Novel preparation method of L-serine

Abstract

The present invention relates to a novel method for preparing L-serine, and more specifically, to a novel method for preparing L-serine from glycine without using toxic substances or microorganisms such as Escherichia coli.

Description

Technical Field

[0001] The present invention relates to a novel preparation method of L-serine. Background Art

[0002] Serine is an important amino acid for metabolizing substances, as it is required for the biosynthesis of purines and pyrimidines. Serine is a precursor of many amino acids, such as glycine, cysteine, and bacterial tryptophan. Serine is also a precursor of many other metabolites, such as folic acid and sphingolipids (which are the main donors of fragments composed of single carbon atoms in biosynthesis).

[0003] Wherein, especially, L-serine participates in multiple metabolic systems, plays an important role in the normal growth and development of nerve cells.L-serine plays a vital role in cell proliferation by synthesizing the base as deoxyribonucleic acid (DNA) material.L-serine is also used as a precursor in the synthesis of glutathione, so L-serine plays an important role in anti-oxidation.The phospholipids such as phosphatidylserine (phosphatidylserine), sphingosine (sphingosine) derived from L-serine are the main components surrounding the myelin sheath of nerve cells, so that neural information can be quickly transmitted.In addition, L-serine can activate the N-methyl-D-aspartate (NMDA) receptor in neurotransmitter.

[0004] Meanwhile, conventionally, L-serine is prepared by fermentation or by first preparing a DL-serine mixture using mercury and then obtaining pure L-serine through optical separation.

[0005] Biochemical Engineering / Biotechnology Progress (Advances in Biochemical Engineering / Biotechnology, 79 (2003) 1) discloses a method for preparing L-serine, which uses Hyphomicrobium sp. NCIB10099 (a Gram-negative bacterium) to produce 45g / L of L-serine with 100g / L of glycine and 88g / L of methanol over 3 days. However, this method is not commercially used because of the problem of bacterial strains and the economic efficiency of reduction, making it unsuitable for drug or food applications.

[0006] Another known method is disclosed in U.S. Pat. No. 5,382,517, which describes a method for preparing L-serine by fermenting a glycine and formaldehyde solution (485 g / L) with E. coli MT-10350 at 50°C for 35 hours with a yield of 89%. However, the use of E. coli in this manner is not suitable for health products or drugs. In addition, the method using fermentation requires additional analysis of harmful bacteria during drug approval, thus requiring management difficulties such as the need for a separate clean area. In addition, formaldehyde has a serious carcinogenicity and an extremely low permissible residual level, so formaldehyde cannot be used for pharmaceuticals in Korea.

[0007] In addition, Org.Synth.1940,20,81 discloses a method for preparing DL-serine, such as Figure 1 As shown. According to this method, an overall yield of about 30% to 40% can be achieved based on mercuric acetate. However, concerns are raised about the potential residue of mercury, which is a Class I managed heavy metal that is highly toxic to humans. In addition, the use of corrosive substances such as HBr requires expensive equipment without corrosion risks, and there are challenges associated with leakage risks and increased wastewater costs, making large-scale production difficult. In addition, the strong odor of acrylates used as starting materials severely limits their industrial use.

[0008] Figure 1 .Preparation method of L-serine

[0009]

[0010] Another method for preparing serine disclosed in Chinese Patent Publication No. 105037060 involves reacting ammonium carbonate and ethylene glycol in an aqueous solution under a high pressure of 3 MPa. However, the reaction under high pressure requires specialized high pressure equipment and there is a huge risk of explosion, making it unsuitable for an industrial manufacturing method.

[0011] In addition, Japanese Patent Publication No. 1993-140056 describes a method for obtaining racemic serine, which uses an expensive platinum catalyst to react highly flammable oxygen with aminodiol. This method also has difficulties in economic feasibility and industrial production stability.

[0012] The Bulletin of the Chemical Society of Japan (2002), 75(6), 1383-1384) describes a method for preparing racemic serine from glycine using a Cu(II) complex as a catalyst. Figure 2 The Cu complex shown has a very complicated synthesis process, making it unsuitable for commercial use in the industrial production of serine.

