Novel method for producing l-serine

Abstract

The present invention relates to a novel method for synthesizing L-serine from glycine without using toxic substances or bacterial strains such as Escherichia coli.

Description

A novel method for producing L-serine

[0001] The present invention relates to a novel method for producing L-serine.

[0002] Serine is a metabolically important amino acid, essential for the biosynthesis of purines and pyrimidines. Serine is a precursor to several amino acids, including tryptophan in bacteria, as well as glycine and cysteine. Serine is also a precursor to numerous other metabolites, including sphingolipids and folic acid, which are major donors of one-carbon fragments in biosynthesis.

[0003] Among them, L-serine in particular is involved in many metabolic systems and plays an important role in the normal growth and development of nerve cells. L-serine plays an essential role in cell proliferation by synthesizing the bases that become the materials of deoxyribonucleic acid (DNA), and also plays an important role in antioxidant activity by acting as a precursor for glutathione synthesis. Phospholipids such as phosphatidylserine and sphingosine, derived from L-serine, are major components of the myelin sheath that surrounds nerve cells, enabling the rapid transmission of nerve information. In addition, it activates the N-methyl-D-aspartate (NMDA) receptor, a neurotransmitter.

[0004] Meanwhile, L-serine has been manufactured mostly through fermentation, or by using a method of manufacturing a DL-serine mixture using mercury, etc., and then obtaining pure L-serine through optical resolution.

[0005] Advances in Biochemical Engineering / Biotechnology 79 (2003) 1 discloses a method for producing serine by using Hyphomicrobium sp. NCIB10099, a type of gram-negative bacterium, to produce 45 g / L L-serine from 100 g / L glycine together with 88 g / L methanol over 3 days. However, this method is not applied industrially due to problems with the strain used and low commercial efficiency for use as a pharmaceutical or food product.

[0006] Another known manufacturing method is disclosed in U.S. Patent No. 5,382,517, which discloses a method of fermenting 485 g / L of glycine and formaldehyde aqueous solution at 50°C for 35 hours using E. coli MT-10350 to obtain a yield of 89%. However, this method using E. coli is not suitable for use as a health functional food or pharmaceutical. In addition, the method using fermentation requires additional analysis tests for harmful bacteria when obtaining pharmaceutical approval, which requires a separate clean area, making management difficult. In addition, formaldehyde has a very low residual tolerance and is a serious carcinogen, making it a substance that cannot be used in the manufacture of pharmaceuticals in Korea.

[0007] In addition, Org. Synth. 1940, 20, 81 discloses a method for producing DL-serine as shown in Figure 1 below. According to this method, an overall yield of about 30 to 40% can be achieved based on mercuric acetate, but the presence of residual mercury, a Class 1 controlled heavy metal that is highly toxic to the human body, is a problem, and expensive manufacturing equipment without the risk of corrosion is required due to the use of a highly corrosive substance such as HBr, and there is a risk of leakage during operation and an increase in wastewater costs, making it difficult to mass-produce, and there is a problem that industrial use is extremely limited due to the very strong odor of acrylate used as a starting material.

[0008] Figure 1. Synthesis method of DL-serine

[0009]

[0010] Another method for producing serine disclosed in Chinese Patent Publication No. 105037060 discloses a method for producing serine by reacting ammonium carbonate and ethylene glycol in an aqueous solution at a high pressure of 3 MPa. However, the reaction at high pressure requires separate high-pressure equipment and has a high risk of explosion, so this is also not suitable as an industrial production method.

[0011] In addition, Japanese Patent Publication No. 1993-140056 discloses a method for obtaining racemic serine by reacting highly flammable oxygen with aminodiol using expensive platinum as a catalyst, but this also has difficulties in economic feasibility and stability for industrial production.

[0012] Bulletin of the Chemical Society of Japan (2002), 75(6), 1383-1384 discloses a method for producing racemic serine from glycine using a Cu(II) complex as a catalyst. However, the Cu complex used here has a structure as shown in Figure 2 below, and the production method is very difficult for commercial use, making it unsuitable for application to the industrial production method of serine.

[0013] Figure 2. Cu complex

[0014]

[0015] (Patent Document 1) U.S. Patent No. 5,382,517

[0016] (Patent Document 2) Chinese Publication Patent No. 105037060

[0017] (Patent Document 3) Japanese Patent Publication No. 1993-140056

[0018] (Non-patent Document 1) Advances in Biochemical Engineering / Biotechnology 79 (2003) 1

[0019] (Non-patent document 2) Org. Synth. 1940, 20, 81

[0020] (Non-patent Document 3) Bulletin of the Chemical Society of Japan (2002), 75(6), 1383-1384

[0021] Accordingly, the inventors of the present invention developed a novel method for synthesizing L-serine from glycine without using toxic substances or fungi such as E. coli, and sought to provide a novel method for producing L-serine that can be applied industrially.

