．２倍モルの量を用いるのが好ましい。
反応に用いられる溶媒としては、酢酸エチル、アセトニトリル、テトラヒドロフラン、ジオキサン、クロロホルム、Ｎ，Ｎ－ジメチルホルムアミド等の有機溶媒があげられる。
これら溶媒は混合して用いてもよく、また水との混合溶媒も用いてもよい。 Here, N-protected glutamic anhydride can be produced by a conventional method such as treating N-protected glutamic acid with a dehydrating agent such as dicyclohexylcarbodiimide (DDC) or acetic anhydride.
The reaction of the above dipeptide with N-protected glutamic anhydride is, for example, 0.5 to 2-fold mol, preferably 0.8 to 1.2-fold mol of dipeptide with respect to N-protected glutamic anhydride. In the presence of 2 to 2 moles, preferably 0.5 to 1.2 moles of N-hydroxysuccinimide (HOSu) in a solvent at −20 to 80 ° C., preferably 0 to 50 ° C. for 30 minutes. It should be done for ~ 24 hours.
At this time, the reaction is preferably carried out in the presence of an alkali metal carbonate. Alkali metal carbonates include normal salts (sodium carbonate, potassium carbonate, etc.) or bicarbonates (sodium bicarbonate, potassium bicarbonate, etc.). Preferred are sodium hydrogen carbonate and potassium hydrogen carbonate. The alkali metal carbonate is preferably used in an amount of 1.0 to 1.2 times the amount of N-protected glutamic acid.
Examples of the solvent used in the reaction include organic solvents such as ethyl acetate, acetonitrile, tetrahydrofuran, dioxane, chloroform and N, N-dimethylformamide.
These solvents may be used as a mixture, or a mixed solvent with water may be used.
上記ジペプチドとＮ－保護グルタミン酸無水物の反応は、例えば、Ｎ－保護グルタミン酸無水物に対して、０．５～２倍モル、好ましくは０．８～１．２倍モルのジペプチドを、０．２～２倍モル、好ましくは０．５～１．２倍モルのＮ－ヒドロキシコハク酸イミド（ＨＯＳｕ）の存在下に、溶媒中、－２０～８０℃、好ましくは０～５０℃で３０分～２４時間行うのがよい。
この際、反応を炭酸アルカリ金属塩の存在下で行うのが好ましい。炭酸アルカリ金属塩には、正塩（炭酸ナトリウム、炭酸カリウムなど）又は、炭酸水素塩（炭酸水素ナトリウム、炭酸水素カリウムなど）が含まれる。好ましくは炭酸水素ナトリム、炭酸水素カリウムである。炭酸アルカリ金属塩は、Ｎ－保護グルタミン酸に対して、１．０～１．２倍モルの量を用いるのが好ましい。
反応に用いられる溶媒としては、酢酸エチル、アセトニトリル、テトラヒドロフラン、ジオキサン、クロロホルム、Ｎ，Ｎ－ジメチルホルムアミド等の有機溶媒があげられる。
これら溶媒は混合して用いてもよく、また水との混合溶媒も用いてもよい。 Here, N-protected glutamic anhydride can be produced by a conventional method such as treating N-protected glutamic acid with a dehydrating agent such as dicyclohexylcarbodiimide (DDC) or acetic anhydride.
The reaction of the above dipeptide with N-protected glutamic anhydride is, for example, 0.5 to 2-fold mol, preferably 0.8 to 1.2-fold mol of dipeptide with respect to N-protected glutamic anhydride. In the presence of 2 to 2 moles, preferably 0.5 to 1.2 moles of N-hydroxysuccinimide (HOSu) in a solvent at −20 to 80 ° C., preferably 0 to 50 ° C. for 30 minutes. It should be done for ~ 24 hours.
At this time, the reaction is preferably carried out in the presence of an alkali metal carbonate. Alkali metal carbonates include normal salts (sodium carbonate, potassium carbonate, etc.) or bicarbonates (sodium bicarbonate, potassium bicarbonate, etc.). Preferred are sodium hydrogen carbonate and potassium hydrogen carbonate. The alkali metal carbonate is preferably used in an amount of 1.0 to 1.2 times the amount of N-protected glutamic acid.
Examples of the solvent used in the reaction include organic solvents such as ethyl acetate, acetonitrile, tetrahydrofuran, dioxane, chloroform and N, N-dimethylformamide.
These solvents may be used as a mixture, or a mixed solvent with water may be used.
上記の方法で得たトリペプチドのＮ脱保護は、常法により行う。例えば、ジペプチドの場合について説明したのと同様に、保護基がベンジルオキシカルボニル基の場合は、上記の方法で得たトリペプチド反応液をアルゴン置換し、パラジウム炭素を加えて、水素置換してＮ脱保護させるのがよい。
さらに、グルタミン酸γ－トリペプチドのγ体の比率を高めるために、粗グルタミン酸γ－トリペプチドを水に溶解しメタノールを加えて、晶析して、α体を除去するのがよい。又、副生するα体を、保護基の除去前でも除去後でも、結晶化、クロマトグラフィーなどの常用の方法によりγ－体から分離してもよい。
尚、本発明の方法により製造されるＺ－γ－グルタミルバリルグリシンの塩としては、ナトリウム塩、カリウム塩などがあげられる。
以下に、実施例を挙げて本発明をさらに詳しく説明するが、これらは本発明を限定するものではない。 N deprotection of the tripeptide obtained by the above method is performed by a conventional method. For example, as described in the case of dipeptide, when the protecting group is a benzyloxycarbonyl group, the tripeptide reaction solution obtained by the above method is replaced with argon, palladium carbon is added, and hydrogen is replaced with N. It should be deprotected.
Further, in order to increase the ratio of γ form of glutamic acid γ-tripeptide, it is preferable to dissolve the crude glutamic acid γ-tripeptide in water and add methanol to crystallize to remove the α form. Further, the by-product α-form may be separated from the γ-form by a conventional method such as crystallization or chromatography before or after removal of the protecting group.
Examples of the salt of Z-γ-glutamylvalylglycine produced by the method of the present invention include sodium salt and potassium salt.
Hereinafter, the present invention will be described in more detail with reference to examples, but these do not limit the present invention.
さらに、グルタミン酸γ－トリペプチドのγ体の比率を高めるために、粗グルタミン酸γ－トリペプチドを水に溶解しメタノールを加えて、晶析して、α体を除去するのがよい。又、副生するα体を、保護基の除去前でも除去後でも、結晶化、クロマトグラフィーなどの常用の方法によりγ－体から分離してもよい。
尚、本発明の方法により製造されるＺ－γ－グルタミルバリルグリシンの塩としては、ナトリウム塩、カリウム塩などがあげられる。
以下に、実施例を挙げて本発明をさらに詳しく説明するが、これらは本発明を限定するものではない。 N deprotection of the tripeptide obtained by the above method is performed by a conventional method. For example, as described in the case of dipeptide, when the protecting group is a benzyloxycarbonyl group, the tripeptide reaction solution obtained by the above method is replaced with argon, palladium carbon is added, and hydrogen is replaced with N. It should be deprotected.
Further, in order to increase the ratio of γ form of glutamic acid γ-tripeptide, it is preferable to dissolve the crude glutamic acid γ-tripeptide in water and add methanol to crystallize to remove the α form. Further, the by-product α-form may be separated from the γ-form by a conventional method such as crystallization or chromatography before or after removal of the protecting group.
Examples of the salt of Z-γ-glutamylvalylglycine produced by the method of the present invention include sodium salt and potassium salt.
Hereinafter, the present invention will be described in more detail with reference to examples, but these do not limit the present invention.
実施例１ Ｖａｌ－Ｇｌｙの合成
下記のスキーム１に基づいて、Ｖａｌ－Ｇｌｙを合成した。
スキーム１
Example 1 Synthesis of Val-Gly Based on the following scheme 1, Val-Gly was synthesized.
Scheme 1
下記のスキーム１に基づいて、Ｖａｌ－Ｇｌｙを合成した。
スキーム１
Scheme 1
(1)Ｚ（ベンジルオキシカルボニル）－バリンのヒドロキシコハク酸イミドエステルの合成
Ｚ－バリン２０ｇ（７９．６ｍｍｏｌ）を酢酸エチル１２０ｍｌに溶解し、Ｎ－ヒドロキシコハク酸イミド（ＨＯＳｕ）９．１８ｇ（１ｅｑ）を加えて５℃に冷却した。Ｎ，Ｎ－ジシクロヘキシルカルボジイミド１６．４２ｇ（１ｅｑ）を酢酸エチル５０ｍｌに溶解して、１０℃以下でゆっくりと加えた。５℃で３０分、室温（２５℃）で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により分離し、酢酸エチル３０ｍｌで洗浄した。目的のＺ－バリンコハク酸イミドエステルを酢酸エチル溶液として得た。 (1) Synthesis of hydroxysuccinimide ester of Z (benzyloxycarbonyl) -valine Z-valine 20 g (79.6 mmol) was dissolved in 120 ml of ethyl acetate, and N-hydroxysuccinimide (HOSu) 9.18 g (1 eq) ) And cooled to 5 ° C. 16.42 g (1 eq) of N, N-dicyclohexylcarbodiimide was dissolved in 50 ml of ethyl acetate and slowly added at 10 ° C. or lower. The mixture was stirred at 5 ° C. for 30 minutes and at room temperature (25 ° C.) overnight. The precipitated dicyclohexylurea was separated by filtration and washed with 30 ml of ethyl acetate. The desired Z-valine succinimide ester was obtained as an ethyl acetate solution.
Ｚ－バリン２０ｇ（７９．６ｍｍｏｌ）を酢酸エチル１２０ｍｌに溶解し、Ｎ－ヒドロキシコハク酸イミド（ＨＯＳｕ）９．１８ｇ（１ｅｑ）を加えて５℃に冷却した。Ｎ，Ｎ－ジシクロヘキシルカルボジイミド１６．４２ｇ（１ｅｑ）を酢酸エチル５０ｍｌに溶解して、１０℃以下でゆっくりと加えた。５℃で３０分、室温（２５℃）で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により分離し、酢酸エチル３０ｍｌで洗浄した。目的のＺ－バリンコハク酸イミドエステルを酢酸エチル溶液として得た。 (1) Synthesis of hydroxysuccinimide ester of Z (benzyloxycarbonyl) -valine Z-valine 20 g (79.6 mmol) was dissolved in 120 ml of ethyl acetate, and N-hydroxysuccinimide (HOSu) 9.18 g (1 eq) ) And cooled to 5 ° C. 16.42 g (1 eq) of N, N-dicyclohexylcarbodiimide was dissolved in 50 ml of ethyl acetate and slowly added at 10 ° C. or lower. The mixture was stirred at 5 ° C. for 30 minutes and at room temperature (25 ° C.) overnight. The precipitated dicyclohexylurea was separated by filtration and washed with 30 ml of ethyl acetate. The desired Z-valine succinimide ester was obtained as an ethyl acetate solution.
