JP4290844B2 - Method for producing theanine - Google Patents

Description

【００２１】
この結果、Ｚ−グルタミン酸無水物を得るためには、懸濁溶液の温度を非常にゆっくり（約３０分間かけて）５５℃まで上昇させ、この温度で２０分間程度攪拌し続けるのがよいことが判明した。
【００２２】
【発明の効果】
本発明の製造は、ペプチド結合の出発物質として無水物を用いることで、カルボキシル保護基の導入・脱離工程を省くことができ、より一層少ない工程で高純度のテアニンを得ることができる。しかも、エチルアミンの導入は、無水物のアミノリシスによる開環反応で行うから、縮合剤を必要とせず、人体に対して安全であるばかりか、製造コストを非常に安価にすることができる。
また、アミノ基の保護基がＺ基１個だけで済み、しかもＺ基の脱離反応も接触還元（水素添加）で簡単に行うことができるメリットがある。
更に、人体に有害な有機溶媒や金属をほとんど使用してないから、食品添加物や医薬品原料として好適に利用することができる。[0001]
[Industrial application fields]
The present invention relates to a method for producing L-glutamic acid-γ-ethylamide (L-theanine) by peptide synthesis, and particularly to a method for producing theanine that can be safely used as a food additive.
[0002]
[Prior art and problems to be solved by the invention]
Theanine, which is known as a delicious component of tea, is known for its usefulness such as caffeine excitation suppression, blood pressure lowering, relaxation, and brain function improvement. However, the amount of theanine contained in the tea leaves is only 0.5 to 2% by weight, which is very small compared to 10 to 15% by weight of tea polyphenol. Therefore, industrially using theanine extracted from tea leaves is not practical in terms of quantity and cost, and there has been a demand for a cheap and safe method for producing theanine as a food additive or a pharmaceutical raw material.
[0003]
As a conventional chemical synthesis method for theanine, as a laboratory production method, glutamic acid is shaken with absolute ethanol in the presence of hydrochloric acid, excess hydrochloric acid and ethanol are removed at low temperature, and the precipitated hydrochloride is dissolved in methanol. The solution is added with the calculated amount of ammonia and cooled to obtain L-glutamic acid-γ-ethyl ester, which is reacted with chlorocarboxylic acid benzyl ester to give N-carboxybenzyl-L-glutamic acid-γ-ethyl ester, A method is disclosed in which this is reacted with ethylamine to form N-protected carboxybenzyl-L-glutamic acid-γ-ethylamide, and then added with syrup under a platinum catalyst to obtain L-glutamic acid-γ-ethylamide (L-theanine). (Saito Yajiro et al .: Nikko Kagaku 23,269 (1950)).
As an industrial production method, for example, in JP-A-5-70419 and the like, the γ-carboxyl group of L-glutamic acid is protected by benzylation, and the amino group is protected by tritylation, which is used as a starting material for peptide synthesis. A method for producing theanine is disclosed.
[0004]
However, in many of the conventional production methods, even if high-purity theanine can be obtained, the number of steps is complicated and the yield is low, and the theanine obtained is actually expensive. For example, when glutamic acid is used as a starting material for peptide synthesis, it was common to protect the γ-carboxyl group with a benzyl ester. However, it is difficult to remove the benzyl group, so the number of steps increases. However, there have been problems such as a long process time or severe reaction conditions.
On the other hand, in consideration of the use of the obtained theanine as a food additive or a pharmaceutical raw material, a production method is desired that not only increases the purity, but also uses a condensing agent or metal harmful to the human body as much as possible.
[0005]
In order to provide a method for producing theanine that can obtain high-purity theanine in a high yield with fewer steps and that uses as little an organic solvent or metal that is harmful to the human body as much as possible. No. 26263, a glutamic acid derivative (Boc-) in which the amino group at the α-position of glutamic acid is protected with a tertiary butyloxycarbonyl group (Boc group) and the carboxyl group is protected with a tertiary butyl ester group (OtBu group). Glu-OtBu) is used as a starting material for peptide synthesis, dissolved in a solvent, a condensing agent and a water-soluble active ester reagent are added to this solution to precipitate a urea derivative, and further, ethylamine hydrochloride and a pH adjuster A mixed liquid was added to add tert-butyloxycarbonyl-γ-ethylamidoglutamic acid tert-butyl ester (B oc-Glu (NHC2H5) -OtBu) was obtained, and this was acid-treated to disclose a theanine production method in which the α-position amino group and carboxyl group OtBu group were eliminated.
