Process for the preparation of n-acetyl-l-tyrosine

Abstract

This invention proposes a method for preparing N-acetyl-L-tyrosine, comprising: acylation reaction of acetic anhydride and L-tyrosine under alkaline conditions to obtain an acylated solution; adjusting the pH of the acylated solution with an alkaline solution to increase the pH, maintaining the reaction temperature, and then adjusting the pH of the resulting reaction solution with an acid solution to obtain a reaction solution containing N-acetyl-L-tyrosine; and purifying the reaction solution containing N-acetyl-L-tyrosine. The N-acetyl-L-tyrosine prepared by this method has advantages such as high yield, low content of related substances, and high optical purity. Furthermore, this method has low energy consumption and low production cost, making it suitable for large-scale production applications.

Description

Technical Field

[0001] This invention relates to the field of amino acid production. Specifically, this invention relates to a method for preparing N-acetyl-L-tyrosine. Background Technology

[0002] N-acetyl-L-tyrosine is an important fine organic chemical intermediate, and is the compound shown in Formula 1.

[0003]

[0004] N-acetyl-L-tyrosine has a wide range of applications: it is used as a substitute for tyrosine to overcome the disadvantage of tyrosine's low solubility, as a nutritional supplement; as a pharmaceutical and pharmaceutical intermediate; it is widely used in cosmetics due to its whitening and anti-ultraviolet effects; other chemical industries and fields; and biotechnology fields such as cell culture.

[0005] Current production processes for N-acetyl-L-tyrosine suffer from drawbacks such as high content of related substances, low yield, low optical purity, high energy consumption, and high production costs. Therefore, further research is needed to develop optimal methods for preparing N-acetyl-L-tyrosine. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention proposes a method for preparing N-acetyl-L-tyrosine. The N-acetyl-L-tyrosine prepared using this method has advantages such as high yield, low content of related substances, and high optical purity. Furthermore, this method consumes less energy and has low production costs, making it suitable for large-scale production applications.

[0007] It should be noted that this invention is based on the following discoveries of the inventors:

[0008] The inventors attempted to prepare N-acetyl-L-tyrosine using the following method: 1 part by weight of L-tyrosine was added to a sodium hydroxide solution, and sodium hydroxide solution and 1-1.5 parts by weight (2-3 times the molar amount) of acetic anhydride were added simultaneously at 0-5℃. The temperature was slowly raised to 20-25℃ and the reaction was maintained at this temperature for 40-60 minutes. Sulfuric acid was added at 0-5℃ to adjust the pH. The mixture was then filtered, concentrated, and crystallized to obtain N-acetyl-L-tyrosine.

[0009] Tyrosine has a large side chain group and strong steric hindrance. The inventors found that a conversion rate of 2 times or more equivalent to acetic anhydride is required to achieve a good conversion rate. However, when 2 times or more equivalent to acetic anhydride is added, the phenolic hydroxyl groups in tyrosine are also acylated, generating impurities such as O,N-diacetyl-L-tyrosine, which affects product crystallization (in cases of high impurity content, it may even prevent crystallization, resulting in an oily appearance) and affects the product's appearance (causing the product to appear brownish-yellow). Furthermore, in the presence of acid anhydride / acetic acid, prolonged heat treatment easily generates a large amount of optical isomers of N-acetyl-L-tyrosine, reducing the optical purity of the final product and affecting product quality. In addition, the process uses sulfuric acid to adjust the pH, and sulfate is difficult to remove, resulting in a high residual sulfate ion content and high residue on ignition. At the same time, the process of adding industrial sulfuric acid dropwise to aqueous solution is highly exothermic, easily generating impurities. Sulfuric acid is also a commonly used racemic reagent for amino acids, which can cause amino acids and amino acid derivatives to undergo or partially undergo racemic reactions.

[0010] The inventors attempted to prepare N-acetyl-L-tyrosine using the following method: Deionized water, liquid alkali, tyrosine, and acetyl chloride were fed into a chemical reaction vessel in a weight ratio of (6-7):1:1:(1-2) (the molar ratio of tyrosine to acetyl chloride was 1:2.3 or higher). After sealing, the mixture was evacuated to a vacuum and gradually heated to 55-59°C under stirring conditions. The reaction was carried out for 2.0-2.2 hours. The pH was adjusted with hydrochloric acid, and the mixture was concentrated, crystallized, and purified.

[0011] This process uses acetyl chloride as the acylation reagent. The inventors discovered that acetyl chloride is extremely reactive, making the reaction violent and difficult to control, and easily generating more impurities. This not only results in low yield but also affects the crystallization and appearance of the product. Similarly, the process is carried out at high temperatures for a long time, which generates a large amount of optical isomers of N-acetyl-L-tyrosine, reducing the optical purity of the product. These optical isomers cannot be removed by recrystallization, thus affecting the quality of the final product.

[0012] The inventors attempted to prepare N-acetyl-L-tyrosine using the following method: without adding an acid-binding agent, tyrosine was directly acetylated using acetic anhydride in an aqueous system, and the mixture was kept under reflux for 4-5 hours. The purified product was obtained by extraction with acetone and precipitation with ethyl acetate, with a yield of 88%.