(2)バリルグリシンの合成(Ｒｕｎ１)
グリシン ６．２８ｇ（１．０５ｅｑ）に水５５ｍｌを加えて溶解し、４Ｍ水酸化ナトリウム水溶液でｐＨ９．０に調整した。この溶液にＺ－バリンのヒドロキシコハク酸イミドエステルの酢酸エチル溶液を室温（２０－２５℃）で１時間かけて滴下、その後１時間撹拌した。その間４Ｍ水酸化ナトリウム水溶液でｐＨ８．５－９．５未満に調整した。６Ｍ塩酸を加えて撹拌、ｐH８に調整した。反応液をアルゴン置換後、５０％ｗｅｔ１０％パラジウム炭素０．４ｇを加えて、水素置換して一晩室温で撹拌した。アルゴン置換後、パラジウム炭素を分離により除いた。反応液を分層して、バリルグリシンの水溶液を得た。収率９５．６％
(２－１) バリルグリシンの合成（Ｒｕｎ２～６）
Ｒｕｎ１の条件を変化させて、バリルグリシンを合成した。変更した合成条件と結果をまとめて、表１に示す。
表１
(2) Synthesis of valylglycine (Run1)
55 ml of water was added to 6.28 g (1.05 eq) of glycine and dissolved, and the pH was adjusted to 9.0 with 4M aqueous sodium hydroxide solution. To this solution, an ethyl acetate solution of Z-valine hydroxysuccinimide ester was added dropwise at room temperature (20-25 ° C.) over 1 hour, followed by stirring for 1 hour. Meanwhile, the pH was adjusted to less than 8.5-9.5 with 4M aqueous sodium hydroxide solution. 6M hydrochloric acid was added and stirred to adjust the pH to 8. The reaction solution was purged with argon, 0.4 g of 50% wet 10% palladium carbon was added, and the mixture was purged with hydrogen and stirred overnight at room temperature. After purging with argon, palladium on carbon was removed by separation. The reaction solution was separated to obtain an aqueous solution of valylglycine. Yield 95.6%
(2-1) Synthesis of valylglycine (Run 2-6)
Valylglycine was synthesized by changing the conditions of Run1. The modified synthesis conditions and results are summarized in Table 1.
Table 1
グリシン ６．２８ｇ（１．０５ｅｑ）に水５５ｍｌを加えて溶解し、４Ｍ水酸化ナトリウム水溶液でｐＨ９．０に調整した。この溶液にＺ－バリンのヒドロキシコハク酸イミドエステルの酢酸エチル溶液を室温（２０－２５℃）で１時間かけて滴下、その後１時間撹拌した。その間４Ｍ水酸化ナトリウム水溶液でｐＨ８．５－９．５未満に調整した。６Ｍ塩酸を加えて撹拌、ｐH８に調整した。反応液をアルゴン置換後、５０％ｗｅｔ１０％パラジウム炭素０．４ｇを加えて、水素置換して一晩室温で撹拌した。アルゴン置換後、パラジウム炭素を分離により除いた。反応液を分層して、バリルグリシンの水溶液を得た。収率９５．６％
(２－１) バリルグリシンの合成（Ｒｕｎ２～６）
Ｒｕｎ１の条件を変化させて、バリルグリシンを合成した。変更した合成条件と結果をまとめて、表１に示す。
表１
55 ml of water was added to 6.28 g (1.05 eq) of glycine and dissolved, and the pH was adjusted to 9.0 with 4M aqueous sodium hydroxide solution. To this solution, an ethyl acetate solution of Z-valine hydroxysuccinimide ester was added dropwise at room temperature (20-25 ° C.) over 1 hour, followed by stirring for 1 hour. Meanwhile, the pH was adjusted to less than 8.5-9.5 with 4M aqueous sodium hydroxide solution. 6M hydrochloric acid was added and stirred to adjust the pH to 8. The reaction solution was purged with argon, 0.4 g of 50% wet 10% palladium carbon was added, and the mixture was purged with hydrogen and stirred overnight at room temperature. After purging with argon, palladium on carbon was removed by separation. The reaction solution was separated to obtain an aqueous solution of valylglycine. Yield 95.6%
(2-1) Synthesis of valylglycine (Run 2-6)
Valylglycine was synthesized by changing the conditions of Run1. The modified synthesis conditions and results are summarized in Table 1.
Table 1
尚、Ｒｕｎ６は、次のようにして行った。
グリシン１．５７ｇ（１．０５ｅｑ）に水２５ｍｌを加えて溶解し、炭酸水素ナトリウム５．１ｇ（３．０ｅｑ）を加えて撹拌した。この溶液にＺ－バリンのヒドロキシコハク酸イミドエステルの酢酸エチル溶液（１９．９ｍｍｏｌ）を加えて４０℃で１８時間撹拌した。６Ｍ塩酸を加えて撹拌、ｐH８に調整した。反応液をアルゴン置換し、５０％ｗｅｔ１０％パラジウム炭素０．０５ｇを加えて、水素置換して一晩室温で撹拌した。反応液をアルゴン置換後、パラジウム炭素を分離により除いた。反応液を分層して、バリルグリシンの水溶液を得た。収率８７．４％ Run 6 was performed as follows.
To 1.57 g (1.05 eq) of glycine was dissolved by adding 25 ml of water, and 5.1 g (3.0 eq) of sodium bicarbonate was added and stirred. To this solution, an ethyl acetate solution (19.9 mmol) of hydroxysuccinimide ester of Z-valine was added and stirred at 40 ° C. for 18 hours. 6M hydrochloric acid was added and stirred to adjust the pH to 8. The reaction solution was purged with argon, 0.05 g of 50% wet 10% palladium on carbon was added, purged with hydrogen, and stirred overnight at room temperature. After replacing the reaction solution with argon, palladium carbon was removed by separation. The reaction solution was separated to obtain an aqueous solution of valylglycine. Yield 87.4%
グリシン１．５７ｇ（１．０５ｅｑ）に水２５ｍｌを加えて溶解し、炭酸水素ナトリウム５．１ｇ（３．０ｅｑ）を加えて撹拌した。この溶液にＺ－バリンのヒドロキシコハク酸イミドエステルの酢酸エチル溶液（１９．９ｍｍｏｌ）を加えて４０℃で１８時間撹拌した。６Ｍ塩酸を加えて撹拌、ｐH８に調整した。反応液をアルゴン置換し、５０％ｗｅｔ１０％パラジウム炭素０．０５ｇを加えて、水素置換して一晩室温で撹拌した。反応液をアルゴン置換後、パラジウム炭素を分離により除いた。反応液を分層して、バリルグリシンの水溶液を得た。収率８７．４％ Run 6 was performed as follows.
To 1.57 g (1.05 eq) of glycine was dissolved by adding 25 ml of water, and 5.1 g (3.0 eq) of sodium bicarbonate was added and stirred. To this solution, an ethyl acetate solution (19.9 mmol) of hydroxysuccinimide ester of Z-valine was added and stirred at 40 ° C. for 18 hours. 6M hydrochloric acid was added and stirred to adjust the pH to 8. The reaction solution was purged with argon, 0.05 g of 50% wet 10% palladium on carbon was added, purged with hydrogen, and stirred overnight at room temperature. After replacing the reaction solution with argon, palladium carbon was removed by separation. The reaction solution was separated to obtain an aqueous solution of valylglycine. Yield 87.4%
実施例２ γ－グルタミルバリルグリシン（γ－Ｇｌｕ－Ｖａｌ－Ｇｌｙ）の合成
まず、下記のスキーム２に基づいて、Ｚ－Ｇｌｕ（Ｖａｌ－Ｇｌｙ）を合成した。
スキーム２
Example 2 Synthesis of γ-Glutamylvalylglycine (γ-Glu-Val-Gly) First, Z-Glu (Val-Gly) was synthesized based on the following scheme 2.
Scheme 2
まず、下記のスキーム２に基づいて、Ｚ－Ｇｌｕ（Ｖａｌ－Ｇｌｙ）を合成した。
スキーム２
Scheme 2
(3)Ｚ－γ－グルタミルバリルグリシンの合成（粗Ｚ－γ－グルタミルバリルグリシン結晶単離）
Ｚ－グルタミン酸２４．６３ｇ（１．１ｅｑ ｖｓ Ｚ－Ｖａｌ）に酢酸エチル１４８ｍｌを加えて撹拌、５℃に冷却した。Ｎ，Ｎ－ジシクロヘキシルカルボジイミド１８．１ｇ（１ｅｑ）を酢酸エチル６２ｍｌに溶解して、１０℃以下でゆっくりと加えた。５℃で３０分、室温（２５℃）で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により分離し、酢酸エチル３７ｍｌで洗浄した。Ｚ－グルタミン酸無水物の酢酸エチル溶液を得た。
実施例１のＲｕｎ３で製造したバリルグリシン水溶液（ＨＯＳｕを含む）に炭酸水素ナトリウム７．３６ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を加えて溶解し、そこにＺ－グルタミン酸無水物の酢酸エチル溶液を１．５時間かけて室温で滴下した。滴下終了後１時間撹拌し、分層、Ｚ－γ－グルタミルバリルグリシンの水溶液を得た。収率８４．８％ γ／α＝９６．４／３．６
水２４０ｍｌに６Ｍ塩酸３４．５ｍｌ（２．６ｅｑ）を加えて撹拌し、そこへ上記で得たＺ－γ－グルタミルバリルグリシン水溶液を９時間かけてゆっくりと滴下、滴下後一晩室温で撹拌した。析出した結晶を分離、水１２０ｍｌで洗浄し、ｗｅｔ晶７８．９５ｇ得た。４０℃で一晩減圧乾燥し１３．８２ｇの粗Ｚ－γ－グルタミルバリルグリシンの結晶（Ａ晶）を得た。収率６８．８％ γ／α＝９７．２／２．８ (3) Synthesis of Z-γ-glutamylvalylglycine (isolation of crude Z-γ-glutamylvalylglycine crystal)
148 ml of ethyl acetate was added to 24.63 g (1.1 eq vs. Z-Val) of Z-glutamic acid, and the mixture was stirred and cooled to 5 ° C. 18.1 g (1 eq) of N, N-dicyclohexylcarbodiimide was dissolved in 62 ml of ethyl acetate and slowly added at 10 ° C. or lower. The mixture was stirred at 5 ° C. for 30 minutes and at room temperature (25 ° C.) overnight. The precipitated dicyclohexylurea was separated by filtration and washed with 37 ml of ethyl acetate. An ethyl acetate solution of Z-glutamic anhydride was obtained.