[0022] In order to achieve the above object, the present invention provides a method for producing L-serine, comprising the steps of a) chelating glycine and a copper compound to form a glycine-copper complex, b) reacting a carbon source with the complex of step a) to form a complex in which hydroxymethyl is introduced, c) treating the complex of step b) with a reducing agent to form DL-serine, and d) optically resolving the DL-serine of step c) to isolate L-serine.

[0023] The method for producing L-serine of the present invention is a method capable of synthesizing L-serine from glycine without using toxic substances or fungi such as E. coli, and has the characteristic of being industrially applicable.

[0024] Figure 1 illustrates one embodiment of a method for producing L-serine according to the present invention.

[0025] Figure 2 shows the results of analyzing L-serine obtained according to the present invention using differential scanning calorimetry (DSC).

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0027] The present invention relates, in one aspect, to a method for producing L-serine.

[0028] Embodiments of the present invention include:

[0029] a) A step of chelating glycine and a copper compound to form a glycine-copper complex;

[0030] b) a step of reacting a carbon source with the complex of step a) to form a complex into which hydroxymethyl is introduced;

[0031] c) a step of treating the complex of step b) with a reducing agent to form DL-serine; and

[0032] d) A step of optically resolving DL-serine of step c) to separate L-serine.

[0033] The present invention relates to a method for producing L-serine, and the method according to the present invention is illustrated in FIG. 1.

[0034] In the present invention, “L-serine” is a type of amino acid that constitutes proteins, is a monoamide of glutamic acid, and means an L-amino acid having a structure represented by the following chemical formula 1.

[0035] [Chemical Formula 1]

[0036]

[0037] In the present invention, “purified” means that when the isolate is separated, the isolate is at least 90% pure, preferably at least 95% pure, more preferably at least 99% pure, and most preferably at least 99.9% pure.

[0038] In the present invention, step a) is a step of forming a glycine-copper complex by chelating glycine and a copper compound.

[0039] The above step a) may be performed by adding glycine, copper, base and purified water.

[0040] The copper forming a complex with glycine in the above step a) is a copper complex that is generally used in various industrial manufacturing methods and is readily available, and may be used as a mixture of any one or more selected from the group consisting of CuSO4, CuOAc2, CuCl2, CuI, CuBr, CuBr2 and hydrates thereof.

[0041] The glycine used in the above step a) can be used in an amount of 5 kg to 700 kg, 10 kg to 600 kg, 20 kg to 500 kg, 30 kg to 400 kg, 40 kg to 300 kg, 50 kg to 200 kg, 60 kg to 150 kg, or 70 kg to 100 kg, depending on the size of the manufacturing device. The amount of the copper compound can be 0.3 to 1 equivalent, preferably 0.4 to 0.7 equivalent, and most preferably 0.45 to 0.55 equivalent relative to the amount of glycine used.

[0042] The base used in the above step a) may be at least one selected from the group consisting of ammonia water, ammonia gas, sodium hydroxide, potassium hydroxide, calcium hydroxide, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and mixtures thereof. The amount of the base may be 0.6 to 4 equivalents, preferably 0.8 to 3 equivalents, and most preferably 1.0 to 2.5 equivalents relative to the amount of glycine.

[0043] At this time, the amount of purified water may be 6 to 30 times the weight of glycine, preferably 4 to 20 times, and most preferably 3 to 10 times, but is not limited thereto. If purified water is used in excessive amounts, handling during the process becomes difficult, and the amount of wastewater discarded after the process increases. Therefore, using the minimum amount is important for ensuring economic feasibility.

[0044] In addition, since increasing the temperature makes it difficult to form a glycine-copper complex and lowers the yield, a temperature of room temperature to 40°C is preferred.

[0045] The glycine-copper complex of step a) above may have a structure represented by the following chemical formula 2.

[0046] [Chemical Formula 2]

[0047]

[0048] In the present invention, step b) is a step of reacting a carbon source with the complex of step a) to form a complex into which hydroxymethyl is introduced.

[0049] The above step b) may be performed by adding a carbon source, a base, and purified water.

[0050] Specifically, step b) is a process of introducing one carbon atom into the complex of step a) and then introducing a hydroxyl group to convert it into a copper complex of racemic serine having a structure of the following chemical formula 3.

[0051] [Chemical Formula 3]

[0052]

[0053] The base used in the above step b) may be at least one selected from the group consisting of ammonia water, ammonia gas, sodium hydroxide, potassium hydroxide, calcium hydroxide, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and mixtures thereof.

