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L-Theanine (5 g) (Sun<strong>[3081-61-6]theanine</strong>) was slurried in dry MeOH (50 ml). Then 5.145 g of Cl2SO were added drop-wise over 5 minutes (part way through the addition all of the solids dissolve to give a clear colourless solution) and the resulting solution was held for 12 hours at 20 C. to allow the reaction shown in scheme (II) to occur:
Examples; EXAMPLE 1; This example demonstrates the manufacture of a compound of the invention (N-ethyl-3- [5- (2-ethylcarbamoyl-ethyl) -3, 6-dioxo- piperazin-2-yl] -propionamide; see formula (4)).; L-Theanine (5 g) (Sun<strong>[3081-61-6]theanine</strong>) was slurried in dry MeOH (50 ml). Then 5.145 g of CI2SO were added drop-wise over 5 minutes (part way through the addition all of the solids dissolve to give a clear colourless solution) and the resulting solution was held for 12 hours at 200C to allow the reaction shown in scheme (II) to occur:; MeOH, SO2 and HCl were then removed under vacuum to leave a thick clear oil to which dry MeOH (50 ml) was added. The pH was then raised by adding MeONa while testing pH by intermittently spotting onto wet indicator paper. After adding 3.17 g of MeONa, the pH had increased to >11, which resulted in the reaction shown in scheme (III) :; The resulting solution was held for 12 hours at 200C to allow the reaction shown in scheme (IV) to occur:The resulting suspension was then filtered to remove NaCl and the filtrate subjected to vacuum to remove MeOH. The product was a white solid.The product was analysed with LC-MS (Liquid-Chromatography coupled Mass Spectroscopy) under the following conditions:Column: Phenomonex Gemini 5mu C18 IIOA 150mm x 2.00mmSolvents: A: 0.1% Formic Acid / WaterB: 0.1% Formic Acid / AcetonitrileFlow rate: 0.2 ml / minuteColumn Temperature: 300C Stop Time: 25 minutes; Mass Spectrometer Detection:Selected Ion Recording at m/z 175.6 for Theanine. Selected Ion Recording at m/z 313.5 for Theanine dimer .The results showed that the product was free from <strong>[3081-61-6]theanine</strong> (retention time of <strong>[3081-61-6]theanine</strong> = 3.7 min) and had a characteristic new peak with a retention time of around 7.4 min.The high resolution proton NMR spectrum of the product in MeOD-d4 is shown in Figure 1.
With boric acid;glutaminase; In water; at 30℃; for 22h;pH 11;
0.3 M glutamine and 1.5 M methylamine hydrochloride were reacted in the presence of 0.3 U glutaminase (commercially available) at 30 C. for 22 hours in a buffer solution of 0.05 M boric acid (pH 11), whereby 225 nm theanine was obtained. Reaction liquid was applied to Dowex 50×8 columnar chromatgraphy and Dowex 1×2 columnar chromatography (both made by Muromachi Chemical Co., Ltd.) thereby to be processed by ethanol, whereby an object substance is isolated from the reaction liquid. The isolated substance was applied to an amino acid analyzer (made by Hitachi Co.) and paper chromatography. Since the isolated substance behaved in the same way as a standard substance, it was recognized as L-theanine. When the isolated substance was processed by hydrolysis using hydrochloric acid or glutaminase, glutamine acid and ethylamine were produced in a ratio of 1:1. Thus, since the isolated substance was hydrolyzed by glutaminase, it was shown that ethylamine was gamma-ethylamine of glutamine acid. Furthermore, it was confirmed on the basis of glutamate dehydrogenase that glutamine acid produced by hydrolysis was L-glutamine acid. As a result, 8.5 g theanine was obtained.
glutaminase; In water; at 30℃; for 22h;pH 11;Aqueous boric buffer;
Example 1; Production of Theanine by an Enzymatic Method; 0.3 M glutamine and 1.5 M ethylamine hydrochloride in 0.05 M boric acid buffer (pH11) were reacted in the presence of 0.3 U glutaminase (commercial product) at 30 C. for 22 hours, and 225 nmol of L-theanine was obtained. Subsequently, the reaction solution was applied to Dowex 50×8 and Dowex 1×2 column chromatography (MUROMACHI CHEMICALS INC.), followed by ethanol treatment, to isolate the objective substance from the reaction solution. The isolated substance was analyzed by an amino acid analyzer (Hitachi, Ltd.) and paper chromatography, and was confirmed to be L-theanine due to the same pattern of movement as that of the standard substance. Hydrolysis treatment with hydrochloric acid or glutaminase produced glutamic acid and ethylamine at a ratio of 1:1. Thus, the isolated substance was hydrolyzed by glutaminase, demonstrating the binding of ethylamine at the gamma position of glutamic acid. L-glutamic acid generated by hydrolysis was confirmed by glutamate dehydrogenase. Thus, 8.5 g of L-theanine was obtained.