7.36 g (1.0 eq vs. Z-Glu) of sodium hydrogen carbonate was added to and dissolved in the valylglycine aqueous solution (including HOSu) produced in Run 3 of Example 1, and an ethyl acetate solution of Z-glutamic anhydride was added thereto. It was dripped at room temperature over 1.5 hours. After completion of the dropping, the mixture was stirred for 1 hour to obtain a layer separation and an aqueous solution of Z-γ-glutamylvalylglycine. Yield 84.8% γ / α = 96.4 / 3.6
To 240 ml of water, 34.5 ml (2.6 eq) of 6M hydrochloric acid was added and stirred. The Z-γ-glutamylvalylglycine aqueous solution obtained above was slowly added dropwise over 9 hours, and the mixture was stirred overnight at room temperature. . The precipitated crystals were separated and washed with 120 ml of water to obtain 78.95 g of wet crystals. It was dried under reduced pressure at 40 ° C. overnight to obtain 13.82 g of crude Z-γ-glutamylvalylglycine crystals (crystal A). Yield 68.8% γ / α = 97.2 / 2.8
Ｚ－グルタミン酸２４．６３ｇ（１．１ｅｑ ｖｓ Ｚ－Ｖａｌ）に酢酸エチル１４８ｍｌを加えて撹拌、５℃に冷却した。Ｎ，Ｎ－ジシクロヘキシルカルボジイミド１８．１ｇ（１ｅｑ）を酢酸エチル６２ｍｌに溶解して、１０℃以下でゆっくりと加えた。５℃で３０分、室温（２５℃）で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により分離し、酢酸エチル３７ｍｌで洗浄した。Ｚ－グルタミン酸無水物の酢酸エチル溶液を得た。
実施例１のＲｕｎ３で製造したバリルグリシン水溶液（ＨＯＳｕを含む）に炭酸水素ナトリウム７．３６ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を加えて溶解し、そこにＺ－グルタミン酸無水物の酢酸エチル溶液を１．５時間かけて室温で滴下した。滴下終了後１時間撹拌し、分層、Ｚ－γ－グルタミルバリルグリシンの水溶液を得た。収率８４．８％ γ／α＝９６．４／３．６
水２４０ｍｌに６Ｍ塩酸３４．５ｍｌ（２．６ｅｑ）を加えて撹拌し、そこへ上記で得たＺ－γ－グルタミルバリルグリシン水溶液を９時間かけてゆっくりと滴下、滴下後一晩室温で撹拌した。析出した結晶を分離、水１２０ｍｌで洗浄し、ｗｅｔ晶７８．９５ｇ得た。４０℃で一晩減圧乾燥し１３．８２ｇの粗Ｚ－γ－グルタミルバリルグリシンの結晶（Ａ晶）を得た。収率６８．８％ γ／α＝９７．２／２．８ (3) Synthesis of Z-γ-glutamylvalylglycine (isolation of crude Z-γ-glutamylvalylglycine crystal)
148 ml of ethyl acetate was added to 24.63 g (1.1 eq vs. Z-Val) of Z-glutamic acid, and the mixture was stirred and cooled to 5 ° C. 18.1 g (1 eq) of N, N-dicyclohexylcarbodiimide was dissolved in 62 ml of ethyl acetate and slowly added at 10 ° C. or lower. The mixture was stirred at 5 ° C. for 30 minutes and at room temperature (25 ° C.) overnight. The precipitated dicyclohexylurea was separated by filtration and washed with 37 ml of ethyl acetate. An ethyl acetate solution of Z-glutamic anhydride was obtained.
7.36 g (1.0 eq vs. Z-Glu) of sodium hydrogen carbonate was added to and dissolved in the valylglycine aqueous solution (including HOSu) produced in Run 3 of Example 1, and an ethyl acetate solution of Z-glutamic anhydride was added thereto. It was dripped at room temperature over 1.5 hours. After completion of the dropping, the mixture was stirred for 1 hour to obtain a layer separation and an aqueous solution of Z-γ-glutamylvalylglycine. Yield 84.8% γ / α = 96.4 / 3.6
To 240 ml of water, 34.5 ml (2.6 eq) of 6M hydrochloric acid was added and stirred. The Z-γ-glutamylvalylglycine aqueous solution obtained above was slowly added dropwise over 9 hours, and the mixture was stirred overnight at room temperature. . The precipitated crystals were separated and washed with 120 ml of water to obtain 78.95 g of wet crystals. It was dried under reduced pressure at 40 ° C. overnight to obtain 13.82 g of crude Z-γ-glutamylvalylglycine crystals (crystal A). Yield 68.8% γ / α = 97.2 / 2.8
なお、Ｚ－γ－グルタミルバリルグリシンには以下のＡ晶～Ｆ晶の結晶多形があることを見出した。これらのうち、結晶としては、Ｅ晶が好ましい。主要ピーク［回折角（２θ）］、製造例と共に以下に示す。又、これらのＡ晶～Ｆ晶の粉末Ｘ線結晶回折パターンを、図１～６に示す。
Ａ晶：酸性晶析した結晶（１０．９、１６．３、１９．２、１９．７、２０．３）
Ｂ晶：メタノールでスラリー洗浄して得られる結晶（６．０、７．３，８．３，１５．０，２０．０）
(３)で得た粗Ｚ－γ－グルタミルバリルグリシンのＡ晶（０．３ｇ）にメタノール１．５ｍｌを加えて２０℃で３日間撹拌した。結晶を分離、４０℃で一晩減圧乾燥し０．２ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｂ晶）を得た。
Ｃ晶：Ａ晶をエタノールで再晶析した結晶（６．１、１５．１、１７．７、２４．６、２６．９）
(３)で得た粗Ｚ－γ－グルタミルバリルグリシンのＡ晶（１．０ｇ）にエタノール１０ｍｌを加えて３０℃で３時間、さらに５０℃で１時間撹拌した。その後６時間かけて２０℃まで冷却し、一晩２０℃で撹拌した。結晶を分離、４０℃で一晩減圧乾燥し０．８５ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｃ晶）を得た。 It has been found that Z-γ-glutamylvalylglycine has the following crystal polymorphs of A crystal to F crystal. Of these, the crystal E is preferred as the crystal. The main peak [diffraction angle (2θ)] and production examples are shown below. The powder X-ray crystal diffraction patterns of these A crystals to F crystals are shown in FIGS.
Crystal A: Crystals crystallized acidic (10.9, 16.3, 19.2, 19.7, 20.3)
Crystal B: Crystal obtained by slurry washing with methanol (6.0, 7.3, 8.3, 15.0, 20.0)
1.5 ml of methanol was added to the crystal A (0.3 g) of the crude Z-γ-glutamylvalylglycine obtained in (3) and stirred at 20 ° C. for 3 days. The crystals were separated and dried under reduced pressure at 40 ° C. overnight to obtain 0.2 g of crystals of Z-γ-glutamylvalylglycine (crystal B).
Crystal C: Crystal obtained by recrystallizing Crystal A from ethanol (6.1, 15.1, 17.7, 24.6, 26.9)
Ethanol (10 ml) was added to the crude Z-γ-glutamylvalylglycine crystal A (1.0 g) obtained in (3), and the mixture was stirred at 30 ° C. for 3 hours and further at 50 ° C. for 1 hour. Thereafter, the mixture was cooled to 20 ° C. over 6 hours and stirred at 20 ° C. overnight. The crystals were separated and dried under reduced pressure overnight at 40 ° C. to obtain 0.85 g of Z-γ-glutamylvalylglycine crystals (C crystals).
Ａ晶：酸性晶析した結晶（１０．９、１６．３、１９．２、１９．７、２０．３）
Ｂ晶：メタノールでスラリー洗浄して得られる結晶（６．０、７．３，８．３，１５．０，２０．０）
(３)で得た粗Ｚ－γ－グルタミルバリルグリシンのＡ晶（０．３ｇ）にメタノール１．５ｍｌを加えて２０℃で３日間撹拌した。結晶を分離、４０℃で一晩減圧乾燥し０．２ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｂ晶）を得た。
Ｃ晶：Ａ晶をエタノールで再晶析した結晶（６．１、１５．１、１７．７、２４．６、２６．９）
(３)で得た粗Ｚ－γ－グルタミルバリルグリシンのＡ晶（１．０ｇ）にエタノール１０ｍｌを加えて３０℃で３時間、さらに５０℃で１時間撹拌した。その後６時間かけて２０℃まで冷却し、一晩２０℃で撹拌した。結晶を分離、４０℃で一晩減圧乾燥し０．８５ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｃ晶）を得た。 It has been found that Z-γ-glutamylvalylglycine has the following crystal polymorphs of A crystal to F crystal. Of these, the crystal E is preferred as the crystal. The main peak [diffraction angle (2θ)] and production examples are shown below. The powder X-ray crystal diffraction patterns of these A crystals to F crystals are shown in FIGS.
Crystal A: Crystals crystallized acidic (10.9, 16.3, 19.2, 19.7, 20.3)
Crystal B: Crystal obtained by slurry washing with methanol (6.0, 7.3, 8.3, 15.0, 20.0)
1.5 ml of methanol was added to the crystal A (0.3 g) of the crude Z-γ-glutamylvalylglycine obtained in (3) and stirred at 20 ° C. for 3 days. The crystals were separated and dried under reduced pressure at 40 ° C. overnight to obtain 0.2 g of crystals of Z-γ-glutamylvalylglycine (crystal B).
Crystal C: Crystal obtained by recrystallizing Crystal A from ethanol (6.1, 15.1, 17.7, 24.6, 26.9)
Ethanol (10 ml) was added to the crude Z-γ-glutamylvalylglycine crystal A (1.0 g) obtained in (3), and the mixture was stirred at 30 ° C. for 3 hours and further at 50 ° C. for 1 hour. Thereafter, the mixture was cooled to 20 ° C. over 6 hours and stirred at 20 ° C. overnight. The crystals were separated and dried under reduced pressure overnight at 40 ° C. to obtain 0.85 g of Z-γ-glutamylvalylglycine crystals (C crystals).
Ｄ晶：Ｂ晶をエタノールで再晶析した結晶（６．６、６．７、１９．０、１９．５、３１．９）
粗Ｚ－γ－グルタミルバリルグリシンのＢ晶（６．０ｇ）にエタノール１０８ｍｌを加えて６５℃で１時間撹拌した。その後１０時間かけて１０℃まで冷却し、一晩１０℃で撹拌した。結晶を分離、４０℃で一晩減圧乾燥し３．８ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｄ晶）を得た。
Ｅ晶：Ａ晶をメタノールで再晶析した結晶（８．５、１４．１、１８．０、２１．６、２２．９）
上記(３)で得た粗Ｚ－γ－グルタミルバリルグリシンにメタノール７３．３ｍｌを加えて４０℃で３時間撹拌した。その後３時間かけて１０℃まで冷却し、一晩１０℃で撹拌した。結晶を分離、メタノール１２ｍｌで洗浄、４０℃で一晩減圧乾燥し８．０ｇのＺ－γ－グルタミルバリルグリシンの結晶を得た。収率４５．５％ γ／α＝９９．９／０．１
1Ｈ ＮＭＲ（ＤＭＳＯ－ｄ６，４００ＭＨｚ）：δ０．８３（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；０．８６（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；１．７０－１．８１（１Ｈ，ｍ）；１．９３－１．９８（２Ｈ，ｍ）；２．１８－２．３６（２Ｈ，ｍ）；３．６９（１Ｈ，ｄｄ，Ｊ＝１７．５，５．８Ｈｚ）；３．７７（１Ｈ，ｄｄ，Ｊ＝１７．５，５．９）；３．９０－４．０２（１Ｈ，ｍ）；４．１８（１Ｈ，ｄｄ，Ｊ＝８．９，６．７Ｈｚ）；５．０３（１Ｈ，ｓ）；７．２６－７．４２（５Ｈ，ｍ）；７．５８（１Ｈ，ｄ，Ｊ＝７．９Ｈｚ）；７．８４（１Ｈ，ｄ，Ｊ＝８．９Ｈｚ）；８．２４（１Ｈ，ｄｄ，Ｊ＝５．９，５．８Ｈｚ）
13Ｃ ＮＭＲ（ＤＭＳＯ－ｄ６，４００ＭＨｚ）：δ１８．３，１９．５，２７．３，３０．８，３２．０，４０．９，５３．８，５７．８，６５．８，１２８．１，１２８．２，１２８．７，１３７．４，１５６．５，１７１．５，１７１．７８，１７１．８４，１７４．０
Ｍａｓｓ（Ｍ＋Ｈ）：４３８ Ｍａｓｓ（Ｍ－Ｈ）：４３６
Ｆ晶：Ｂ晶を酸性で再晶析した結晶（６．６、１３．５、１５．２、２０．９）
粗Ｚ－γ－グルタミルバリルグリシンのＢ晶（１．０ｇ）にメタノール８ｍｌとＺ－グルタミン酸（３５ｍｇ）を加えて４０℃で１時間撹拌した。その後３時間かけて１０℃まで冷却し、一晩１０℃で撹拌した。結晶を分離、４０℃で一晩減圧乾燥し０．３５ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｆ晶）を得た。 Crystal D: Crystal obtained by recrystallizing Crystal B from ethanol (6.6, 6.7, 19.0, 19.5, 31.9)
108 B of ethanol was added to B crystal (6.0 g) of crude Z-γ-glutamylvalylglycine, and the mixture was stirred at 65 ° C. for 1 hour. Thereafter, the mixture was cooled to 10 ° C. over 10 hours and stirred at 10 ° C. overnight. The crystals were separated and dried under reduced pressure at 40 ° C. overnight to obtain 3.8 g of Z-γ-glutamylvalylglycine crystals (crystal D).