[0013] The inventors discovered that excessive use of acetic anhydride produces more impurities, such as O,N-diacetyl-L-tyrosine, resulting in a lower yield. Prolonged reflux at high temperatures leads to the formation of a large number of optical isomers of N-acetyl-L-tyrosine, negatively impacting the quality of the final product.

[0014] The inventors attempted to prepare N-acetyl-L-tyrosine using the following methods: catalytic hydrogenation of 2-acetamide-3-(4-hydroxyphenyl)-2-acrylic acid; or hydrolysis of acetyltyrosine methyl ester with lithium hydroxide to obtain acetyltyrosine. However, both of these methods are costly, cumbersome, and have no industrial value.

[0015] The inventors attempted to prepare N-acetyl-L-tyrosine using the following method: Acetyltyrosine ethyl ester was resolved by microbial reaction in an ethanol solution at 25°C for 48 hours, thus decomposing the ester group to obtain high-optical-purity acetyltyrosine. This method is time-consuming and has high material costs.

[0016] To address the above issues, the inventors conducted in-depth research on the production process of N-acetyl-L-tyrosine and obtained a method for preparing N-acetyl-L-tyrosine. This method has the advantages of high yield, low content of related substances, and high optical purity. Moreover, this method has low energy consumption and low production cost, making it suitable for large-scale production applications.

[0017] This invention provides a method for preparing N-acetyl-L-tyrosine. According to an embodiment of the invention, the method includes: acylation reaction of acetic anhydride and L-tyrosine under alkaline conditions to obtain an acylated solution; adjusting the pH of the acylated solution with an alkaline solution to increase the pH, maintaining the reaction temperature, and then adjusting the pH of the resulting reaction solution with an acid solution to obtain a reaction solution containing N-acetyl-L-tyrosine; and purifying the reaction solution containing N-acetyl-L-tyrosine.

[0018] As mentioned earlier, the inventors initially attempted to use twice or more acetic anhydride, but this resulted in the acylation of the phenolic hydroxyl groups in tyrosine, generating the impurity O,N-diacetyl-L-tyrosine, which affected product crystallization and appearance. Subsequently, the inventors discovered that by reducing the amount of acetic anhydride used, i.e., to less than twice the amount of acetic anhydride added, the formation of O,N-diacetyl-L-tyrosine and its optical isomers could be reduced. Simultaneously, the relatively weak alkalinity of the system during the acylation reaction also reduced the formation of O,N-diacetyl-L-tyrosine and its optical isomers. Then, after the acetic anhydride was consumed, the pH of the reaction solution was increased, allowing the impurities O,N-diacetyl-L-tyrosine and O-acetyl-L-tyrosine generated by the acylation reaction to decompose into N-acetyl-L-tyrosine and L-tyrosine, respectively, under stronger conditions. This improved the reaction yield, reduced the content of related substances, and improved product quality. Finally, by adjusting the pH of the resulting reaction solution with acid, N-acetyl-L-tyrosine could be obtained. Finally, purification is performed to further improve the purity of the product.

[0019] According to embodiments of the present invention, the above method may further have the following additional technical features:

[0020] According to embodiments of the present invention, the molar ratio of L-tyrosine to acetic anhydride is 1:(0.9–1.5). A sufficient tyrosine acylation reaction requires at least two equivalents of acetic anhydride. The inventors have found that if the amount of acetic anhydride added is two times or more, the phenolic hydroxyl groups in tyrosine are also acylated, generating the impurity O,N-diacetyl-L-tyrosine, which affects product crystallization and appearance. Furthermore, through extensive experiments, the inventors have found that using the above-mentioned ratio of L-tyrosine to acetic anhydride, and reducing the amount of acetic anhydride used, can further reduce the amount of O,N-diacetyl-L-tyrosine generated, thereby improving yield and product quality. In some preferred embodiments, the molar ratio of L-tyrosine to acetic anhydride is 1:(0.9–1.2).

[0021] According to an embodiment of the present invention, the alkaline solution is selected from sodium hydroxide solution, and the acid solution is selected from hydrochloric acid. Under the above alkaline conditions, the acetic anhydride reaction can be promoted to complete, generating N-acetyl-L-tyrosine salt, which is then reacted in the acid solution to obtain N-acetyl-L-tyrosine. If other types of acids are used, such as sulfuric acid, the content of inorganic impurities in the product will be higher, and sulfate ions are more difficult to remove than chloride ions. Therefore, using hydrochloric acid can reduce the content of inorganic salts, reduce the residue on ignition, and improve product quality. At the same time, the process of adding industrial sulfuric acid dropwise to aqueous solution is highly exothermic and easily generates impurities. Sulfuric acid is also a commonly used racemic reagent for amino acids, which can cause amino acids and amino acid derivatives to undergo or partially undergo racemic reactions.