[0013] Figure 2 .Cu complex

[0014]

[0015] Prior art literature

[0016] (Patent Document 1) U.S. Patent No. 5382517

[0017] (Patent Document 2) China Published Patent No. 105037060

[0018] (Patent Document 3) Japanese Patent Publication No. 1993-140056

[0019] (Non-patent document 1) Biochemical Engineering / Biotechnology Progress 79 (2003) 1

[0020] (Non-patent document 2) Org. Synth. 1940, 20, 81

[0021] (Non-patent document 3) Bulletin of the Chemical Society of Japan (2002), 75(6), 1383-1384. Summary of the invention

[0022] Technical issues

[0023] The inventors of the present invention have attempted to provide a novel method for synthesizing L-serine from glycine without using toxic substances or microorganisms such as Escherichia coli.

[0024] Technical Solution

[0025] In order to achieve the above object, the present invention provides a method for preparing L-serine, comprising: a) a step of chelating glycine and a copper compound to form a glycine-copper complex; b) a step of reacting the complex of step a) with a carbon source to form a complex into which a hydroxymethyl group 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 optically separating the DL-serine of step c) to collect L-serine.

[0026] Advantageous Effects of the Invention

[0027] The method for preparing L-serine of the present invention is a method for synthesizing L-serine from glycine without using toxic substances or fungi such as Escherichia coli, and has industrial applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 One embodiment of the method for producing L-serine according to the present invention is shown.

[0029] Figure 2 The results of analyzing L-serine obtained by the preparation method of the present invention using Differential Scanning Calorimeter (DSC) are shown. DETAILED DESCRIPTION

[0030] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as understood by those skilled in the art to which the invention belongs. Generally, the terms used in this specification are well known and commonly used in the art.

[0031] One aspect of the present invention relates to a method for preparing L-serine.

[0032] An embodiment of the present invention relates to a method for producing L-serine, comprising: a) chelating glycine and a copper compound to form a glycine-copper complex; b) reacting the complex of step a) with a carbon source to form a complex into which a hydroxymethyl group is introduced; c) treating the complex of step b) with a reducing agent to form DL-serine; and d) optically separating the DL-serine of step c) to collect L-serine. An embodiment of the preparation method of the present invention is Figure 1 Shown in.

[0033] L-serine used in the present invention is a compound represented by the following Chemical Formula 1.

[0034] [Chemical formula 1]

[0035]

[0036] In the present specification, the term "purified" means that when the isolate is isolated, the purity of the isolate is 90% or more, preferably 95% or more, more preferably 99% or more, and most preferably 99.9% or more.

[0037] In the present invention, the step a) is a step of chelating glycine and a copper compound to form a glycine-copper complex.

[0038] The step a) can be performed by adding glycine, copper, a base and purified water.

[0039] The copper that forms a complex with glycine in the step a) can be any one or a mixture of more than one selected from the group consisting of CuSO4, CuOAc2, CuCl2, CuI, CuBr, CuBr2 and hydrates thereof, as a copper complex that is generally used in various industrial preparation methods and is easily available.

[0040] The glycine used in the step a) can be used in an amount of 5kg to 700kg, 10kg to 600kg, 20kg to 500kg, 30kg to 400kg, 40kg to 300kg, 50kg to 200kg, 60kg to 150kg or 70kg to 100kg, depending on the size of the preparation 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.

[0041] The base used in the step a) can be 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 can 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.

[0042] At this time, the amount of purified water can be 6 to 30 times, preferably 4 to 20 times, and most preferably 3 to 10 times the weight of glycine, but is not limited thereto. In the case of excessive use of purified water, the process is difficult to handle, and the amount of wastewater discarded after the process increases. Therefore, using the minimum amount of purified water is important to ensure economic efficiency.

[0043] In addition, when the temperature is increased, it becomes difficult to form a glycine-copper complex, and the yield decreases. Therefore, the temperature is preferably between room temperature and 40°C.

[0044] The glycine-copper complex of step a) may have a structure of the following Chemical Formula 2.

[0045] [Chemical formula 2]

[0046]

[0047] In the present invention, step b) is a step of reacting the complex obtained in step a) with a carbon source to form a complex having a hydroxymethyl group introduced therein.

[0048] The step b) can be performed by adding a carbon source, an alkali and purified water.

[0049] Specifically, the step b) is a process of introducing a carbon into the complex of the 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.