Crystal E: Crystal obtained by recrystallizing Crystal A from methanol (8.5, 14.1, 18.0, 21.6, 22.9)
73.3 ml of methanol was added to the crude Z-γ-glutamylvalylglycine obtained in (3) above and stirred at 40 ° C. for 3 hours. Thereafter, the mixture was cooled to 10 ° C. over 3 hours and stirred at 10 ° C. overnight. The crystals were separated, washed with 12 ml of methanol, and dried under reduced pressure at 40 ° C. overnight to obtain 8.0 g of Z-γ-glutamylvalylglycine crystals. Yield 45.5% γ / α = 99.9 / 0.1
1 H NMR (DMSO-d6, 400 MHz): δ 0.83 (3H, d, J = 6.8 Hz); 0.86 (3H, d, J = 6.8 Hz); 1.70-1.81 (1H , M); 1.93-1.98 (2H, m); 2.18-2.36 (2H, m); 3.69 (1H, dd, J = 17.5, 5.8 Hz); 3 .77 (1H, dd, J = 17.5, 5.9); 3.90-4.02 (1H, m); 4.18 (1H, dd, J = 8.9, 6.7 Hz); 5.03 (1H, s); 7.26-7.42 (5H, m); 7.58 (1H, d, J = 7.9 Hz); 7.84 (1H, d, J = 8.9 Hz) ); 8.24 (1H, dd, J = 5.9, 5.8 Hz)
13 C NMR (DMSO-d6, 400 MHz): δ 18.3, 19.5, 27.3, 30.8, 32.0, 40.9, 53.8, 57.8, 65.8, 128.1 , 128.2, 128.7, 137.4, 156.5, 171.5, 171.78, 171.84, 174.0
Mass (M + H): 438 Mass (M−H): 436
Crystal F: Crystal obtained by recrystallizing Crystal B (6.6, 13.5, 15.2, 20.9)
8 ml of methanol and Z-glutamic acid (35 mg) were added to B crystals (1.0 g) of crude Z-γ-glutamylvalylglycine, and the mixture was stirred at 40 ° C. for 1 hour. Thereafter, the mixture was cooled to 10 ° C. over 3 hours and stirred at 10 ° C. overnight. The crystals were separated and dried under reduced pressure at 40 ° C. overnight to obtain 0.35 g of Z-γ-glutamylvalylglycine crystals (F crystals).
粗Ｚ－γ－グルタミルバリルグリシンのＢ晶（６．０ｇ）にエタノール１０８ｍｌを加えて６５℃で１時間撹拌した。その後１０時間かけて１０℃まで冷却し、一晩１０℃で撹拌した。結晶を分離、４０℃で一晩減圧乾燥し３．８ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｄ晶）を得た。
Ｅ晶：Ａ晶をメタノールで再晶析した結晶（８．５、１４．１、１８．０、２１．６、２２．９）
上記(３)で得た粗Ｚ－γ－グルタミルバリルグリシンにメタノール７３．３ｍｌを加えて４０℃で３時間撹拌した。その後３時間かけて１０℃まで冷却し、一晩１０℃で撹拌した。結晶を分離、メタノール１２ｍｌで洗浄、４０℃で一晩減圧乾燥し８．０ｇのＺ－γ－グルタミルバリルグリシンの結晶を得た。収率４５．５％ γ／α＝９９．９／０．１
1Ｈ ＮＭＲ（ＤＭＳＯ－ｄ６，４００ＭＨｚ）：δ０．８３（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；０．８６（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；１．７０－１．８１（１Ｈ，ｍ）；１．９３－１．９８（２Ｈ，ｍ）；２．１８－２．３６（２Ｈ，ｍ）；３．６９（１Ｈ，ｄｄ，Ｊ＝１７．５，５．８Ｈｚ）；３．７７（１Ｈ，ｄｄ，Ｊ＝１７．５，５．９）；３．９０－４．０２（１Ｈ，ｍ）；４．１８（１Ｈ，ｄｄ，Ｊ＝８．９，６．７Ｈｚ）；５．０３（１Ｈ，ｓ）；７．２６－７．４２（５Ｈ，ｍ）；７．５８（１Ｈ，ｄ，Ｊ＝７．９Ｈｚ）；７．８４（１Ｈ，ｄ，Ｊ＝８．９Ｈｚ）；８．２４（１Ｈ，ｄｄ，Ｊ＝５．９，５．８Ｈｚ）
13Ｃ ＮＭＲ（ＤＭＳＯ－ｄ６，４００ＭＨｚ）：δ１８．３，１９．５，２７．３，３０．８，３２．０，４０．９，５３．８，５７．８，６５．８，１２８．１，１２８．２，１２８．７，１３７．４，１５６．５，１７１．５，１７１．７８，１７１．８４，１７４．０
Ｍａｓｓ（Ｍ＋Ｈ）：４３８ Ｍａｓｓ（Ｍ－Ｈ）：４３６
Ｆ晶：Ｂ晶を酸性で再晶析した結晶（６．６、１３．５、１５．２、２０．９）
粗Ｚ－γ－グルタミルバリルグリシンのＢ晶（１．０ｇ）にメタノール８ｍｌとＺ－グルタミン酸（３５ｍｇ）を加えて４０℃で１時間撹拌した。その後３時間かけて１０℃まで冷却し、一晩１０℃で撹拌した。結晶を分離、４０℃で一晩減圧乾燥し０．３５ｇのＺ－γ－グルタミルバリルグリシンの結晶（Ｆ晶）を得た。 Crystal D: Crystal obtained by recrystallizing Crystal B from ethanol (6.6, 6.7, 19.0, 19.5, 31.9)
108 B of ethanol was added to B crystal (6.0 g) of crude Z-γ-glutamylvalylglycine, and the mixture was stirred at 65 ° C. for 1 hour. Thereafter, the mixture was cooled to 10 ° C. over 10 hours and stirred at 10 ° C. overnight. The crystals were separated and dried under reduced pressure at 40 ° C. overnight to obtain 3.8 g of Z-γ-glutamylvalylglycine crystals (crystal D).
Crystal E: Crystal obtained by recrystallizing Crystal A from methanol (8.5, 14.1, 18.0, 21.6, 22.9)
73.3 ml of methanol was added to the crude Z-γ-glutamylvalylglycine obtained in (3) above and stirred at 40 ° C. for 3 hours. Thereafter, the mixture was cooled to 10 ° C. over 3 hours and stirred at 10 ° C. overnight. The crystals were separated, washed with 12 ml of methanol, and dried under reduced pressure at 40 ° C. overnight to obtain 8.0 g of Z-γ-glutamylvalylglycine crystals. Yield 45.5% γ / α = 99.9 / 0.1
1 H NMR (DMSO-d6, 400 MHz): δ 0.83 (3H, d, J = 6.8 Hz); 0.86 (3H, d, J = 6.8 Hz); 1.70-1.81 (1H , M); 1.93-1.98 (2H, m); 2.18-2.36 (2H, m); 3.69 (1H, dd, J = 17.5, 5.8 Hz); 3 .77 (1H, dd, J = 17.5, 5.9); 3.90-4.02 (1H, m); 4.18 (1H, dd, J = 8.9, 6.7 Hz); 5.03 (1H, s); 7.26-7.42 (5H, m); 7.58 (1H, d, J = 7.9 Hz); 7.84 (1H, d, J = 8.9 Hz) ); 8.24 (1H, dd, J = 5.9, 5.8 Hz)
13 C NMR (DMSO-d6, 400 MHz): δ 18.3, 19.5, 27.3, 30.8, 32.0, 40.9, 53.8, 57.8, 65.8, 128.1 , 128.2, 128.7, 137.4, 156.5, 171.5, 171.78, 171.84, 174.0
Mass (M + H): 438 Mass (M−H): 436
Crystal F: Crystal obtained by recrystallizing Crystal B (6.6, 13.5, 15.2, 20.9)
8 ml of methanol and Z-glutamic acid (35 mg) were added to B crystals (1.0 g) of crude Z-γ-glutamylvalylglycine, and the mixture was stirred at 40 ° C. for 1 hour. Thereafter, the mixture was cooled to 10 ° C. over 3 hours and stirred at 10 ° C. overnight. The crystals were separated and dried under reduced pressure at 40 ° C. overnight to obtain 0.35 g of Z-γ-glutamylvalylglycine crystals (F crystals).
次いで、下記のスキーム３に基づいて、γ－Ｇｌｕ-Ｖａｌ－Ｇｌｙを合成し、単離・精製した。
スキーム３
Next, γ-Glu-Val-Gly was synthesized, isolated and purified based on the following scheme 3.