[0022] According to an embodiment of the present invention, during the acylation reaction, the pH of the reaction solution is controlled at 8-10. The pH of the acylation solution is then adjusted to 10-12 using an alkaline solution, and subsequently the pH of the reaction solution containing N-acetyl-L-tyrosine is adjusted to 1.5-2.2. The inventors have discovered through extensive experiments that conducting the acylation reaction under weakly alkaline conditions (pH 8-10) can reduce the large-scale formation of O,N-diacetyl-L-tyrosine and its optical isomer. Then, by increasing the pH of the system, O,N-diacetyl-L-tyrosine is converted to N-acetyl-L-tyrosine via alkaline hydrolysis, and O-acetyl-L-tyrosine is converted to L-tyrosine via alkaline hydrolysis, thereby improving the reaction yield and product quality. Finally, the pH of the reaction solution is adjusted to acidity using an acidic solution to convert the generated N-acetyl-L-tyrosine salt to N-acetyl-L-tyrosine.

[0023] According to embodiments of the present invention, the acylation reaction takes 20–60 minutes (e.g., 20–40 minutes); the holding reaction takes 15–30 minutes at a temperature of 60–70°C. The inventors have found that under these acylation reaction conditions, the acetic anhydride reaction can be completed. Using the above-mentioned holding reaction conditions, it is possible to better convert O,N-diacetyl-L-tyrosine to N-acetyl-L-tyrosine and O-acetyl-L-tyrosine to L-tyrosine, further reducing the amount of optical isomers formed and improving the optical purity of the product.

[0024] According to an embodiment of the present invention, the purification process includes: concentrating and crystallizing the reaction solution containing N-acetyl-L-tyrosine, collecting the crystals to obtain crude N-acetyl-L-tyrosine I; extracting crude N-acetyl-L-tyrosine I and collecting the extract; and concentrating the extract to obtain crude N-acetyl-L-tyrosine II. As mentioned above, in order to reduce the formation of O,N-diacetyl-L-tyrosine and optical isomers, the amount of acetic anhydride used is reduced, resulting in a small amount of residual unreacted L-tyrosine in the acylation solution. The L-tyrosine can be separated by extraction and reused in the acylation reaction, thus improving the yield.

[0025] According to embodiments of the present invention, the extractant used in the extraction process is an alcohol solvent, preferably methanol or ethanol. N-acetyl-L-tyrosine is soluble in alcohol solvents, while L-tyrosine has low solubility in alcohol solvents and forms a precipitate. Thus, separation can be achieved. Furthermore, only one high-quality mother liquor is produced, requiring no additional treatment. The alcohol solvent can be reused repeatedly and directly added to the acylation reaction as a solvent, enabling reuse and correspondingly improving the yield, thus possessing good economic value.

[0026] According to embodiments of the present invention, the extraction temperature is 40–70°C. This can further improve the reaction yield. In some preferred embodiments, the extraction temperature is 50–70°C.

[0027] According to embodiments of the present invention, the amount of extractant added is 2 to 10 times the weight of the crude N-acetyl-L-tyrosine I. This can further improve the reaction yield. In some preferred embodiments, the amount of extractant added is 2 to 4 times the weight of the crude N-acetyl-L-tyrosine I.

[0028] According to an embodiment of the present invention, the method further includes: washing the insoluble matter obtained from the extraction process with water and collecting the insoluble matter. The insoluble matter after extraction is washed with water to remove inorganic salts, acids, etc., leaving tyrosine, which can be reused in the reaction, achieving reuse, avoiding material waste, and improving the reaction yield in continuous production.

[0029] According to an embodiment of the present invention, the method further includes decolorizing the crude N-acetyl-L-tyrosine II to obtain a decolorized solution; crystallizing the decolorized solution, collecting the crystals, washing with water to obtain refined N-acetyl-L-tyrosine. This can further improve the product's color, reduce the content of optical isomer impurities, improve the product's optical purity, and thus improve product quality.

[0030] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0032] Figure 1 This is a process flow diagram for producing N-acetyl-L-tyrosine, provided as an embodiment of the present invention. Detailed Implementation

[0033] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0034] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0035] Example 1

[0036] In this embodiment, N-acetyl-L-tyrosine was prepared according to the following steps, and the process flow diagram is shown below. Figure 1 :

[0037] Step 1: Disperse 100g of L-tyrosine in 200ml of water and stir quickly to avoid clumping.

[0038] Step 2: Add 30% sodium hydroxide solution dropwise until all L-tyrosine is dissolved, pH=12.05.

[0039] Step 3: Add 59.2 g (1.05 molar amount) of acetic anhydride dropwise over 30 minutes, while simultaneously adding 30% sodium hydroxide solution to maintain the pH between 8 and 10.

[0040] Step 4: After adding acetic anhydride, add sodium hydroxide solution dropwise to adjust the pH to 11.50, and keep warm at 60℃ for 20 minutes.

[0041] Step 5: Add industrial hydrochloric acid to adjust the pH of the solution to 1.72.

[0042] Step 6: Control the vacuum to above 0.08 MPa and concentrate the reaction solution at 80°C under reduced pressure until it is basically in a solid state.

[0043] Step 7: After cooling to 4℃ for full crystallization, the material is discharged to obtain crude N-acetyl-L-tyrosine I (a mixture containing acetyltyrosine, tyrosine, inorganic salts, and a small amount of acid / water). The mother liquor is discarded.