[0054] The amount of purified water used in the above step b) may be 6 to 25 times, preferably 5 to 20 times, and most preferably 4 to 15 times the weight of the glycine copper complex having the structure of chemical formula 2, but is not limited thereto.

[0055] The carbon source used in the step b) may be at least one selected from the group consisting of a diluted formaldehyde solution, paraformaldehyde, 1,3-dioxolane, 1.3-dioxane, and hexamidine, and preferably paraformaldehyde or hexamidine. The formaldehyde may be used as a diluted solution in the form of a 15 to 35% aqueous solution, but is not limited thereto. The carbon source may be 1 to 5.0 equivalents, preferably 1.5 to 4.0 equivalents, and most preferably 2.0 to 3.5 equivalents, relative to the content of the glycine copper complex having the structure of Chemical Formula 2, but is not limited thereto.

[0056] The reaction temperature is at room temperature, but for smooth reaction, the temperature may be raised to room temperature or lower than 40°C and stirred, and the reaction time may be 1 to 18 hours, preferably 2 to 14 hours, and most preferably 3 to 12 hours.

[0057] In the present invention, step c) is a step of forming DL-serine by treating the complex of step b) with a reducing agent.

[0058] The reducing agent used in the above step c) may be sodium borohydride (NaBH4), lithium aluminum hydride (LiAlH4), or sodium sulfide (Na2S), and most preferably sodium sulfide. The sodium sulfide may be used in an amount of 1 to 5 equivalents, preferably 1.2 to 4.0 equivalents, and most preferably 1.5 to 3.5 equivalents relative to the content of the copper complex of racemic serine having the structure of chemical formula 3, but is not limited thereto.

[0059] The solvent used in the above step c) may be at least one selected from the group consisting of purified water, methanol, ethanol, isopropanol, butanol, and hexanol, preferably purified water, methanol, ethanol, or isopropanol, and most preferably purified water or methanol. In addition, when using the organic solvent, water may be mixed and used in an amount of 10 to 40% of the total amount of solvent used to facilitate smooth stirring. The total amount of solvent used may be 6 to 30 times, preferably 5 to 20 times, and most preferably 4 to 12 times the weight of the copper complex of racemic serine having the structure of chemical formula 3, but is not limited thereto.

[0060] The reaction temperature is room temperature, but for smooth reaction, the temperature can be raised to room temperature or lower than 50℃ and stirred. The reaction time can be 3 to 24 hours, preferably 4 to 18 hours, and most preferably 6 to 12 hours.

[0061] After performing step c), a step of cooling by refluxing with purified water may be further included to completely remove the copper removed from the complex. The amount of purified water used at this time may be 2 to 10 times the amount of DL-serine obtained in step c), and preferably 4 to 7 times.

[0062] In the present invention, step d) is a step of optically resolving DL-serine of step c) to separate L-serine.

[0063] In the above step d), in order to selectively obtain only L-serine, various acids that are optical resolution promoting substances can be used to obtain L-serine-acid salts of the following chemical formula 4.

[0064] [Chemical Formula 4]

[0065]

[0066] The acid used in the above step d) may be at least one selected from the group consisting of tartaric acid, malic acid, mandelic acid, and camphor sulphonic acid, or at least one selected from the group consisting of (+)-tartaric acid, (-)-malic acid, (-)-mandelic acid, and (+)-camphor-10-sulfonic acid. The acid may be 0.85 to 1.5 equivalents, preferably 0.90 to 1.25 equivalents, most preferably 0.95 to 1.15 equivalents of the racemic DL-serine obtained in step c), and the reaction temperature may be 40 to 100°C, preferably 50 to 90°C, most preferably 55 to 85°C.

[0067] The reaction solvent in the above step d) may be at least one selected from the group consisting of ethyl acetate, toluene, methyl-t-butyl ether, acetic acid, acetone, tetrahydrofuran, isopropanol, methanol, and ethanol, and preferably ethyl acetate, acetic acid, or isopropanol. The amount of solvent used is 6 to 20 times the weight of DL-serine, preferably 5 to 15 times, and most preferably 4 to 12 times, but is not limited thereto. The reaction time may be 1 to 12 hours, preferably 2 to 10 hours, and most preferably 3 to 6 hours.