Scheme 3
スキーム３
Scheme 3
(４-1)γ－グルタミルバリルグリシン（単離・精製されたＺ体を使用）
Ｚ－γ－グルタミルバリルグリシン（Ｅ晶）７．５ｇに水８０ｍｌ、メタノール８０ｍｌを加えて懸濁し、アルゴン置換した。５０％ｗｅｔ１０％パラジウム炭素０．２ｇを加えて水素置換し、４０℃で一晩撹拌した。反応液をアルゴン置換後、パラジウム炭素を分離により除いた。ろ過液を約５０ｍｌまで減圧濃縮した。ミリポア（０．４５μｍ）ろ過により不溶物を分離、ろ液を約１８．５ｇまで減圧濃縮した。濃縮液を５０℃に加温し撹拌した。メタノール５６ｍｌを３０分かけて滴下し、滴下後５０℃で２時間撹拌後、室温で一晩撹拌した。析出した結晶を分離し、メタノール８ｍｌで洗浄した。４０℃で一晩減圧乾燥して４．５６ｇのγ－グルタミルバリルグリシンの結晶を得た。収率３７．６％ ｆｒｏｍ Ｚ－Ｖａｌ γ／α＝９９．９／０．１
1Ｈ ＮＭＲ（Ｄ２Ｏ，４００ＭＨｚ）：δ０．８８（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；０．８９（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；１．９９－２．１０（３Ｈ，ｍ）；２．４０－２．５０（２Ｈ，ｍ）；３．７２（１Ｈ，ｔ，Ｊ＝６．４Ｈｚ）；３．８６（１Ｈ，ｄ，Ｊ＝１７．８Ｈｚ）；３．９１（１Ｈ，ｄ，Ｊ＝１７．８Ｈｚ）；４．０８（１Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；
13Ｃ ＮＭＲ（Ｄ２Ｏ，４００ＭＨｚ）：δ１７．７，１８．７，２６．５，３０．３，３１．５，４１．７，５４．２，６０．０，１７３．９，１７４．０，１７４．５，１７５．３
Ｍａｓｓ（Ｍ＋Ｈ）：３０４ Ｍａｓｓ（Ｍ－Ｈ）：３０２ (4-1) γ-Glutamylvalylglycine (using isolated and purified Z form)
To 7.5 g of Z-γ-glutamylvalylglycine (crystal E), 80 ml of water and 80 ml of methanol were added and suspended, and the atmosphere was replaced with argon. Hydrogen substitution was performed by adding 0.2 g of 50% wet 10% palladium carbon, and the mixture was stirred at 40 ° C. overnight. After replacing the reaction solution with argon, palladium carbon was removed by separation. The filtrate was concentrated under reduced pressure to about 50 ml. Insoluble matters were separated by Millipore (0.45 μm) filtration, and the filtrate was concentrated under reduced pressure to about 18.5 g. The concentrate was warmed to 50 ° C. and stirred. Methanol (56 ml) was added dropwise over 30 minutes, and after the addition, the mixture was stirred at 50 ° C. for 2 hours and then stirred at room temperature overnight. The precipitated crystals were separated and washed with 8 ml of methanol. It was dried under reduced pressure at 40 ° C. overnight to obtain 4.56 g of γ-glutamylvalylglycine crystals. Yield 37.6% from Z-Val γ / α = 99.9 / 0.1
1 H NMR (D 2 O, 400 MHz): δ 0.88 (3H, d, J = 6.8 Hz); 0.89 (3H, d, J = 6.8 Hz); 1.99-2.10 (3H, m 2.40-2.50 (2H, m); 3.72 (1H, t, J = 6.4Hz); 3.86 (1H, d, J = 17.8Hz); 3.91 (1H) , D, J = 17.8 Hz); 4.08 (1H, d, J = 6.8 Hz);
13 C NMR (D 2 O, 400 MHz): δ 17.7, 18.7, 26.5, 30.3, 31.5, 41.7, 54.2, 60.0, 173.9, 174.0, 174 .5, 175.3
Mass (M + H): 304 Mass (M−H): 302
Ｚ－γ－グルタミルバリルグリシン（Ｅ晶）７．５ｇに水８０ｍｌ、メタノール８０ｍｌを加えて懸濁し、アルゴン置換した。５０％ｗｅｔ１０％パラジウム炭素０．２ｇを加えて水素置換し、４０℃で一晩撹拌した。反応液をアルゴン置換後、パラジウム炭素を分離により除いた。ろ過液を約５０ｍｌまで減圧濃縮した。ミリポア（０．４５μｍ）ろ過により不溶物を分離、ろ液を約１８．５ｇまで減圧濃縮した。濃縮液を５０℃に加温し撹拌した。メタノール５６ｍｌを３０分かけて滴下し、滴下後５０℃で２時間撹拌後、室温で一晩撹拌した。析出した結晶を分離し、メタノール８ｍｌで洗浄した。４０℃で一晩減圧乾燥して４．５６ｇのγ－グルタミルバリルグリシンの結晶を得た。収率３７．６％ ｆｒｏｍ Ｚ－Ｖａｌ γ／α＝９９．９／０．１
1Ｈ ＮＭＲ（Ｄ２Ｏ，４００ＭＨｚ）：δ０．８８（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；０．８９（３Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；１．９９－２．１０（３Ｈ，ｍ）；２．４０－２．５０（２Ｈ，ｍ）；３．７２（１Ｈ，ｔ，Ｊ＝６．４Ｈｚ）；３．８６（１Ｈ，ｄ，Ｊ＝１７．８Ｈｚ）；３．９１（１Ｈ，ｄ，Ｊ＝１７．８Ｈｚ）；４．０８（１Ｈ，ｄ，Ｊ＝６．８Ｈｚ）；
13Ｃ ＮＭＲ（Ｄ２Ｏ，４００ＭＨｚ）：δ１７．７，１８．７，２６．５，３０．３，３１．５，４１．７，５４．２，６０．０，１７３．９，１７４．０，１７４．５，１７５．３
Ｍａｓｓ（Ｍ＋Ｈ）：３０４ Ｍａｓｓ（Ｍ－Ｈ）：３０２ (4-1) γ-Glutamylvalylglycine (using isolated and purified Z form)
To 7.5 g of Z-γ-glutamylvalylglycine (crystal E), 80 ml of water and 80 ml of methanol were added and suspended, and the atmosphere was replaced with argon. Hydrogen substitution was performed by adding 0.2 g of 50% wet 10% palladium carbon, and the mixture was stirred at 40 ° C. overnight. After replacing the reaction solution with argon, palladium carbon was removed by separation. The filtrate was concentrated under reduced pressure to about 50 ml. Insoluble matters were separated by Millipore (0.45 μm) filtration, and the filtrate was concentrated under reduced pressure to about 18.5 g. The concentrate was warmed to 50 ° C. and stirred. Methanol (56 ml) was added dropwise over 30 minutes, and after the addition, the mixture was stirred at 50 ° C. for 2 hours and then stirred at room temperature overnight. The precipitated crystals were separated and washed with 8 ml of methanol. It was dried under reduced pressure at 40 ° C. overnight to obtain 4.56 g of γ-glutamylvalylglycine crystals. Yield 37.6% from Z-Val γ / α = 99.9 / 0.1
1 H NMR (D 2 O, 400 MHz): δ 0.88 (3H, d, J = 6.8 Hz); 0.89 (3H, d, J = 6.8 Hz); 1.99-2.10 (3H, m 2.40-2.50 (2H, m); 3.72 (1H, t, J = 6.4Hz); 3.86 (1H, d, J = 17.8Hz); 3.91 (1H) , D, J = 17.8 Hz); 4.08 (1H, d, J = 6.8 Hz);
13 C NMR (D 2 O, 400 MHz): δ 17.7, 18.7, 26.5, 30.3, 31.5, 41.7, 54.2, 60.0, 173.9, 174.0, 174 .5, 175.3
Mass (M + H): 304 Mass (M−H): 302
(４-2)γ－グルタミルバリルグリシン（Ｚ体単離・未精製）
ｗｅｔ粗Ｚ－γ－グルタミルバリルグリシン４４．０ｇ（ｎｅｔ１２．０８ｇ、γ／α＝９６．１／３．９）に水５４ｍｌ、酢酸エチル６０．４ｍｌ、メタノール３０．２ｍｌを加えて懸濁し、アルゴン置換した。５０％ｗｅｔ１０％パラジウム炭素０．２４ｇを加えて水素置換し、４０℃で一晩撹拌した。反応液をアルゴン置換後パラジウム炭素を分離により除いた。ろ過液を分層し、水層を約６０ｍｌまで減圧濃縮した。ミリポア（０．４５μｍ）ろ過により不溶物を分離、ろ液を約３０ｇまで５０℃以下で減圧濃縮した。濃縮液を５０℃に加温し、種付けをして１時間室温で撹拌した。メタノール５４．４ｍｌを３０分かけて滴下し、滴下後５０℃で２時間、２０℃で一晩撹拌した。析出した結晶を分離し、メタノール１２．１ｍｌで洗浄した。４０℃で一晩減圧乾燥して６．８８ｇのγ－グルタミルバリルグリシンの結晶を得た。収率５７．０％ ｆｒｏｍ Ｚ－Ｖａｌ γ／α＝９９．２／０．８であった。 (4-2) γ-Glutamylvalylglycine (Z-isolated / unpurified)
Wet crude Z-γ-glutamylvalylglycine 44.0 g (net 12.08 g, γ / α = 96.1 / 3.9) was added with water 54 ml, ethyl acetate 60.4 ml, methanol 30.2 ml and suspended. Replaced. 50% wet 10% palladium carbon 0.24g was added, hydrogen substitution was carried out, and it stirred at 40 degreeC overnight. After replacing the reaction solution with argon, palladium carbon was removed by separation. The filtrate was separated into layers, and the aqueous layer was concentrated under reduced pressure to about 60 ml. Insoluble matters were separated by Millipore (0.45 μm) filtration, and the filtrate was concentrated under reduced pressure to 50 g or less to about 30 g. The concentrate was warmed to 50 ° C., seeded and stirred for 1 hour at room temperature. Methanol (54.4 ml) was added dropwise over 30 minutes. After the addition, the mixture was stirred at 50 ° C. for 2 hours and at 20 ° C. overnight. The precipitated crystals were separated and washed with 12.1 ml of methanol. Drying under reduced pressure at 40 ° C. overnight gave 6.88 g of γ-glutamylvalylglycine crystals. The yield was 57.0% from Z-Val γ / α = 99.2 / 0.8.
ｗｅｔ粗Ｚ－γ－グルタミルバリルグリシン４４．０ｇ（ｎｅｔ１２．０８ｇ、γ／α＝９６．１／３．９）に水５４ｍｌ、酢酸エチル６０．４ｍｌ、メタノール３０．２ｍｌを加えて懸濁し、アルゴン置換した。５０％ｗｅｔ１０％パラジウム炭素０．２４ｇを加えて水素置換し、４０℃で一晩撹拌した。反応液をアルゴン置換後パラジウム炭素を分離により除いた。ろ過液を分層し、水層を約６０ｍｌまで減圧濃縮した。ミリポア（０．４５μｍ）ろ過により不溶物を分離、ろ液を約３０ｇまで５０℃以下で減圧濃縮した。濃縮液を５０℃に加温し、種付けをして１時間室温で撹拌した。メタノール５４．４ｍｌを３０分かけて滴下し、滴下後５０℃で２時間、２０℃で一晩撹拌した。析出した結晶を分離し、メタノール１２．１ｍｌで洗浄した。４０℃で一晩減圧乾燥して６．８８ｇのγ－グルタミルバリルグリシンの結晶を得た。収率５７．０％ ｆｒｏｍ Ｚ－Ｖａｌ γ／α＝９９．２／０．８であった。 (4-2) γ-Glutamylvalylglycine (Z-isolated / unpurified)
Wet crude Z-γ-glutamylvalylglycine 44.0 g (net 12.08 g, γ / α = 96.1 / 3.9) was added with water 54 ml, ethyl acetate 60.4 ml, methanol 30.2 ml and suspended. Replaced. 50% wet 10% palladium carbon 0.24g was added, hydrogen substitution was carried out, and it stirred at 40 degreeC overnight. After replacing the reaction solution with argon, palladium carbon was removed by separation. The filtrate was separated into layers, and the aqueous layer was concentrated under reduced pressure to about 60 ml. Insoluble matters were separated by Millipore (0.45 μm) filtration, and the filtrate was concentrated under reduced pressure to 50 g or less to about 30 g. The concentrate was warmed to 50 ° C., seeded and stirred for 1 hour at room temperature. Methanol (54.4 ml) was added dropwise over 30 minutes. After the addition, the mixture was stirred at 50 ° C. for 2 hours and at 20 ° C. overnight. The precipitated crystals were separated and washed with 12.1 ml of methanol. Drying under reduced pressure at 40 ° C. overnight gave 6.88 g of γ-glutamylvalylglycine crystals. The yield was 57.0% from Z-Val γ / α = 99.2 / 0.8.