[0044] Step 8: Extract N-acetyl-L-tyrosine by mixing the obtained crude N-acetyl-L-tyrosine product I with 3 times its volume of ethanol (95%) at 60°C.

[0045] Step 9: Concentrate the ethanol solution obtained in Step 8 under reduced pressure to recover the solvent, and crystallize to obtain crude N-acetyl-L-tyrosine II. The recovered ethanol can be reused.

[0046] Step 10: Add crude N-acetyl-L-tyrosine II and an equal volume of purified water to a decolorization container. Stir and heat to 65°C to dissolve. Add activated carbon (0.5% of the solid weight of crude N-acetyl-L-tyrosine II) for decolorization for 10 minutes. Filter after decolorization. Cool the filtrate to crystallize. Cool the filtrate to 4°C and stir until crystals are completely precipitated. Filter and dry to obtain 100.05 g of purified N-acetyl-L-tyrosine. In continuous production, the crystallization mother liquor from the refining process can be reused in step 1.

[0047] After extraction in steps 11 and 8, the resulting filter residue is dissolved in water to remove inorganic salts and some impurities. The remaining insoluble substance is L-tyrosine, which can be reused in step 1 for continuous production.

[0048] Structural identification of the N-acetyl-L-tyrosine concentrate obtained in this embodiment: 1 H NMR(D2O,400MHz)δ2.07(s,3H,COCH3),2.82-2.86(m,1H,ArCHH),2.96-3.01(m,1H,Ar CHH), 3.67-3.70 (m, 1H, CHCOO), 6.70 (d, J = 8.0Hz, 2H, ArH), 7.01 (d, J = 8.0Hz, 2H, ArH).

[0049] Example 2

[0050] In this embodiment, N-acetyl-L-tyrosine was prepared according to the following steps:

[0051] Step 1: Disperse 500g of L-tyrosine in 1 liter of water and stir quickly to avoid clumping.

[0052] Step 2: Add 30% sodium hydroxide solution dropwise until all L-tyrosine is dissolved, pH = 11.80.

[0053] Step 3: Add 296.3 g (1.05 molar amount) of acetic anhydride dropwise over 30 minutes, while simultaneously adding 30% sodium hydroxide solution to maintain the pH between 8 and 10.

[0054] Step 4: After adding acetic anhydride, adjust the pH to 11.22 with sodium hydroxide solution and keep warm at 60℃ for 20 minutes.

[0055] Step 5: Add industrial hydrochloric acid to adjust the pH of the solution to 1.90.

[0056] Step 6: Control the vacuum to above 0.08 MPa and concentrate the reaction solution under reduced pressure at 60-80°C until it is basically in a solid state.

[0057] Step 7: After cooling to 4°C and allowing for full crystallization, the material is discharged to obtain crude N-acetyl-L-tyrosine I, and the mother liquor is discarded.

[0058] Step 8: Extract N-acetyl-L-tyrosine from the obtained crude N-acetyl-L-tyrosine product I by mixing it with 3 times its volume of ethanol (95%) at 60°C.

[0059] Step 9: Concentrate the ethanol solution obtained in Step 8 under reduced pressure to recover the solvent, and crystallize to obtain crude N-acetyl-L-tyrosine II. The recovered ethanol can be reused.

[0060] Step 10: Add crude N-acetyl-L-tyrosine II and an equal volume of purified water to a decolorization container. Stir and heat to 65°C to dissolve. Add activated carbon (0.5% of the solid weight of crude N-acetyl-L-tyrosine II) for decolorization for 10 minutes. Filter after decolorization. Cool the filtrate to crystallize. Cool the filtrate to 4°C and stir until crystals are completely precipitated. Filter and dry to obtain 499.85 g of purified N-acetyl-L-tyrosine. In continuous production, the crystallization mother liquor from the refining process can be reused in step 1.

[0061] After extraction in steps 11 and 8, the resulting filter residue is dissolved in water to remove inorganic salts and some impurities. The remaining insoluble substance is L-tyrosine, which can be reused in step 1 for continuous production.

[0062] Example 3

[0063] In this embodiment, N-acetyl-L-tyrosine was prepared according to the following steps:

[0064] Step 1: Disperse 100g of L-tyrosine in 100ml of water and stir quickly to avoid clumping.

[0065] Step 2: Add the high-quality mother liquor from Example 1 and the insoluble matter (L-tyrosine) after washing to the reaction vessel.

[0066] Step 3: Add 30% sodium hydroxide solution dropwise until all L-tyrosine is dissolved, pH=12.25.

[0067] Step 4: Add 59.2 g (1.05 molar amount) of acetic anhydride dropwise over 30 minutes, while simultaneously adding 30% sodium hydroxide solution to maintain the pH between 8 and 10.

[0068] Step 5: After adding acetic anhydride, adjust the pH to 10.99 with sodium hydroxide solution and keep warm at 60℃ for 30 minutes.

[0069] Step 6: Add industrial hydrochloric acid to adjust the pH of the solution to 1.87.

[0070] Step 7: Control the vacuum to above 0.08 MPa and concentrate the reaction solution at 80°C under reduced pressure until it is basically in a solid state.