[0068] In the process of filtering the produced L-serine acid salt and washing away the excess acid, all filtration methods possible in industrial production methods, such as vacuum filtration, centrifugation, and filter dryer, can be used. An organic solvent can be used in an amount of 1 to 8 times, preferably 2 to 7 times, and most preferably 3 to 6 times the amount of DL-serine used. At this time, the organic solvent preferably has a moisture content of 1% or less in order to prevent a decrease in yield. The organic solvent may be at least one selected from the group consisting of acetone, methanol, ethanol, isopropanol, butanol, hexanol, ethyl acetate, toluene, methyl-t-butyl ether, methylene chloride, cyclohexane, and pentane, and preferably acetone, methanol, ethanol, or isopropanol.

[0069] In order to obtain only pure L-serine, a process of neutralizing the L-serine acid salt of chemical formula 4 using a base may be additionally included. At this time, the usable base may be at least one selected from the group consisting of ammonia water, ammonia gas, sodium hydroxide, potassium hydroxide, calcium hydroxide, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and mixtures thereof. The pH at this time may be 6 to 7.

[0070] The drying temperature may be between 20 and 80°C, preferably between 30 and 70°C, and most preferably between 40 and 60°C, and a vacuum or hot air drying method may be used, but is not limited thereto.

[0071] Hereinafter, the present invention will be described in detail using examples and other means to aid understanding. However, the examples according to the present invention may be modified in various ways, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0072] Example 1. Preparation of L-glycine copper complex

[0073] Example 1.1. Preparation of L-glycine copper complex using anhydrous CuSO4

[0074] 75.0 kg of glycine (obtained from EOS, China) and 1000 L of purified water were placed in a reactor at room temperature and stirred for 10 minutes. Then, 156 kg of anhydrous CuSO4 (obtained from Pure & Applied, China) and 80 kg of NaOH (obtained from Heungchang Chemical) were added and stirred at room temperature for 6 hours. After completion of the reaction, the temperature was lowered to approximately 10°C, and the formed solid was filtered under reduced pressure and washed three times with 100 kg of purified water. The resulting solid was vacuum-dried at 50°C for 10 hours to obtain the target compound, a blue solid (yield: 83.5%).

[0075] Example 1.2. 5-Hydrate CuSO4 (CuSO 4· Preparation of L-glycine copper complex using H2O)

[0076] 75.0 kg of glycine (purchased from EOS, China) and 1200 L of purified water were placed in a reactor at room temperature and stirred for 10 minutes. After that, CuSO 4· 249 kg of H2O (purchased from Pure & Applied, China) and 80 kg of NaOH (purchased from Heungchang Chemical) were added and stirred at room temperature for 6 hours. After completion of the reaction, the temperature was lowered to approximately 10°C, and the formed solid was filtered under reduced pressure and washed three times with 100 kg of purified water. After vacuum drying at 50°C for 10 hours, the target compound, a blue solid, was obtained (yield: 82.0%).

[0077] Example 2. Preparation of DL-serine copper complex

[0078] Example 2-1. Preparation of DL-serine copper complex

[0079] In Example 1, 60 kg of the glycine copper complex obtained, 15 kg of paraformaldehyde (obtained from EOS, China), and 500 kg of purified water were placed in a reactor at room temperature and stirred for 10 minutes. Thereafter, 45 kg of NaOH was slowly added while maintaining the room temperature. After stirring at room temperature for 1 hour, the mixture was stirred for 12 hours while increasing the temperature to approximately 40°C, filtered, and cooled to approximately room temperature. The formed solid was filtered under reduced pressure and washed three times with 100 kg of purified water. The resulting solid was vacuum-dried at 50°C for 10 hours to obtain a blue solid as the target compound (yield: 77.0%).

[0080] Example 2-2. Preparation of DL-serine copper complex

[0081] The target compound, a blue solid, was obtained in the same manner as Example 2-1, except that 12.5 kg of hexamidine (imported from Reliable chem, China) was used instead of paraformaldehyde in Example 2-1 (yield: 74.0%).

[0082] Example 3. Preparation of DL-serine

[0083] 30 kg of the material obtained in Example 2 and 240 kg of purified water were placed in a reactor at room temperature and stirred for 10 minutes, and then 8.8 kg of sodium sulfide (Na2S) (obtained from EOS, China) was slowly added while maintaining the temperature at room temperature. After stirring at room temperature for 2 hours, the mixture was stirred at a temperature of about 45°C for 8 hours, and then cooled to about 5°C. After adding the formed solid to 150 kg of purified water, the mixture was heated to reflux for 2 hours, and then slowly cooled to about 5°C. After filtration under reduced pressure, the mixture was washed once with 10 kg of purified water and twice with 20 kg of ethanol. The resulting mixture was vacuum dried at 50°C for 10 hours to obtain a white solid as the target compound (yield: 79%).