(４-3)γ－グルタミルバリルグリシン（Ｚ体非単離・抽出）
Ｚ－グルタミン酸２４．６４ｇ（１．１ｅｑ）に酢酸エチル１２０ｍｌを加えて５℃に冷却、ジシクロヘキシルカルボジイミド１８．１ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を酢酸エチル４０ｍｌに溶解して加えた。５℃で３０分、室温で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により除き、酢酸エチル４０ｍｌで洗浄、Ｚ－グルタミン酸無水物の酢酸エチル溶液を得た。
バリルグリシン水溶液（ＨＯＳｕを含む）に炭酸水素ナトリウム７．３６ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を加え撹拌し、上記で調製したＺ－グルタミン酸無水物の酢酸エチル溶液を１．５時間かけて滴下した。滴下後１時間室温で撹拌し、ＴＨＦ７０ｍｌを加えて４０℃に加温した。濃塩酸２２ｍｌを加えてｐＨ３．０に調整し抽出した。分層し酢酸エチル層を水１００ｍｌで洗浄した。酢酸エチル層に水１２０ｍｌ、５０％ｗｅｔ１０％パラジウム炭素０．７５ｇを加えて水素置換し、４０℃で撹拌脱保護した。反応終了後、パラジウム炭素をろ過し分層した。水層を１００ｍｌまで減圧濃縮しミリポアろ過（０．４５μｍ）により不溶物を除き、再度５０ｇまで減圧濃縮した。５０℃に加温しメタノール６０ｍｌを滴下、終了後５０℃で３時間、室温で一晩撹拌、晶析した。析出した結晶を分離（ｗｅｔ２５．９３ｇ）し、メタノール３０ｍｌで洗浄した。５０℃で一晩減圧乾燥して、γ－グルタミルバリルグリシンの結晶１７．７３ｇを得た。収率６７．４％ γ／α＝９９．６／０．４ (4-3) γ-Glutamylvalylglycine (Z body non-isolated / extracted)
120 ml of ethyl acetate was added to 24.64 g (1.1 eq) of Z-glutamic acid and cooled to 5 ° C., and 18.1 g (1.0 eq vs Z-Glu) of dicyclohexylcarbodiimide was dissolved in 40 ml of ethyl acetate and added. Stir at 5 ° C. for 30 minutes and at room temperature overnight. The precipitated dicyclohexylurea was removed by filtration and washed with 40 ml of ethyl acetate to obtain a solution of Z-glutamic anhydride in ethyl acetate.
7.36 g (1.0 eq vs. Z-Glu) of sodium bicarbonate was added to an aqueous valylglycine solution (including HOSu) and stirred, and the ethyl acetate solution of Z-glutamic anhydride prepared above was added dropwise over 1.5 hours. did. After the dropwise addition, the mixture was stirred at room temperature for 1 hour, 70 ml of THF was added, and the mixture was heated to 40 ° C. Concentrated hydrochloric acid (22 ml) was added to adjust to pH 3.0 and extraction was performed. The layers were separated, and the ethyl acetate layer was washed with 100 ml of water. To the ethyl acetate layer, 120 ml of water and 0.75 g of 50% wet 10% palladium carbon were added to replace with hydrogen, followed by deprotection with stirring at 40 ° C. After completion of the reaction, palladium carbon was filtered and separated. The aqueous layer was concentrated to 100 ml under reduced pressure, insoluble material was removed by Millipore filtration (0.45 μm), and the mixture was again concentrated to 50 g under reduced pressure. The mixture was heated to 50 ° C., and 60 ml of methanol was added dropwise. The precipitated crystals were separated (wet 25.93 g) and washed with 30 ml of methanol. Drying under reduced pressure at 50 ° C. overnight gave 17.73 g of γ-glutamylvalylglycine crystals. Yield 67.4% γ / α = 99.6 / 0.4
Ｚ－グルタミン酸２４．６４ｇ（１．１ｅｑ）に酢酸エチル１２０ｍｌを加えて５℃に冷却、ジシクロヘキシルカルボジイミド１８．１ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を酢酸エチル４０ｍｌに溶解して加えた。５℃で３０分、室温で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により除き、酢酸エチル４０ｍｌで洗浄、Ｚ－グルタミン酸無水物の酢酸エチル溶液を得た。
バリルグリシン水溶液（ＨＯＳｕを含む）に炭酸水素ナトリウム７．３６ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を加え撹拌し、上記で調製したＺ－グルタミン酸無水物の酢酸エチル溶液を１．５時間かけて滴下した。滴下後１時間室温で撹拌し、ＴＨＦ７０ｍｌを加えて４０℃に加温した。濃塩酸２２ｍｌを加えてｐＨ３．０に調整し抽出した。分層し酢酸エチル層を水１００ｍｌで洗浄した。酢酸エチル層に水１２０ｍｌ、５０％ｗｅｔ１０％パラジウム炭素０．７５ｇを加えて水素置換し、４０℃で撹拌脱保護した。反応終了後、パラジウム炭素をろ過し分層した。水層を１００ｍｌまで減圧濃縮しミリポアろ過（０．４５μｍ）により不溶物を除き、再度５０ｇまで減圧濃縮した。５０℃に加温しメタノール６０ｍｌを滴下、終了後５０℃で３時間、室温で一晩撹拌、晶析した。析出した結晶を分離（ｗｅｔ２５．９３ｇ）し、メタノール３０ｍｌで洗浄した。５０℃で一晩減圧乾燥して、γ－グルタミルバリルグリシンの結晶１７．７３ｇを得た。収率６７．４％ γ／α＝９９．６／０．４ (4-3) γ-Glutamylvalylglycine (Z body non-isolated / extracted)
120 ml of ethyl acetate was added to 24.64 g (1.1 eq) of Z-glutamic acid and cooled to 5 ° C., and 18.1 g (1.0 eq vs Z-Glu) of dicyclohexylcarbodiimide was dissolved in 40 ml of ethyl acetate and added. Stir at 5 ° C. for 30 minutes and at room temperature overnight. The precipitated dicyclohexylurea was removed by filtration and washed with 40 ml of ethyl acetate to obtain a solution of Z-glutamic anhydride in ethyl acetate.
7.36 g (1.0 eq vs. Z-Glu) of sodium bicarbonate was added to an aqueous valylglycine solution (including HOSu) and stirred, and the ethyl acetate solution of Z-glutamic anhydride prepared above was added dropwise over 1.5 hours. did. After the dropwise addition, the mixture was stirred at room temperature for 1 hour, 70 ml of THF was added, and the mixture was heated to 40 ° C. Concentrated hydrochloric acid (22 ml) was added to adjust to pH 3.0 and extraction was performed. The layers were separated, and the ethyl acetate layer was washed with 100 ml of water. To the ethyl acetate layer, 120 ml of water and 0.75 g of 50% wet 10% palladium carbon were added to replace with hydrogen, followed by deprotection with stirring at 40 ° C. After completion of the reaction, palladium carbon was filtered and separated. The aqueous layer was concentrated to 100 ml under reduced pressure, insoluble material was removed by Millipore filtration (0.45 μm), and the mixture was again concentrated to 50 g under reduced pressure. The mixture was heated to 50 ° C., and 60 ml of methanol was added dropwise. The precipitated crystals were separated (wet 25.93 g) and washed with 30 ml of methanol. Drying under reduced pressure at 50 ° C. overnight gave 17.73 g of γ-glutamylvalylglycine crystals. Yield 67.4% γ / α = 99.6 / 0.4
(４-4)γ－グルタミルバリルグリシンの精製
上記γ－グルタミルバリルグリシンの結晶に水７０ｍｌを加えて６０℃に加熱溶解した。不溶物をミリポアろ過（０．４５μｍ）し６２ｇまで減圧濃縮した。５０℃に加熱しメタノール８８ｍｌを加えて種晶を添加、５０℃で３時間、室温で一晩撹拌、晶析した。析出した結晶を分離（ｗｅｔ１８．２ｇ）し、メタノール３５ｍｌで洗浄した。５０℃で一晩減圧乾燥して、γ－グルタミルバリルグリシンの結晶１４．９ｇを得た。収率５８．０％ γ／α＝９９．９／０．１
γ－グルタミルバリルグリシンの結晶の粉末Ｘ線結晶回折パターンを図７に示す。 (4-4) Purification of γ-glutamylvalylglycine 70 ml of water was added to the above crystals of γ-glutamylvalylglycine and dissolved at 60 ° C. by heating. The insoluble material was filtered through Millipore (0.45 μm) and concentrated under reduced pressure to 62 g. After heating to 50 ° C. and adding 88 ml of methanol, seed crystals were added, and the mixture was stirred and crystallized at 50 ° C. for 3 hours and at room temperature overnight. The precipitated crystals were separated (wet 18.2 g) and washed with 35 ml of methanol. Drying under reduced pressure at 50 ° C. overnight gave 14.9 g of γ-glutamylvalylglycine crystals. Yield 58.0% γ / α = 99.9 / 0.1
FIG. 7 shows a powder X-ray crystal diffraction pattern of the crystals of γ-glutamylvalylglycine.
上記γ－グルタミルバリルグリシンの結晶に水７０ｍｌを加えて６０℃に加熱溶解した。不溶物をミリポアろ過（０．４５μｍ）し６２ｇまで減圧濃縮した。５０℃に加熱しメタノール８８ｍｌを加えて種晶を添加、５０℃で３時間、室温で一晩撹拌、晶析した。析出した結晶を分離（ｗｅｔ１８．２ｇ）し、メタノール３５ｍｌで洗浄した。５０℃で一晩減圧乾燥して、γ－グルタミルバリルグリシンの結晶１４．９ｇを得た。収率５８．０％ γ／α＝９９．９／０．１
γ－グルタミルバリルグリシンの結晶の粉末Ｘ線結晶回折パターンを図７に示す。 (4-4) Purification of γ-glutamylvalylglycine 70 ml of water was added to the above crystals of γ-glutamylvalylglycine and dissolved at 60 ° C. by heating. The insoluble material was filtered through Millipore (0.45 μm) and concentrated under reduced pressure to 62 g. After heating to 50 ° C. and adding 88 ml of methanol, seed crystals were added, and the mixture was stirred and crystallized at 50 ° C. for 3 hours and at room temperature overnight. The precipitated crystals were separated (wet 18.2 g) and washed with 35 ml of methanol. Drying under reduced pressure at 50 ° C. overnight gave 14.9 g of γ-glutamylvalylglycine crystals. Yield 58.0% γ / α = 99.9 / 0.1
FIG. 7 shows a powder X-ray crystal diffraction pattern of the crystals of γ-glutamylvalylglycine.