[0071] Step 8: After cooling to 4℃ for full crystallization, the material is discharged to obtain crude N-acetyl-L-tyrosine I (a mixture containing N-acetyl-L-tyrosine, L-tyrosine, inorganic salts, and a small amount of acid / water). The mother liquor is discarded.

[0072] Step 9: Extract N-acetyl-L-tyrosine by mixing the obtained crude N-acetyl-L-tyrosine product I with 3 times its volume of ethanol (95%) at 60°C.

[0073] Step 10: Concentrate the ethanol solution obtained in Step 8 under reduced pressure to recover the solvent, and crystallize to obtain crude N-acetyl-L-tyrosine II. The recovered ethanol can be reused.

[0074] Step 11: Add crude N-acetyl-L-tyrosine II and an equal volume of purified water to a decolorization container. Stir and heat to 65°C to dissolve. Add activated carbon (0.5% of the solid weight of crude N-acetyl-L-tyrosine II) for decolorization for 10 minutes. Filter after decolorization. Cool the filtrate to crystallize. Cool the filtrate to 4°C and stir until crystals are completely precipitated. Filter and dry to obtain 118.15 g of purified N-acetyl-L-tyrosine. In continuous production, the crystallization mother liquor from the refining process can be reused in step 1.

[0075] After extraction in steps 12 and 8, the resulting filter residue is dissolved in water to remove inorganic salts and some impurities. The remaining insoluble substance is L-tyrosine, which can be reused in step 1 for continuous production.

[0076] Comparative Example 1

[0077] In this comparative example, N-acetyl-L-tyrosine was prepared according to the following steps:

[0078] Step 1: Suspend 100g of L-tyrosine in 200ml of water and stir quickly to avoid clumping.

[0079] Step 2: Add 30% sodium hydroxide solution dropwise until pH = 12.10 (L-tyrosine is completely dissolved).

[0080] Step 3: Add 59.2 g (1.05 molar amount) of acetic anhydride dropwise, while simultaneously adding 30% sodium hydroxide solution to maintain the pH between 8 and 10.

[0081] Step 4: After adding the acetic anhydride, adjust the pH to 11.75 with sodium hydroxide solution and keep warm for 20 minutes.

[0082] Step 5: Adjust the pH to 1.52 with hydrochloric acid.

[0083] Step 6: Control the vacuum to above 0.08 MPa and concentrate the reaction solution under reduced pressure at 80°C until crystals just begin to precipitate.

[0084] Step 7: After cooling to 4°C and allowing for full crystallization, the material is discharged to obtain crude N-acetyl-L-tyrosine, and the mother liquor is discarded.

[0085] Step 8: Add crude N-acetyl-L-tyrosine and purified water to a decolorization container. Stir and heat to 65°C, add 0.5% activated carbon for decolorization for 10 minutes, filter after decolorization, cool the filtrate to crystallize, cool the filtrate to 4°C and stir until the crystals are completely precipitated, filter, and dry to obtain 93.52 grams of purified N-acetyl-L-tyrosine.

[0086] Comparative Example 2

[0087] In this comparative example, N-acetyl-L-tyrosine was prepared according to the following steps:

[0088] Step 1: Disperse 100g of L-tyrosine in 200ml of water and stir quickly to avoid clumping.

[0089] Step 2: Add 30% sodium hydroxide solution dropwise until all L-tyrosine is dissolved, pH=12.05.

[0090] Step 3: Add 59.2 g (1.05 molar amount) of acetic anhydride dropwise over 30 minutes, while simultaneously adding 30% sodium hydroxide solution to maintain the pH between 8 and 10.

[0091] Step 4: After adding acetic anhydride, add sodium hydroxide solution dropwise to adjust the pH to 11.50, and keep warm at 60℃ for 20 minutes.

[0092] Step 5: Add industrial sulfuric acid to adjust the pH of the solution to 1.72.

[0093] Step 6: Control the vacuum to above 0.08 MPa and concentrate the reaction solution at 80°C under reduced pressure until it is basically in a solid state.

[0094] Step 7: After cooling to 4℃ for full crystallization, the material is discharged to obtain crude N-acetyl-L-tyrosine I (a mixture containing acetyltyrosine, tyrosine, inorganic salts, and a small amount of acid / water). The mother liquor is discarded.

[0095] Step 8: Extract N-acetyl-L-tyrosine by mixing the obtained crude N-acetyl-L-tyrosine product I with 3 times its volume of ethanol (95%) at 60°C.

[0096] Step 9: Concentrate the ethanol solution obtained in Step 8 under reduced pressure to recover the solvent, and crystallize to obtain crude N-acetyl-L-tyrosine II. The recovered ethanol can be reused.

[0097] Step 10: Add crude N-acetyl-L-tyrosine II and an equal volume of purified water to a decolorization container. Stir and heat to 65°C to dissolve. Add activated carbon (0.5% of the solid weight of crude N-acetyl-L-tyrosine II) for decolorization for 10 minutes. Filter after decolorization. Cool the filtrate to crystallize. Cool the filtrate to 4°C and stir until crystals are completely precipitated. Filter and dry to obtain 102.22 g of purified N-acetyl-L-tyrosine. In continuous production, the crystallization mother liquor from the refining process can be reused in step 1.