[0050] [Chemical formula 3]

[0051]

[0052] The base used in the step b) may be any one or more selected from the group consisting of aqueous ammonia, ammonia gas, sodium hydroxide, potassium hydroxide, calcium hydroxide, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and mixtures thereof.

[0053] The amount of purified water used in the step b) can be 6 to 25 times, preferably 5 to 20 times, and most preferably 4 to 15 times, compared to the weight of the copper glycine complex having the structure of Chemical Formula 2, but is not limited thereto.

[0054] The carbon source used in the b) step can be selected from any one or more of the group consisting of formaldehyde dilute solution, paraformaldehyde, 1,3-dioxolane, 1.3-dioxane and hexamidine, and can be preferably paraformaldehyde or hexamidine. The formaldehyde can be used as a dilute solution, but not limited to a 15% to 35% aqueous solution. The carbon source can be 1 to 5.0 equivalents, preferably 1.5 to 4.0 equivalents, and most preferably 2.0 to 3.5 equivalents compared to the content of the glycine copper complex having the structure of Chemical Formula 2, but is not limited thereto.

[0055] Regarding the reaction temperature, although the reaction can be carried out at room temperature, in order to facilitate the reaction, the reaction temperature can be raised to room temperature to below 40°C and stirred. The reaction time can be 1 to 18 hours, preferably 2 to 14 hours, and most preferably 3 to 12 hours.

[0056] In the present invention, step c) is a step of treating the complex obtained in step b) with a reducing agent to form DL-serine.

[0057] The reducing agent used in the step c) may be sodium borohydride (NaBH4), lithium aluminum hydride (LiAlH4) or sodium sulfide (Na2S), and most preferably may be sodium sulfide. The content of the copper complex of the racemic serine having the structure of Chemical Formula 3 may be 1 to 5 equivalents, preferably 1.2 to 4.0 equivalents, and most preferably 1.5 to 3.5 equivalents, but is not limited thereto.

[0058] The solvent used in the step c) may be one or more 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 the organic solvent is used, in order to stir smoothly, 10% to 40% of water in the total amount of the solvent may be mixed for use. The total amount of the 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.

[0059] Regarding the reaction temperature, although the reaction can be carried out at room temperature, for a smooth reaction, the reaction temperature may be raised to room temperature to 50° C. or less and stirred. The reaction time may be 3 to 24 hours, preferably 4 to 18 hours, and most preferably 6 to 12 hours.

[0060] After the step c), in order to completely remove the copper removed from the complex, a step of reflux cooling using purified water may be further included. The amount of purified water used at this time may be 2 to 10 times, preferably 4 to 7 times, the amount of DL-serine obtained in the step c).

[0061] In the present invention, step d) is a step of optically separating the DL-serine obtained in step c) to collect L-serine.

[0062] In the step d), in order to selectively obtain only L-serine, various acids as optical separation promoting substances may be used to obtain L-serine monoacid salt of the following Chemical Formula 4.

[0063] [Chemical formula 4]

[0064]

[0065] The acid used in the step d) may be any one or more selected from the group consisting of tartaric acid, malic acid, mandelic acid and camphorsulfonic acid, or one or more 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, and most preferably 0.95 to 1.15 equivalents of the racemic DL-serine obtained in the step c), and the reaction temperature may be 40°C to 100°C, preferably 50°C to 90°C, and most preferably 55°C to 85°C.

[0066] The reaction solvent in the step d) can be any one or more selected from the group consisting of ethyl acetate, toluene, methyl tert-butyl ether, acetic acid, acetone, tetrahydrofuran, isopropanol, methanol and ethanol, and can be preferably ethyl acetate, acetic acid or isopropanol. The amount of solvent used is 6 to 20 times, preferably 5 to 15 times, and most preferably 4 to 12 times, compared to the weight of DL-serine, but is not limited thereto. The reaction time can be 1 to 12 hours, preferably 2 to 10 hours, and most preferably 3 to 6 hours.