[0006]
According to the present invention, by using a method different from the above disclosed method, specifically, a method using glutamic anhydride as a starting material, inexpensive and high-purity theanine can be obtained in a high yield with fewer steps, and the human body. It is intended to provide a method for producing theanine that uses as little as possible a condensing agent or metal harmful to the environment.
[0007]
[Means for Solving the Problems]
For this purpose, the present invention provides an N-protected glutamic anhydride in which the amino group of glutamic acid is protected by selectively ethylamidating the γ position of the anhydride using dimethyl sulfoxide (hereinafter abbreviated as “DMSO”). N-protected theanine, and the N-protecting group is eliminated to give theanine.
[0008]
This production method is characterized in that glutamic anhydride is used as a starting material for peptide synthesis and that the γ position of the anhydride is selectively ethylamidated using DMSO. If glutamic anhydride is used as a starting material, only one protecting group is required, so that the step of removing the protecting group can be reduced compared to a starting material in which both the amino group and the carboxyl group are protected. Furthermore, since this glutamic anhydride can perform ring opening and aminolysis without using a condensing agent by using DMSO, not only can the production cost be reduced by that amount, but also safety to the human body. Can also be increased. Furthermore, by using DMSO, the γ position can be selectively amidated, that is, the γ position can be more advantageously ethylamidated than the α position, and the purity and yield of theanine can be dramatically increased.
[0009]
In the present invention, “glutamic acid anhydride in which the amino group of glutamic acid is protected” used as a starting material for peptide synthesis is a Z-glutamic acid anhydride in which the amino group at the α-position of glutamic acid is protected with a benzyloxycarbonyl group (Z group). It is preferable to use (Z-Glu-O). Thus, when the Z group is an amino protecting group, not only the protecting group can be easily introduced and removed, but also racemization can be suppressed, and the crystallinity of Z-glutamic acid is good. Moreover, the protecting group can be removed by a mild, harmless and clean method such as catalytic reduction.
However, the amino protecting group used in the present invention is not limited to the Z group. In addition, those used as amino protecting groups in normal peptide synthesis, such as tertiary butyloxycarbonyl (Boc) group, other urethane type protecting groups, acyl type protecting groups such as formyl group and tosyl group, trityl group Such alkyl-type protecting groups can also be used. The Boc group is advantageous in that there is no fear of esterification.
[0010]
In the present invention, specific methods for selectively ethylamidating the γ-position of glutamic anhydride include, for example, Z-glutamic anhydride (Z-Glu-O), ethylamine hydrochloride and triethylamine or sodium bicarbonate. A method of adding it to DMSO together with a mixed solution of can preferably be exemplified. The anhydride opens in DMSO and undergoes aminolysis with ethylamine. At this time, ethylamine is introduced more preferentially at the γ position than at the α position. In fact, experiments have confirmed that γ-form can be advantageously obtained at a ratio of γ-form: α-form = 3: 1 or more. However, the present inventor has also found that the α-form and the γ-form can be separated by using silica gel chromatography. Further, by using ethylamine hydrochloride instead of anhydrous ethylamine, there is also an advantage that the reaction can proceed under very mild conditions as in normal peptide synthesis.
Either triethylamine or sodium hydrogen carbonate can be used, but sodium hydrogen carbonate is superior from the viewpoint of the generation of by-products and the safety to the human body.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Here, a method for producing theanine, which is performed through the following four steps using Z-glutamic anhydride as a starting material for peptide synthesis, will be described.