[0098] After extraction in steps 11 and 8, the resulting filter residue is dissolved in water to remove inorganic salts and some impurities. The remaining insoluble substance is L-tyrosine, which can be reused in step 1 for continuous production.

[0099] Comparative Example 3

[0100] In this comparative example, N-acetyl-L-tyrosine was prepared according to the following steps:

[0101] Step 1: Suspend 100g of L-tyrosine in 200ml of water and stir quickly to avoid clumping.

[0102] Step 2: Add 30% sodium hydroxide solution dropwise until pH = 12.00 (L-tyrosine is completely dissolved).

[0103] Step 3: Add 118.5 g (2 molar amounts) of acetic anhydride, while simultaneously adding 30% sodium hydroxide solution to maintain the pH between 8 and 10.

[0104] Step 4: After adding acetic anhydride, adjust the pH to 11.55 with sodium hydroxide solution and keep warm at 60℃ for 20 minutes.

[0105] Step 5: Adjust the pH to 1.51 with hydrochloric acid.

[0106] Step 6: Control the vacuum to above 0.08 MPa and concentrate the reaction solution at 80°C under reduced pressure until it is basically in a solid state.

[0107] Step 7: After cooling to 4°C and allowing for full crystallization, the material is discharged to obtain crude N-acetyl-L-tyrosine I, and the mother liquor is discarded.

[0108] Step 8: The solid from the previous step is slurried with 3 times the amount of ethanol (95%) at 60°C to extract N-acetyl-L-tyrosine.

[0109] Step 9: The ethanol solution obtained in the previous step is concentrated under reduced pressure to recover the solvent, and crystallization is performed to obtain crude N-acetyl-L-tyrosine II.

[0110] Step 10: Add crude N-acetyl-L-tyrosine II and purified water to a decolorization container. Stir and heat to 65°C to dissolve. Add 0.5% activated carbon for decolorization for 10 minutes. Filter after decolorization. Cool the filtrate to 4°C and stir until crystals are completely precipitated. Filter and dry to obtain 102.12 g of purified N-acetyl-L-tyrosine.

[0111] Comparative Example 4

[0112] In this comparative example, N-acetyl-L-tyrosine was prepared according to the following steps:

[0113] Step 1: Suspend 100g of L-tyrosine in 200ml of water and stir quickly to avoid clumping.

[0114] Step 2: Add 30% sodium hydroxide solution dropwise until pH = 12.12 (L-tyrosine is completely dissolved).

[0115] Step 3: Add 237 grams (4 molar amounts) of acetic anhydride, while simultaneously adding 30% sodium hydroxide solution to maintain the pH between 8 and 10.

[0116] Step 4: After adding acetic anhydride, adjust the pH to 10.88 with sodium hydroxide solution and keep warm at 60℃ for 20 minutes.

[0117] Step 5: Adjust the pH to 1.50 with hydrochloric acid.

[0118] Step 6: Control the vacuum to above 0.08 MPa and concentrate the reaction solution at 80°C under reduced pressure until it is basically in a solid state.

[0119] Step 7: After cooling to 4°C and allowing for full crystallization, the material is discharged to obtain crude N-acetyl-L-tyrosine I, and the mother liquor is discarded.

[0120] Step 8: The solid from the previous step is slurried with 3 times the amount of ethanol (95%) at 60°C to extract N-acetyl-L-tyrosine.

[0121] Step 9: The ethanol solution obtained in the previous step is concentrated under reduced pressure to recover the solvent, and crystallization is performed to obtain crude N-acetyl-L-tyrosine II.

[0122] Step 10: Add crude N-acetyl-L-tyrosine II and purified water to a decolorization container. Stir and heat to 65°C to dissolve. Add 0.5% activated carbon for decolorization for 10 minutes. Filter after decolorization. Cool the filtrate to crystallize. Cool the filtrate to 4°C and stir until the crystals are completely precipitated. Filter and dry to obtain 103.11 grams of purified N-acetyl-L-tyrosine.

[0123] Comparative Example 5

[0124] In this comparative example, N-acetyl-L-tyrosine was prepared according to the following steps:

[0125] 1) Add 60g of purified water, 10g of 40% sodium hydroxide solution, 100g of L-tyrosine and 20g of acetyl chloride to the reaction vessel. While stirring, gradually increase the temperature to 55℃ and stir for 2.0 hours. Monitor the endpoint according to the ninhydrin reaction. After the reaction is completed, send the reaction solution to the crystallization vessel.

[0126] 2) When the pH of the reaction solution was adjusted to 4.35 with industrial hydrochloric acid, the addition of hydrochloric acid was stopped, the temperature was lowered to 5°C, and after standing for 24 hours, crystallization was completed. After filtration, crude N-acetyl-L-tyrosine was obtained.