実施例３ ノルバリルグリシン及びγ－グルタミルノルバリルグリシンの合成
(1)Ｚ（ベンジルオキシカルボニル）－ノルバリンのヒドロキシコハク酸イミドエステルの合成
Ｚ－ノルバリン１０ｇ（３９．８ｍｍｏｌ）を酢酸エチル６０ｍｌに溶解し、Ｎ－ヒドロキシコハク酸イミド（ＨＯＳｕ）４．５９ｇ（１ｅｑ）を加えて５℃に冷却した。Ｎ，Ｎ－ジシクロヘキシルカルボジイミド８．２１ｇ（１ｅｑ）を酢酸エチル２５ｍｌに溶解して、１０℃以下でゆっくりと加えた。５℃で３０分、室温（２５℃）で一晩撹拌した。
析出したジシクロヘキシル尿素をろ過により分離し、酢酸エチル１５ｍｌで洗浄した。目的のＺ－ノルバリンコハク酸イミドエステルを酢酸エチル溶液として得た。
(2)ノルバリルグリシンの合成
グリシン３．１４ｇ（１．０５ｅｑ）に水２８ｍｌを加えて溶解し、４Ｍ水酸化ナトリウム水溶液でｐＨ９．０に調整した。この溶液にＺ－ノルバリンのヒドロキシコハク酸イミドエステルの酢酸エチル溶液を室温（２０－２５℃）で１時間かけて滴下、その後１時間撹拌した。その間４Ｍ水酸化ナトリウム水溶液でｐＨ９．０－１０．０未満に調整した。６Ｍ塩酸を加えて撹拌、ｐH８に調整した。反応液をアルゴン置換後、５０％ｗｅｔ１０％パラジウム炭素０．２ｇを加えて、水素置換して一晩室温で撹拌した。アルゴン置換後、パラジウム炭素を分離により除いた。反応液を分層して、ノルバリルグリシンの水溶液を得た。収率９０．０％ Example 3 Synthesis of norvalylglycine and γ-glutamylnorvalylglycine
(1) Synthesis of hydroxysuccinimide ester of Z (benzyloxycarbonyl) -norvaline Z-norvaline 10 g (39.8 mmol) was dissolved in 60 ml of ethyl acetate, and 4.59 g (1 eq) of N-hydroxysuccinimide (HOSu) was dissolved. ) And cooled to 5 ° C. 8.21 g (1 eq) of N, N-dicyclohexylcarbodiimide was dissolved in 25 ml of ethyl acetate and slowly added at 10 ° C. or lower. The mixture was stirred at 5 ° C. for 30 minutes and at room temperature (25 ° C.) overnight.
The precipitated dicyclohexylurea was separated by filtration and washed with 15 ml of ethyl acetate. The desired Z-norvaline succinimide ester was obtained as an ethyl acetate solution.
(2) Synthesis of norvalylglycine 28 ml of water was dissolved in 3.14 g (1.05 eq) of glycine, and the pH was adjusted to 9.0 with a 4M aqueous sodium hydroxide solution. To this solution, an ethyl acetate solution of Z-norvaline hydroxysuccinimide ester was added dropwise at room temperature (20-25 ° C.) over 1 hour, followed by stirring for 1 hour. Meanwhile, the pH was adjusted to less than 9.0-10.0 with 4M aqueous sodium hydroxide solution. 6M hydrochloric acid was added and stirred to adjust the pH to 8. The reaction solution was purged with argon, 0.2 g of 50% wet 10% palladium carbon was added, and the mixture was purged with hydrogen and stirred overnight at room temperature. After purging with argon, palladium on carbon was removed by separation. The reaction solution was separated to obtain an aqueous solution of norvalylglycine. Yield 90.0%
(1)Ｚ（ベンジルオキシカルボニル）－ノルバリンのヒドロキシコハク酸イミドエステルの合成
Ｚ－ノルバリン１０ｇ（３９．８ｍｍｏｌ）を酢酸エチル６０ｍｌに溶解し、Ｎ－ヒドロキシコハク酸イミド（ＨＯＳｕ）４．５９ｇ（１ｅｑ）を加えて５℃に冷却した。Ｎ，Ｎ－ジシクロヘキシルカルボジイミド８．２１ｇ（１ｅｑ）を酢酸エチル２５ｍｌに溶解して、１０℃以下でゆっくりと加えた。５℃で３０分、室温（２５℃）で一晩撹拌した。
析出したジシクロヘキシル尿素をろ過により分離し、酢酸エチル１５ｍｌで洗浄した。目的のＺ－ノルバリンコハク酸イミドエステルを酢酸エチル溶液として得た。
(2)ノルバリルグリシンの合成
グリシン３．１４ｇ（１．０５ｅｑ）に水２８ｍｌを加えて溶解し、４Ｍ水酸化ナトリウム水溶液でｐＨ９．０に調整した。この溶液にＺ－ノルバリンのヒドロキシコハク酸イミドエステルの酢酸エチル溶液を室温（２０－２５℃）で１時間かけて滴下、その後１時間撹拌した。その間４Ｍ水酸化ナトリウム水溶液でｐＨ９．０－１０．０未満に調整した。６Ｍ塩酸を加えて撹拌、ｐH８に調整した。反応液をアルゴン置換後、５０％ｗｅｔ１０％パラジウム炭素０．２ｇを加えて、水素置換して一晩室温で撹拌した。アルゴン置換後、パラジウム炭素を分離により除いた。反応液を分層して、ノルバリルグリシンの水溶液を得た。収率９０．０％ Example 3 Synthesis of norvalylglycine and γ-glutamylnorvalylglycine
(1) Synthesis of hydroxysuccinimide ester of Z (benzyloxycarbonyl) -norvaline Z-norvaline 10 g (39.8 mmol) was dissolved in 60 ml of ethyl acetate, and 4.59 g (1 eq) of N-hydroxysuccinimide (HOSu) was dissolved. ) And cooled to 5 ° C. 8.21 g (1 eq) of N, N-dicyclohexylcarbodiimide was dissolved in 25 ml of ethyl acetate and slowly added at 10 ° C. or lower. The mixture was stirred at 5 ° C. for 30 minutes and at room temperature (25 ° C.) overnight.
The precipitated dicyclohexylurea was separated by filtration and washed with 15 ml of ethyl acetate. The desired Z-norvaline succinimide ester was obtained as an ethyl acetate solution.
(2) Synthesis of norvalylglycine 28 ml of water was dissolved in 3.14 g (1.05 eq) of glycine, and the pH was adjusted to 9.0 with a 4M aqueous sodium hydroxide solution. To this solution, an ethyl acetate solution of Z-norvaline hydroxysuccinimide ester was added dropwise at room temperature (20-25 ° C.) over 1 hour, followed by stirring for 1 hour. Meanwhile, the pH was adjusted to less than 9.0-10.0 with 4M aqueous sodium hydroxide solution. 6M hydrochloric acid was added and stirred to adjust the pH to 8. The reaction solution was purged with argon, 0.2 g of 50% wet 10% palladium carbon was added, and the mixture was purged with hydrogen and stirred overnight at room temperature. After purging with argon, palladium on carbon was removed by separation. The reaction solution was separated to obtain an aqueous solution of norvalylglycine. Yield 90.0%
(３)γ－グルタミルノルバリルグリシン（Ｚ体非単離・抽出）
Ｚ－グルタミン酸１２．３１ｇ（１．１ｅｑ）に酢酸エチル５０ｍｌを加えて５℃に冷却、ジシクロヘキシルカルボジイミド９．０ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を酢酸エチル１５ｍｌに溶解して加えた。５℃で３０分、室温で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により除き、酢酸エチル２０ｍｌで洗浄、Ｚ－グルタミン酸無水物の酢酸エチル溶液を得た。
ノルバリルグリシン水溶液（ＨＯＳｕを含む）に炭酸水素ナトリウム３．６８ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を加え撹拌し、上記で調製したＺ－グルタミン酸無水物の酢酸エチル溶液を１．５時間かけて滴下した。滴下後１時間室温で撹拌し、４０℃に加温した。濃塩酸１１ｍｌを加えてｐＨ３．０に調整し抽出した。分層し酢酸エチル層を水５０ｍｌで洗浄した。酢酸エチル層に水５０ｍｌ、メタノール２５ｍｌ、５０％ｗｅｔ１０％パラジウム炭素０．３７ｇを加えて水素置換し、２０～２５℃で撹拌脱保護した。反応終了後、パラジウム炭素をミリポアろ過（０．４５μｍ）により除き、４０ｇまで減圧濃縮した。５０℃に加温しメタノール５０ｍｌを滴下、終了後５０℃で３時間、室温で一晩撹拌、晶析した。析出した結晶を分離し、メタノール１２ｍｌで洗浄した。５０℃で一晩減圧乾燥して、γ－グルタミルノルバリルグリシンの結晶８．０９ｇを得た。収率６７．０％ γ／α＝９９．８／０．２ (3) γ-Glutamylnorvalylglycine (Z-form non-isolated / extracted)
To 12.31 g (1.1 eq) of Z-glutamic acid, 50 ml of ethyl acetate was added and cooled to 5 ° C., and 9.0 g (1.0 eq vs. Z-Glu) of dicyclohexylcarbodiimide was dissolved in 15 ml of ethyl acetate and added. Stir at 5 ° C. for 30 minutes and at room temperature overnight. The precipitated dicyclohexylurea was removed by filtration and washed with 20 ml of ethyl acetate to obtain a solution of Z-glutamic anhydride in ethyl acetate.
3.68 g (1.0 eq vs. Z-Glu) of sodium hydrogen carbonate was added to an aqueous solution of norvalylglycine (including HOSu) and stirred, and the ethyl acetate solution of Z-glutamic anhydride prepared above was added over 1.5 hours. It was dripped. After dropping, the mixture was stirred at room temperature for 1 hour and heated to 40 ° C. Concentrated hydrochloric acid (11 ml) was added to adjust to pH 3.0 and extraction was performed. The layers were separated, and the ethyl acetate layer was washed with 50 ml of water. The ethyl acetate layer was purged with hydrogen by adding 50 ml of water, 25 ml of methanol, and 0.37 g of 50% wet 10% palladium on carbon, and deprotected by stirring at 20 to 25 ° C. After completion of the reaction, palladium carbon was removed by Millipore filtration (0.45 μm), and the mixture was concentrated under reduced pressure to 40 g. The mixture was heated to 50 ° C. and 50 ml of methanol was added dropwise. The precipitated crystals were separated and washed with 12 ml of methanol. After drying under reduced pressure at 50 ° C. overnight, 8.09 g of γ-glutamylnorvalylglycine crystals were obtained. Yield 67.0% γ / α = 99.8 / 0.2
Ｚ－グルタミン酸１２．３１ｇ（１．１ｅｑ）に酢酸エチル５０ｍｌを加えて５℃に冷却、ジシクロヘキシルカルボジイミド９．０ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を酢酸エチル１５ｍｌに溶解して加えた。５℃で３０分、室温で一晩撹拌した。析出したジシクロヘキシル尿素をろ過により除き、酢酸エチル２０ｍｌで洗浄、Ｚ－グルタミン酸無水物の酢酸エチル溶液を得た。
ノルバリルグリシン水溶液（ＨＯＳｕを含む）に炭酸水素ナトリウム３．６８ｇ（１．０ｅｑ ｖｓ Ｚ－Ｇｌｕ）を加え撹拌し、上記で調製したＺ－グルタミン酸無水物の酢酸エチル溶液を１．５時間かけて滴下した。滴下後１時間室温で撹拌し、４０℃に加温した。濃塩酸１１ｍｌを加えてｐＨ３．０に調整し抽出した。分層し酢酸エチル層を水５０ｍｌで洗浄した。酢酸エチル層に水５０ｍｌ、メタノール２５ｍｌ、５０％ｗｅｔ１０％パラジウム炭素０．３７ｇを加えて水素置換し、２０～２５℃で撹拌脱保護した。反応終了後、パラジウム炭素をミリポアろ過（０．４５μｍ）により除き、４０ｇまで減圧濃縮した。５０℃に加温しメタノール５０ｍｌを滴下、終了後５０℃で３時間、室温で一晩撹拌、晶析した。析出した結晶を分離し、メタノール１２ｍｌで洗浄した。５０℃で一晩減圧乾燥して、γ－グルタミルノルバリルグリシンの結晶８．０９ｇを得た。収率６７．０％ γ／α＝９９．８／０．２ (3) γ-Glutamylnorvalylglycine (Z-form non-isolated / extracted)
To 12.31 g (1.1 eq) of Z-glutamic acid, 50 ml of ethyl acetate was added and cooled to 5 ° C., and 9.0 g (1.0 eq vs. Z-Glu) of dicyclohexylcarbodiimide was dissolved in 15 ml of ethyl acetate and added. Stir at 5 ° C. for 30 minutes and at room temperature overnight. The precipitated dicyclohexylurea was removed by filtration and washed with 20 ml of ethyl acetate to obtain a solution of Z-glutamic anhydride in ethyl acetate.