1) Synthesis of Z-glutamic acid 2) Synthesis of Z-glutamic anhydride 3) Synthesis of Z-γ-ethylamidoglutamic acid 4) Removal and purification of protecting group
1) Synthesis of Z-glutamic acid: Z- formation of glutamic acid Z- formation of glutamic acid can employ any currently known method. For example, in addition to the Schotten-Baumann method using benzyloxycarbonyl chloride (Z-Cl), a method using p-nitrophenyl ester (Z-ONp), N-hydroxysuccinimide ester (Z-ONSu) and other active esters Can be mentioned. Commercially available Z-glutamic acid can also be used.
[0013]
As a specific synthesis example, a solution A in which glutamic acid (H-Glu-OH), trimethylamine (NEt3) and water (H2O) are mixed with benzyloxycarbonylsuccinimide (Z-OSu), acetonitrile and triethylamine (NEt3). Solution B mixed with was added over about 20 minutes with stirring. After the reaction was completed, hydrochloric acid was added to the reaction solution to adjust to pH 2, and this was separated and extracted with water / ethyl acetate. Add hydrochloric acid and separate and extract again.The resulting ethyl acetate layer is dried by adding sodium sulfate, filtered, concentrated under reduced pressure to give an oil, and ether is added to crystallize under ice-cooling. The crystal can be obtained by filtration.
[0014]
2) Synthesis of Z-glutamic anhydride Z-glutamic anhydride (Z-Glu-OH) was added to acetic anhydride, heated, and the resulting solution was concentrated to concentrate Z-glutamic anhydride ( Z-Glu-O) can be obtained.
Specifically, Z-glutamic acid (10.4 g) is added and stirred in acetic anhydride (for example, 75 ml) (at this time, the solution is suspended). The temperature of this suspension solution is very slowly (10 minutes to 40 minutes, preferably 25 minutes to 35 minutes, most preferably about 30 minutes), about 50 ° C. to 60 ° C., preferably about 55 ° C. The temperature is raised and stirring is continued at this temperature for about 10 to 30 minutes, preferably 15 to 25 minutes, and most preferably about 20 minutes (solution becomes transparent). After confirming the completion of the reaction by thin layer chromatography (hereinafter abbreviated as “TLC”), the solvent is concentrated and the resulting oily substance is crystallized to obtain Z-glutamic anhydride.
[0015]
3) Synthesis of Z-γ- ethylamidoglutamic acid Z-glutamic anhydride is dissolved in DMSO, while a mixture of ethylamine hydrochloride and triethylamine or sodium bicarbonate is dissolved in DMSO, both solutions are added and stirred at room temperature for 20 minutes. Then, the target Z-γ-ethylamidoglutamic acid can be obtained by performing extraction and purification thereafter. The DMSO used at this time needs to be sufficiently dried with a molecular sieve or the like.
As an extraction / purification method, for example, sodium hydrogen carbonate (saturated 10 ml) and 10 ml of water are added, the aqueous layer is extracted several times (for example, twice) with dichlorometal, and acidified so that the aqueous layer becomes PH1. . Thereafter, extraction was performed several times (for example, 3 times) with ethyl acetate, the ethyl acetate layer was dried over magnesium sulfate, the solvent was removed, and a residual oil was obtained. If this oil is dissolved in a small amount of ethyl acetate and separated and purified by a silica gel column, white crystalline Z-γ-ethylamidoglutamic acid can be obtained. In this case, with ethyl acetate and acetic acid (100: 1), the faster elution is α form (α position is ethylamidation), and the later elution is γ form (γ position is ethylamidation). According to the method, a γ-form in which the γ-position is ethylamidated at a ratio of γ-form: α-form = 3: 1 or more can be obtained more advantageously.
As an elution method, the eluent is eluted only with ethyl acetate, and then eluted with a 5% methanol ethyl acetate solution and further with a methanol solution. After confirming each fraction solution by TLC, the eluent is concentrated. Can also be mentioned.
[0016]
4) Removal of the protecting group and removal of the purified Z group are preferred in that catalytic reduction (hydrogenation) is a mild, harmless and clean method. Specifically, Z-γ-ethylamidoglutamic acid may be catalytically reduced with palladium or the like in a dioxane solution containing hydrochloric acid.