glutaminase; In water; at 30℃; for 22h;pH 11;Aqueous boric buffer;
Example 6; Production of a Mixture of Theanyl-Glutamine and Glutaminyl-Theanine; 0.4 M L-<strong>[3081-61-6]theanine</strong> and 0.4 M L-glutamine in 0.05 M boric acid buffer (ph11) were reacted in the presence of 0.3 U glutaminase (commercial product) at 30 C. for 22 hours to obtain 120 nmol of a mixture of theanyl-glutamine and glutaminyl-<strong>[3081-61-6]theanine</strong>. Subsequently, the reaction solution was applied to Dowex 50×8 and Dowex 1×2 column chromatography (MUROMACHI CHEMICALS INC.) to isolate the objective substance from the reaction solution. Structural analysis was conducted on the theanyl-glutamine and glutaminyl-<strong>[3081-61-6]theanine</strong> by mass spectrum analysis and NMR for confirmation.
glutaminase; In water; at 30℃; for 22h;pH 11;Aqueous boric buffer;
Example 4; Production of Theanyl-Theanine; 0.32 M L-<strong>[3081-61-6]theanine</strong> in 0.05 M boric acid buffer (pH11) was reacted in the presence of 0.3 U glutaminase (commercial product) at 30 C. for 22 hours, and 150 nmol of theanyl-<strong>[3081-61-6]theanine</strong> was obtained. Subsequently, the reaction solution was applied to Dowex 50×8 and Dowex 1×2 column chromatography (MUROMACHI CHEMICALS INC.) to isolate the objective substance from the reaction solution.
With glutaminase; water; boric acid; at 30℃; for 22h;pH 11;Enzymatic reaction;
0.3 M glutamine and 1.5 M methylamine hydrochloride were reacted in the presence of 0.3 U glutaminase (commercially available) at 30°C for 22 hours in a buffer solution of 0.05 M boric acid (pH 11), whereby 225 nm theanine was obtained. Reaction liquid was applied to Dowex 50.x.8 columnar chromatography and Dowex 1.x.2 columnar chromatography (both made by Muromachi Chemical Co., Ltd.) thereby to be processed by ethanol, whereby an object substance is isolated from the reaction liquid. As a result, 8.5 g theanine was obtained. The isolated substance was applied to an amino acid analyzer (made by Hitachi Co.) and paper chromatography. Since the isolated substance behaved in the same way as a standard substance, it was recognized as L-theanine. When the isolated substance was processed by hydrolysis using hydrochloric acid or glutaminase, glutamine acid and ethylamine were produced in a ratio of 1:1. Thus, since the isolated substance was hydrolyzed by glutaminase, it was shown that ethylamine was gamma-ethylamine of glutamine acid. Furthermore, it was confirmed on the basis of glutamate dehydrogenase that glutamine acid produced by hydrolysis was L-glutamine acid.
With glutaminase; at 30℃; for 22h;pH 11;borate buffer; Enzymatic reaction;
By reacting 0.3M glutamine and 1.5M ethylamine hydrochloride in 0.05M borate buffer (pH 11) at 30°C for 22 hours under the presence of 0.3U glutaminase (commercially available product), 225nmol of theanine were obtained. The reaction solution was then subject to chromatography using Dowex 50 x 8 and Dowex 1 x 2 columns (both made by Muromachi Technos Co. , Ltd.) and to ethanol treatment to isolate the target substance from the reaction solution. 8.5g of theanine were thus obtained. The isolated substance was then subject to an amino acid analyzer (made by Hitachi, Ltd.) and paper chromatography and, by exhibition of the same behavior as a standard substance, was confirmed to be L-theanine. Upon hydrolytic treatment with hydrochloric acid or glutaminase, monosodium glutamate and ethylamine were produced at a ratio of 1:1. That the isolated substance is hydrolyzed by glutaminase indicates that ethylamine is bonded to the gamma position of monosodium glutamate. That the monosodium glutamate resulting from hydrolysis is the L-isomer was confirmed by means of monosodium glutamate dehydrogenase.
Example 1: Preparation of N (5) -ethyl-L-glutamine (theanine)5.8 g of N-phthaloyl-L-glutamic acid 5-methyl ester(formula 1: R = Me, Xi = X2 = X3 = X4 = H) was added to 12.9 g of 70% ethylamine solution at 0C and stirred for one hour. Then, the reaction temperature was raised up to20C. After stirring the resulting solution at 20C for 22 hours, the ethylamine existing excessively was removed under reduced pressure. After adding 38.6 g of acetone to the resulting solution, the pH of the solution was regulated as 5 to 6 using acetic acid. Then, the resulting solution was stirred for one hour. Produced solids were filtrated and washed with ethanol . The filtrated white solids were dried to obtain a target compound (3.1 g, 89%).NMR (D2O) delta(ppm) : 3.77(t, IH), 3.20(q, 2H), 2.40(m, 2H), 2.13 (dd, 2H), l.ll(t, 3H) [alpha]20 +8.0 (c=5, H2O)
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