3.68 g (1.0 eq vs. Z-Glu) of sodium hydrogen carbonate was added to an aqueous solution of norvalylglycine (including HOSu) and stirred, and the ethyl acetate solution of Z-glutamic anhydride prepared above was added over 1.5 hours. It was dripped. After dropping, the mixture was stirred at room temperature for 1 hour and heated to 40 ° C. Concentrated hydrochloric acid (11 ml) was added to adjust to pH 3.0 and extraction was performed. The layers were separated, and the ethyl acetate layer was washed with 50 ml of water. The ethyl acetate layer was purged with hydrogen by adding 50 ml of water, 25 ml of methanol, and 0.37 g of 50% wet 10% palladium on carbon, and deprotected by stirring at 20 to 25 ° C. After completion of the reaction, palladium carbon was removed by Millipore filtration (0.45 μm), and the mixture was concentrated under reduced pressure to 40 g. The mixture was heated to 50 ° C. and 50 ml of methanol was added dropwise. The precipitated crystals were separated and washed with 12 ml of methanol. After drying under reduced pressure at 50 ° C. overnight, 8.09 g of γ-glutamylnorvalylglycine crystals were obtained. Yield 67.0% γ / α = 99.8 / 0.2
1Ｈ ＮＭＲ（Ｄ２Ｏ，４００ＭＨｚ）：δ０．８１（３Ｈ，ｔ，Ｊ＝７．４Ｈｚ）； １．２２－１．３８（２Ｈ，ｍ）；１．５５－１．７３（２Ｈ，ｍ）；２．０２－２．１０（２Ｈ，ｍ）；２．３５－２．４８（２Ｈ，ｍ）；３．７２（１Ｈ，ｔ，Ｊ＝６．２ Ｈｚ）；３．８４（１Ｈ，ｄ，Ｊ＝１７．６Ｈｚ）；３．８９（１Ｈ，ｄ，Ｊ＝１８．０Ｈｚ）； ４．２０（１Ｈ，ｄｄ，Ｊ＝５．２，５．６Ｈｚ）；
13Ｃ ＮＭＲ（Ｄ２Ｏ，４００ＭＨｚ）：δ１２．７，１８．４，２６．０，３１．１，３３．０，４１．３，５３．７，５３．８，１７３．５，１７４．８，１７５．０；
Ｍａｓｓ（Ｍ＋Ｈ）：３０４ Ｍａｓｓ（Ｍ－Ｈ）：３０２ 1 H NMR (D 2 O, 400 MHz): δ 0.81 (3H, t, J = 7.4 Hz); 1.22-1.38 (2H, m); 1.55-1.73 (2H, m); 2.02-2.10 (2H, m); 2.35-2.48 (2H, m); 3.72 (1H, t, J = 6.2 Hz); 3.84 (1H, d, J = 17.6 Hz); 3.89 (1 H, d, J = 18.0 Hz); 4.20 (1 H, dd, J = 5.2, 5.6 Hz);
13 C NMR (D 2 O, 400 MHz): δ 12.7, 18.4, 26.0, 31.1, 33.0, 41.3, 53.7, 53.8, 173.5, 174.8, 175 0.0;
Mass (M + H): 304 Mass (M−H): 302
13Ｃ ＮＭＲ（Ｄ２Ｏ，４００ＭＨｚ）：δ１２．７，１８．４，２６．０，３１．１，３３．０，４１．３，５３．７，５３．８，１７３．５，１７４．８，１７５．０；
Ｍａｓｓ（Ｍ＋Ｈ）：３０４ Ｍａｓｓ（Ｍ－Ｈ）：３０２ 1 H NMR (D 2 O, 400 MHz): δ 0.81 (3H, t, J = 7.4 Hz); 1.22-1.38 (2H, m); 1.55-1.73 (2H, m); 2.02-2.10 (2H, m); 2.35-2.48 (2H, m); 3.72 (1H, t, J = 6.2 Hz); 3.84 (1H, d, J = 17.6 Hz); 3.89 (1 H, d, J = 18.0 Hz); 4.20 (1 H, dd, J = 5.2, 5.6 Hz);
13 C NMR (D 2 O, 400 MHz): δ 12.7, 18.4, 26.0, 31.1, 33.0, 41.3, 53.7, 53.8, 173.5, 174.8, 175 0.0;
Mass (M + H): 304 Mass (M−H): 302
Claims (10)
- Ｎ－保護中性アミノ酸のＮ－ヒドロキシコハク酸イミドエステルと、中性アミノ酸とを、ｐＨ８.５～１１.５の条件下で反応させることを特徴とする末端アミノ基が保護されたジペプチドの製造方法。 Production of a dipeptide having a terminal amino group protected, comprising reacting an N-hydroxysuccinimide ester of an N-protected neutral amino acid with a neutral amino acid under conditions of pH 8.5 to 11.5 Method.
- Ｎ－保護中性アミノ酸が、Ｎ－保護バリンもしくはＮ－保護ノルバリンであり、中性アミノ酸がグリシンである、請求項１記載のジペプチドの製造方法。 The method for producing a dipeptide according to claim 1, wherein the N-protected neutral amino acid is N-protected valine or N-protected norvaline, and the neutral amino acid is glycine.
- Ｎ－保護中性アミノ酸が、Ｎ－保護バリンであり、中性アミノ酸がグリシンである、請求項１記載のジペプチドの製造方法。 The method for producing a dipeptide according to claim 1, wherein the N-protected neutral amino acid is N-protected valine and the neutral amino acid is glycine.
- 請求項１～３のいずれか１項記載の方法で得たジペプチドのＮ脱保護を行うジペプチドの製造方法。 A method for producing a dipeptide, comprising N-deprotecting the dipeptide obtained by the method according to any one of claims 1 to 3.
- 請求項４に記載の方法でＮ脱保護して得られたジペプチドを、Ｎ－ヒドロキシコハク酸イミドの存在下に、Ｎ－保護グルタミン酸無水物と反応させることを特徴とするＮ－保護グルタミン酸γ－トリペプチドの製造方法。 A dipeptide obtained by N-deprotection by the method according to claim 4 is reacted with an N-protected glutamic anhydride in the presence of N-hydroxysuccinimide. A method for producing a tripeptide.
- 反応を炭酸アルカリ金属塩の存在下で行う請求項５記載の方法。 The method according to claim 5, wherein the reaction is carried out in the presence of an alkali metal carbonate.
- 請求項５又は６記載の方法で得たトリペプチドのＮ脱保護を行うグルタミン酸γ－トリペプチドの製造方法。 A method for producing a glutamic acid γ-tripeptide, which comprises N-deprotecting the tripeptide obtained by the method according to claim 5 or 6.
- グルタミン酸γ－トリペプチドが、γ－グルタミルバリルグリシンである、請求項７記載の製造方法。 The production method according to claim 7, wherein the glutamic acid γ-tripeptide is γ-glutamylvalylglycine.
- グルタミン酸γ－トリペプチドが、γ－グルタミルノルバリルグリシンである、請求項７記載の製造方法。 The production method according to claim 7, wherein the glutamic acid γ-tripeptide is γ-glutamylnorvalylglycine.
- Ｚ－γ－グルタミルバリルグリシン又はその塩。 Z-γ-glutamyl valylglycine or a salt thereof.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2883960A4 (en) * | 2012-08-10 | 2016-05-18 | Ajinomoto Kk | Method for producing -glutamyl-valyl-glycine crystal |
| CN113969297A (en) * | 2021-10-30 | 2022-01-25 | 福州三合元生物科技有限公司 | Oligomeric gamma-aminobutyric acid and preparation method thereof |
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| JPH08119916A (en) * | 1994-10-20 | 1996-05-14 | Ajinomoto Co Inc | Selective production of n-protected glutamic acid gamma-derivative |
| JP2009514791A (en) * | 2005-11-09 | 2009-04-09 | 味の素株式会社 | Kokumi imparting agent |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08119916A (en) * | 1994-10-20 | 1996-05-14 | Ajinomoto Co Inc | Selective production of n-protected glutamic acid gamma-derivative |
| JP2009514791A (en) * | 2005-11-09 | 2009-04-09 | 味の素株式会社 | Kokumi imparting agent |
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| Title |
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| ANDERSON GW. ET AL.: "The Use of Esters of N-Hydroxysuccinimide in Peptide Synthesis", J. AM. CHEM. SOC., vol. 86, no. 9, 1964, pages 1839 - 1842 * |
| GUARNACCIA R. ET AL.: "Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. III. Co-oligopeptides of L-phenylalanine, L-tryptophan, and Glycine: synthesis and ultraviolet absorption properties", BIOPOLYMERS, vol. 15, no. 6, 1976, pages 1103 - 1117 * |
| TAKEDA K. ET AL.: "An Improved Method for the Synthesis of Active Esters of N-Protected Amino Acids and Subsequent Synthesis of Dipeptides", SYNTHESIS, vol. 1991, no. 9, 1991, pages 689 - 691 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2883960A4 (en) * | 2012-08-10 | 2016-05-18 | Ajinomoto Kk | Method for producing -glutamyl-valyl-glycine crystal |
| US9512177B2 (en) | 2012-08-10 | 2016-12-06 | Ajinomoto Co., Inc. | Method for producing γ-glutamyl-valyl-glycine crystal |
| CN113969297A (en) * | 2021-10-30 | 2022-01-25 | 福州三合元生物科技有限公司 | Oligomeric gamma-aminobutyric acid and preparation method thereof |
| CN113969297B (en) * | 2021-10-30 | 2024-03-26 | 福州三合元生物科技有限公司 | Oligomeric gamma-aminobutyric acid and preparation method thereof |
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