The removal of the Z group can be carried out by acid treatment such as liquid HF, sulfonic acids or HBr / AcOH in addition to the above catalytic reduction.
[0017]
After elimination of the Z group, palladium and the like are filtered, the solvent is concentrated, crystallized with ether, and then purified with an ion exchange resin (cation exchange resin: Amberlite IR-124, Dowex50w-x12). Good.
[0018]
(test)
In this test, the relationship between the reaction time and the yield in the synthesis of Z-Glu anhydride (anhydride) was examined.
[0019]
A suspension was prepared by adding 1.969 mg of Z-glutamic acid (Z-Glu-OH) to 15 ml of acetic anhydride with vigorous stirring, and the temperature of the suspension was changed from room temperature to 55 ° C. for 10 hours with a water bath. Minutes, 30 minutes, 60 minutes). And after all the solid melt | dissolved, it stirred for 20 to 60 minutes, keeping the temperature.
Thereafter, the solvent was concentrated under reduced pressure, and the oil was dried together with sodium hydroxide in an evaporating dish in a vacuum desiccator.
Crystallization was carried out by adding isopropyl ether / petroleum ether (1/1, v / v) little by little while cooling, followed by stirring and then returning to room temperature in 1 hour to cool the resulting crystals. Washed with petroleum ether.
Table 1 below shows the relationship between the temperature raising time, the reaction time, and the yield. In addition,-in the table | surface has shown the result which was not able to be obtained as a crystal | crystallization.
[0020]
[Table 1]

[0021]
As a result, in order to obtain Z-glutamic anhydride, it is preferable to raise the temperature of the suspended solution very slowly (over about 30 minutes) to 55 ° C. and continue stirring at this temperature for about 20 minutes. found.
[0022]
【The invention's effect】
In the production of the present invention, by using an anhydride as a starting material for peptide bonds, it is possible to omit the introduction / desorption step of the carboxyl protecting group, and to obtain highly purified theanine with fewer steps. Moreover, since the introduction of ethylamine is carried out by a ring-opening reaction by aminolysis of an anhydride, it does not require a condensing agent, and is safe for the human body, and can be very inexpensive to produce.
Further, only one Z protecting group is required for the amino group, and the elimination reaction of the Z group can be easily performed by catalytic reduction (hydrogenation).
Furthermore, since almost no organic solvent or metal harmful to the human body is used, it can be suitably used as a food additive or a pharmaceutical raw material.

Claims (4)

N-protected glutamic anhydride in which the amino group of glutamic acid is protected is converted to N-protected theanine by selectively ethylamidating the γ-position of the anhydride using dimethyl sulfoxide, and then removing the N-protecting group. Manufacturing method. Z-glutamic acid anhydride in which the amino group of glutamic acid is protected with benzyloxycarbonyl group (Z group) is selectively ethylamidated with dimethyl sulfoxide to γ-position of the anhydride to form Z-γ-ethylamidoglutamic acid. Then, a method for producing theanine, which comprises the step of removing the benzyloxycarbonyl group (Z group) to form theanine. A method for producing theanine having the following steps 1) to 2).
1) Add Z-glutamic anhydride in which the amino group of glutamic acid is protected with a benzyloxycarbonyl group (Z group) together with a mixture of ethylamine hydrochloride and triethylamine or sodium hydrogen carbonate into dimethyl sulfoxide to add Z-γ-ethylamide. A step of synthesizing glutamic acid,
2) A step of eliminating the Z group of the obtained Z-γ-ethylamidoglutamic acid. The above Z-glutamic acid anhydride is obtained by adding Z-glutamic acid in acetic anhydride and heating this solution to 50 ° C. to 60 ° C. over 10 minutes to 40 minutes , while maintaining the temperature for 10 minutes to 30 minutes. The method for producing theanine according to claim 2 or 3, wherein the reaction solution is obtained by stirring for a minute and concentrating the reaction solution.