[0067] As a process of filtering the generated L-serine-acid salt and washing the excess acid, any filtration method such as reduced pressure filtration, centrifugation, filtration drying, etc. that is feasible in industrial production methods can be used. An organic solvent 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 can be used. At this time, in order to prevent the yield from decreasing, it is preferred that the moisture content of the organic solvent is 1% or less. The organic solvent can be selected from any one or more of the group consisting of acetone, methanol, ethanol, isopropanol, butanol, hexanol, ethyl acetate, toluene, methyl tert-butyl ether, dichloromethane, cyclohexane and pentane, and can be preferably acetone, methanol, ethanol or isopropanol.

[0068] In order to obtain only pure L-serine, the process may further include neutralizing the L-serine monoacid salt of Chemical Formula 4 with a base, wherein the base may be 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.

[0069] The drying temperature may be between 20°C and 80°C, preferably between 30°C and 70°C, and most preferably between 40°C and 60°C. Vacuum or hot air drying may be used but are not limited thereto.

[0070] The present invention will be described in more detail below according to the following examples. However, the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited thereto. The embodiments of the present invention are provided in order to more fully explain the present invention to those of ordinary skill in the art to which the present invention belongs.

[0071] Example 1. Preparation of L-glycine copper complex

[0072] Example 1.1. Preparation of L-glycine copper complex using anhydrous CuSO4

[0073] 75.0 kg of glycine (source: EOS, China) and 1000 L of purified water were added to the reactor at room temperature and stirred for 10 minutes. Then, 156 kg of anhydrous CuSO4 (source: China Yongchun Applied Materials Co., Ltd.) and 80 kg of NaOH (source: Xingchang Chemical) were added and stirred at room temperature for 6 hours. After the reaction was completed, the temperature was lowered to about 10°C, the solid formed was filtered under reduced pressure, and then washed 3 times with 100 kg of purified water. After vacuum drying at 50°C for 10 hours, a blue solid as the target compound was obtained (yield: 83.5%).

[0074] Example 1.2. Preparation of L-glycine copper complex using 5-hydrate CuSO4 (CuSO4·H2O)

[0075] 75.0 kg of glycine (source: EOS, China) and 1200 L of purified water were added to the reactor at room temperature and stirred for 10 minutes. Then, 249 kg of CuSO4·H2O (source: China Yongchun Applied Materials Co., Ltd.) and 80 kg of NaOH (source: Xingchang Chemical) were added and stirred at room temperature for 6 hours. After the reaction was completed, the temperature was lowered to about 10°C, the solid formed was filtered under reduced pressure, and then washed 3 times with 100 kg of purified water. After vacuum drying at 50°C for 10 hours, a blue solid as the target compound was obtained (yield: 82.0%).

[0076] Example 2. Preparation of DL-serine copper complex

[0077] Example 2-1. Preparation of DL-serine copper complex

[0078] 60 kg of the copper glycine complex obtained in Preparation Example 1, 15 kg of paraformaldehyde (source: EOS, China) and 500 kg of purified water were added to the reactor at room temperature and stirred for 10 minutes. Then, 45 kg of sodium hydroxide (NaOH) was slowly added while maintaining the room temperature. After stirring at room temperature for 1 hour, the temperature was raised to about 40°C and stirred for 12 hours, filtered and cooled to about room temperature. After the formed solid was filtered under reduced pressure, it was washed 3 times with 100 kg of purified water. After vacuum drying at 50°C for 10 hours, a blue solid as the target compound was obtained (yield: 77.0%).

[0079] Example 2-2. Preparation of DL-serine copper complex

[0080] The target compound was obtained as a blue solid (yield: 74.0%) in the same manner as in Example 2-1, except that 12.5 kg of Hexamidine (imported by Rayleigh Chemical, China) was used instead of paraformaldehyde in Preparation Example 2-1.

[0081] Example 3. Preparation of DL-serine

[0082] After adding 30 kg of the substance obtained in Preparation Example 2 and 240 kg of purified water to the reactor at room temperature and stirring for 10 minutes, 8.8 kg of sodium sulfide (Na2S) (source: China EOS Company) was slowly added while maintaining the room temperature. After stirring at room temperature for 2 hours, stir at about 45°C for 8 hours, and then cool to about 5°C. After adding the formed solid to 150 kg of purified water, increase the temperature and reflux for 2 hours, gradually lower the temperature and cool to about 5°C. After decompression filtration, wash it once with 10 kg of purified water and twice with 20 kg of ethanol. After vacuum drying at 50°C for 10 hours, a white solid as the target compound was obtained (yield: 79%).