[0127] 3) Add crude N-acetyl-L-tyrosine to 50 ml of purified water and heat while stirring. At the same time, add 0.2% activated carbon and stir for 1 hour to decolorize. Then filter and crystallize at a constant temperature of 5℃ for 24 hours. After filtration, dry the solid at 45℃ for 3.5 hours to obtain 80.02 g of finished product.

[0128] Comparative Example 6

[0129] In this comparative example, N-acetyl-L-tyrosine was prepared according to the following steps:

[0130] 1) Add 600g of purified water, 100g of L-tyrosine and 67.6g of acetic anhydride to the reaction vessel, and gradually raise the temperature to liquid reflux while stirring. Keep the temperature and stir for 4.2 hours. Monitor the endpoint according to the ninhydrin reaction. After the reaction is completed, send the reaction solution to the crystallization kettle.

[0131] 2) Concentrate the reaction solution to dryness;

[0132] 3) N-acetyl-L-tyrosine was extracted with 400 mL of acetone, filtered, and the filtrate was desolventized with 400 mL of ethyl acetate to precipitate N-acetyl-L-tyrosine. After crystallization, the solid was filtered and dried at 45 °C for 3.5 hours to obtain 81.5 g of the product.

[0133] Example 4

[0134] 1. Research on related substances of refined products

[0135] The N-acetyl-L-tyrosine purified products obtained in Examples 1-3 and Comparative Examples 1-6 were tested using the method described in AJI 92 standard. The results of the test of relevant substances are shown in Table 1.

[0136] Table 1 Comparison of HPLC Detection Results of Related Substances in Refined Products

[0137]

[0138] As can be seen from Table 1, the content of related substances in the refined products of Examples 1-3 is significantly different from that in each pair of proportions.

[0139] Comparative Example 1 did not undergo an extraction step, meaning that unreacted tyrosine was not removed, resulting in a significantly higher tyrosine content in the refined product.

[0140] In Comparative Example 2, the pH of the reaction solution was adjusted with sulfuric acid, and the content of relevant substances in the refined product increased significantly.

[0141] Compared with Example 1, Comparative Examples 3 and 4 increased the amount of acetic anhydride used, by 2 times and 4 times the molar amount, respectively. The content of O-acetyl-L-tyrosine, O,N-diacetyl-L-tyrosine and other unknown impurities in the refined products increased significantly, and the content of impurities increased with the increase of acetic anhydride addition.

[0142] Comparative Example 5 used acetyl chloride as the acylation reagent. Due to the high activity of acetyl chloride, the content and composition of related substances in the purified product increased.

[0143] The acylation reaction in Comparative Example 6 was not carried out under alkaline conditions, and the content and composition of related substances in the refined product increased.

[0144] 2. Study on specific rotation and optical isomers of refined products

[0145] The specific rotation and optical isomers of the N-acetyl-L-tyrosine purified products obtained in Examples 1-3 and Comparative Examples 1-6 were detected, and the results are shown in Table 2. The specific rotation was detected using the AJI92 standard, and the optical isomer detection method is described in "A High Performance Liquid Chromatography Detection Method for Optical Isomers of N-acetyl-L-tyrosine" (Patent CN201510100898.4).

[0146] Table 2 Comparison of specific rotation and optical isomer test results of refined products

[0147] sample Specific curl / ° L-type / % Type D / % Example 1 48.5 99.95 0.05 Example 2 48.2 99.92 0.08 Example 3 48.1 99.91 0.09 Comparative Example 1 10.3 99.11 0.89 Comparative Example 2 46.3 97.70 2.30 Comparative Example 3 46.1 97.50 2.50 Comparative Example 4 17.5 63.50 36.5 Comparative Example 5 46.0 95.52 4.48 Comparative Example 6 46.2 96.01 4.99

[0148] As shown in Table 2, the optical purity of the purified products in Examples 1-3 was superior to that of the comparative examples. Specifically, Comparative Example 1, which did not remove unreacted L-tyrosine, had a significantly lower specific rotation than the examples. Comparative Example 2, which used sulfuric acid to adjust the pH of the reaction solution, significantly increased the N-acetyl-D-tyrosine content. Comparative Example 3, which added 2 molar amounts of acetic anhydride, significantly increased the N-acetyl-D-tyrosine content. Comparative Example 4, which added 4 molar amounts of acetic anhydride, significantly decreased the specific rotation and significantly increased the N-acetyl-D-tyrosine content. Comparative Example 5, which used D-type acyl chloride, experienced more vigorous side reactions, increased impurities, and lower optical purity. Comparative Example 6, which did not use an acid-binding agent (sodium hydroxide) to adjust the pH of the system and used acetic anhydride for prolonged incubation, resulted in decreased optical purity.

[0149] 3. Study on the physicochemical properties of refined products

[0150] The physicochemical properties of the N-acetyl-L-tyrosine purified products prepared in Examples 1-3 and Comparative Examples 1-5 were tested using the methods described in AJI 92 standard, and the results are shown in Table 3.