[0084] Example 4. Preparation of L-serine

[0085] 4.1. Production of L-serine using camphorsulfonic acid

[0086] 10 kg of DL-serine obtained in Example 3 and 23.2 kg of L-(-)-camphorsulfonic acid (purchased from EOS, China) were placed in a reactor, and 100 kg of acetic acid (purchased from Samcheon Pure Chemicals) was added and dissolved. The mixture was heated to about 80 to 90°C and stirred for 3 hours. Thereafter, the mixture was cooled to about 5 to 10°C, stirred for 1 hour, and filtered to obtain L-serine-camphorsulfonic acid salt as a white solid. The obtained solid was dissolved in 120 L of anhydrous ethanol, and 28 to 30% ammonia water was added to adjust the pH to about 6.0 to 7.0. The reaction mass was stirred at room temperature for 1.5 hours, and then the temperature was lowered to about 5 to 10°C, stirred for an additional 3 hours, and filtered to obtain a crude L-serine product as a white solid. Afterwards, 15 kg of the above crude product, 1 kg of anhydrous ethanol and purified water were added, stirred at room temperature for 2 hours, filtered, and washed with 2 kg of anhydrous ethanol. After vacuum drying at 50°C for 6 hours, 4.25 kg of L-serine was obtained, and the chemical purity and optical purity of the product were measured to be 99.5% and 99.7%, respectively.

[0087] 4.2. Production of L-serine using tartaric acid

[0088] Using (+)-tartaric acid instead of camphorsulfonic acid, the same method as in Example 4.1 was used to obtain 4.15 kg of L-serine. The chemical purity and optical purity of the product were measured to be 99.4% and 99.7%, respectively.

[0089] Experimental Example 1. Confirmatory Test of L-Serine

[0090] The L-serine obtained in Example 4 was analyzed based on the L-serine analysis method in the United States Pharmacopoeia, and it was confirmed that it all conformed to this.

[0091] When L-serine obtained in Example 4 was tested according to the ATR method of the infrared spectroscopy method in the general test methods of the Korean Pharmacopoeia, 2930, 1676, 1439, 1389, 1257, 1093, and 658 cm -1 It was confirmed that major absorption occurred near each wavelength, and when 1 mL of ninhydrin solution (1 -> 1000) was added to 5 mL of L-serine dissolved in water and heated for 3 minutes, it was confirmed that the solution exhibited a purple color.

[0092] In addition, the obtained L-serine was analyzed using a differential scanning calorimeter (DSC), and it was confirmed that an endothermic peak appeared at 226 degrees, which was consistent with the literature value (see Fig. 2).

Claims

1. a) A step of chelating glycine and a copper compound to form a glycine-copper complex; b) a step of reacting a carbon source with the complex of step a) to form a complex into which hydroxymethyl is introduced; c) a step of treating the complex of step b) with a reducing agent to form DL-serine; and d) a step of separating L-serine by optical resolution of DL-serine of step c) above; A method for producing L-serine, comprising:

2. In paragraph 1, The copper compound of step a) above is CuSO 4 , CuOAc 2 , CuCl 2 , CuI, CuBr, CuBr 2 A method for producing L-serine, wherein the method comprises at least one selected from the group consisting of hydrates thereof.

3. In paragraph 1 or 2, A method for producing L-serine, wherein step a) is performed by adding glycine, copper, a base and purified water.

4. In paragraph 3, A method for producing L-serine, wherein the purified water is added in an amount of 6 to 30 times the weight of glycine.

5. In any one of paragraphs 1 to 4, A method for producing L-serine, wherein the glycine-copper complex of step a) above has a structure represented by the following chemical formula 2 [Chemical formula 2] .

6. In any one of paragraphs 1 to 5, A method for producing L-serine, wherein the step b) is performed by adding a carbon source, a base, and purified water.

7. In any one of paragraphs 1 to 6, A method for producing L-serine, wherein the carbon source is at least one selected from the group consisting of formaldehyde, paraformaldehyde, 1,3-dioxolane, 1,3-dioxane, and hexamidine.

8. In any one of paragraphs 1 to 7, A method for producing L-serine, wherein the complex of step b) above has a structure represented by the following chemical formula 3 [Chemical Formula 3] .

9. In any one of paragraphs 1 to 8, The reducing agent in step c) above is sodium sulfide (Na 2 S) A method for producing L-serine.

10. In any one of paragraphs 1 to 9, A method for producing L-serine, further comprising the step of cooling by refluxing with purified water after performing step c).

11. In any one of paragraphs 1 to 10, A method for producing L-serine, wherein at least one acid selected from the group consisting of tartaric acid, malic acid, mandelic acid, and camphorsulfonic acid is used in step d).

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