[0083] Example 4. Preparation of L-serine

[0084] Example 4-1. Preparation of L-serine using camphorsulfonic acid

[0085] 10 kg of DL-serine and 23.2 kg of L-(-)-camphorsulfonic acid (source: China EOS) obtained in Preparation Example 3 were added to the reactor, and 100 kg of acetic acid (source: Samchun Pure Chemical Co., Ltd.) was added for dissolution. Heat to about 80°C to 90°C and stir for 3 hours. The mixture was then cooled to about 5°C to 10°C, stirred for 1 hour, and filtered to obtain a white solid L-serine-camphorsulfonate. The obtained solid was dissolved in 120 L of anhydrous ethanol, 28 to 30% ammonia water was added, and the pH was adjusted to about 6.0 to 7.0. The reactants were stirred at room temperature for 1.5 hours, the temperature was lowered to about 5°C to 10°C, and further stirred for 3 hours and filtered to obtain a crude L-serine product as a white solid. Then, 15 kg of the crude product, 1 kg of anhydrous ethanol and purified water were added, stirred for 2 hours at room temperature, 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. The chemical purity and optical purity of the product were measured to be 99.5% and 99.7%, respectively.

[0086] Example 4-2. Preparation of L-serine using tartaric acid

[0087] Using (+)-tartaric acid instead of camphorsulfonic acid, 4.15 kg of L-serine was obtained in the same manner as in Preparation Example 4.1. The chemical purity and optical purity of the product were measured to be 99.4% and 99.7%, respectively.

[0088] Experimental Example 1. Confirmation test of L-serine

[0089] The L-serine obtained in Preparation Example 4 was analyzed based on the L-serine analysis method specified in the United States Pharmacopoeia, and it was confirmed that the analysis results all met the requirements.

[0090] The L-serine obtained in Preparation Example 4 was tested by infrared spectroscopy (ATR method) in the Korean Pharmacopoeia General Test Method. It was confirmed that the L-serine at 2930, 1676, 1439, 1389, 1257, 1093, 658 cm -1 Main absorption was observed near each wave number, and it was confirmed that when L-serine was dissolved in water, 1 mL of ninhydrin solution (1->1000) was added to 5 mL and heated for 3 minutes, the solution exhibited purple color.

[0091] In addition, the obtained L-serine was analyzed by differential scanning calorimetry (DSC), and the result confirmed that an endothermic peak appeared at 226°C, which is consistent with the literature value (refer to Figure 2 ).

Claims

1. A method for preparing L-serine, comprising: a) chelating glycine and a copper compound to form a glycine-copper complex; b) reacting the complex obtained in step a) with a carbon source to form a complex into which a hydroxymethyl group is introduced; c) a step of treating the complex obtained in step b) with a reducing agent to form DL-serine; and d) a step of optically separating the DL-serine obtained in step c) to collect L-serine.

2. The method according to claim 1, wherein: The copper compound in step a) is one or more selected from the group consisting of CuSO4, CuOAc2, CuCl2, CuI, CuBr, CuBr2 and hydrates thereof.

3. The method according to claim 1, wherein: Step a) is performed by adding glycine, copper, base and purified water.

4. The method according to claim 3, wherein: Purified water is added in an amount of 6 to 30 times the weight of glycine.

5. The method according to claim 1, wherein: The glycine-copper complex of step a) has the structure of Chemical Formula 2: [Chemical formula 2] 6. The method according to claim 1, wherein: Step b) is carried out by adding a carbon source, a base and purified water.

7. The method according to claim 1, wherein: The carbon source is one or more selected from the group consisting of formaldehyde, paraformaldehyde, 1,3-dioxolane, 1.3-dioxane and hexamidine.

8. The method according to claim 1, wherein: The complex of step b) has the structure of Chemical Formula 3: [Chemical formula 3] 9. The method according to claim 1, wherein: The reducing agent in step c) is sodium sulfide (Na2S).

10. The method according to claim 1, further comprising a step of reflux cooling with purified water after step c).

11. The method according to claim 1, wherein: In step d), at least one selected from the group consisting of tartaric acid, malic acid, mandelic acid, and camphorsulfonic acid is used.

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