[0151] Table 3 Comparison of Physicochemical Test Results of Refined Products

[0152] sample Chloride ions / ppm sulfate ions / ppm Residue on ignition / % Example 1 ＜100 Not detected 0.03 Example 2 ＜100 Not detected 0.02 Example 3 ＜100 Not detected 0.04 Comparative Example 1 500 Not detected 0.09 Comparative Example 2 ＜100 400 0.55 Comparative Example 3 ＜100 Not detected 0.05 Comparative Example 4 ＜100 Not detected 0.05 Comparative Example 5 400 Not detected 0.06

[0153] As shown in Table 3, the purified products of Examples 1-3 had a chloride ion content of <100 ppm, no sulfate ions were detected, and the residue on ignition did not exceed 0.04%, indicating significantly better inorganic impurity content than the comparative examples. Specifically, Comparative Example 1 did not undergo an extraction step, resulting in a significant increase in chloride ion content. Comparative Example 2 used sulfuric acid to adjust the pH of the reaction solution, leading to a high sulfate residue. Comparative Example 5 used acyl chloride as the acylation reagent, resulting in a high chloride ion residue.

[0154] 4. Yield study of refined products

[0155] The yields of the N-acetyl-L-tyrosine purified products obtained in Examples 1-3 and Comparative Examples 1-6 were calculated, and the results are shown in Table 4.

[0156] Table 4 Comparison of the yield of refined products

[0157]

[0158]

[0159] As shown in Table 4, Comparative Example 5 used acyl chloride as the acylation reagent, and Comparative Example 6 did not undergo acylation under alkaline conditions, resulting in a significant decrease in the yield of the purified product. Example 3 adopted a continuous production process to prepare N-acetyl-L-tyrosine. After reusing the purified mother liquor and residual L-tyrosine, the yield was significantly better than that of the comparative examples.

[0160] 5. Research on other substances in refined products

[0161] The contents of other substances in the N-acetyl-L-tyrosine purified products obtained in Examples 1-3 were tested, and the results are shown in Table 5.

[0162] Table 5 Other test data for refined products

[0163] Testing items Example 1 Example 2 Example 3 ammonium salts Not detected Not detected Not detected iron <2ppm <2ppm <2ppm Loss on drying 0.02％ 0.02％ 0.04％ Heavy metals (as lead) <5ppm <5ppm <5ppm arsenic <1ppm <1ppm <1ppm Solvent residue (ethanol) 5ppm 4ppm 7ppm Transmission rate 99.1％ 99.2％ 98.9％ Pyrogen Meets requirements Meets requirements Meets requirements endotoxin <3 EU / g <3 EU / g <3 EU / g

[0164] As can be seen from Table 5, the N-acetyl-L-tyrosine prepared by the method of the present invention not only meets the AJI92 and AJI97 food standards, but also meets the pharmaceutical standards such as EP 9.0 and USP 40. Furthermore, the extremely low impurity level and ultra-high optical purity can also meet the needs of high-end customers.

[0165] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0166] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing N-acetyl-L-tyrosine, characterized by, The application relates to a method for preparing N-acetyl-L-tyrosine. The acylation reaction is carried out under alkaline conditions, and the pH value of the acylation liquid is adjusted by using alkali to increase the pH value and to keep the reaction, and then the pH value of the obtained reaction liquid is adjusted by using acid to obtain a reaction liquid containing N-acetyl-L-tyrosine. The reaction liquid containing N-acetyl-L-tyrosine is purified. During the acylation reaction, the pH value of the reaction liquid is controlled to be 8-10, and the pH value of the acylation liquid is adjusted by using alkali to increase the pH value to 10-12. The molar ratio of the L-tyrosine to acetic anhydride is 1:(0.9-1.5). The temperature of the keeping reaction is 60-70 DEG C, and the keeping reaction time is 15-30 min. The purification treatment comprises the following steps: The reaction liquid containing N-acetyl-L-tyrosine is concentrated and crystallized to collect the crystalline substance and obtain N-acetyl-L-tyrosine crude product I. The N-acetyl-L-tyrosine crude product I is extracted to collect the extraction liquid. The extraction liquid is concentrated to obtain N-acetyl-L-tyrosine crude product II. The extraction agent used in the extraction treatment is an alcohol solvent. The alkali is selected from a sodium hydroxide solution, and the acid is selected from hydrochloric acid.

2. The method of claim 1, wherein, The pH value of the reaction liquid containing N-acetyl-L-tyrosine is 1.5-2.

2.

3. The method of claim 1, wherein, The acylation reaction time is 20-60 min.

4. The method of claim 1, wherein, The extraction agent used in the extraction treatment is methanol or ethanol.

5. The method of claim 1, wherein, The extraction treatment temperature is 40-70 DEG C.

6. The method of claim 1, wherein, The addition amount of the extraction agent used in the extraction treatment is 2-10 times the weight of the N-acetyl-L-tyrosine crude product I.

7. The method of claim 1, wherein, The application further comprises the following steps:

8. The method of claim 1, wherein, The insoluble substance obtained through the extraction treatment is washed with water to collect the insoluble substance. The application further comprises the following steps:

9. The method of claim 1, wherein, The N-acetyl-L-tyrosine crude product II is subjected to decolorization treatment to obtain a decolorized liquid. The decolorized liquid is crystallized to collect the crystalline substance, which is washed with water to obtain N-acetyl-L-tyrosine fine product. ​

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