Preparation method of high-purity Ectoine

Abstract

The invention belongs to the technical field of organic chemistry, and discloses a preparation method of high-purity ectoine, which comprises the following steps: by taking L-glutamine as a raw material, carrying out amino protection under an alkaline condition, carrying out Hofmann degradation carbonyl removal reaction without separation, carrying out secondary Boc protection without separation, and carrying out double-Boc protection on L-2, 4, 6-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5- The method comprises the following steps: extracting L-2, 4-diaminobutyric acid into an organic phase by using an organic solvent, directly introducing a proper amount of hydrogen chloride gas into the combined organic phase, and simultaneously completing deprotection and separating out an L-2, 4-diaminobutyric acid dihydrochloride intermediate. The preparation process has the characteristics of being high in yield, simple and convenient in post-treatment, environment-friendly, high in purity and the like; the separated-out L-2, 4-diaminobutyric acid dihydrochloride solid does not need to be refined and is directly subjected to cyclization reaction with trimethyl orthoacetate to generate the target product Ectoine. The preparation method provided by the invention is low in production cost, efficient and environment-friendly.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis technology and relates to a method for preparing high-purity ectoine. Specifically, it relates to a simple, industrial-grade technical solution for the preparation, separation, and purification of high-purity ectoine in a cosmetic-grade manner. Background Technology

[0002] Ectoine, also known as tetrahydromethylpyrimidine carboxylic acid, is a white crystalline powder. It is a natural, highly soluble, low-molecular-weight zwitterionic cyclic amino acid derivative. It is a compatible solute, also called an extreme solute, first discovered in halophilic bacteria for stabilizing cellular physiological properties and protecting cells from high salinity, high temperature, dehydration, and ultraviolet radiation. Ectoine possesses cell-protective, anti-inflammatory, and anti-allergic activities, exhibiting high efficacy and safety, and is currently mainly used in the cosmetics industry. Multiple studies have shown that ectoine, as an enzyme stabilizer and cell protectant, has properties including protection against stress mediators, natural cell protection, lubrication, and hydration, making it promising for applications in the pharmaceutical and food industries. Currently, it is widely used in the skin and food sectors. Its preparation methods are mainly divided into fermentation and organic synthesis. Compared with traditional fermentation processes, chemical synthesis is more efficient, produces less waste, is more environmentally friendly, and the synthesized ectoine has a lower bioburden, especially in terms of endotoxins, making it easier for the pharmaceutical industry to select for application and new product development.

[0003] Currently, the main method for chemically synthesizing ectoine is to react L-2,4-diaminobutyrate and triethyl orthoacetate in an alcohol solvent at a reaction temperature of 50-80℃, and then obtain the target product through a one-step cyclization process. For example, invention patents JP03031265 A1 and KR2570658 B1; compared to the "one-step method", invention patent CN202310286517.0 uses 2,4-diaminobutyric acid as a raw material, reacts with acetyl chloride to obtain ectoine intermediate, and then reacts with a strong base to obtain crude ectoine, adopting a "two-step method" to prepare ectoine. This process requires anhydrous conditions and uses a strong base reaction, which is more demanding than the "one-step method". In contrast, invention patent CN201810002838.2 uses L-2,4-diaminobutyric acid dihydrochloride protected by acetic anhydride as a raw material and uses a strong base cyclization under anhydrous conditions to obtain the target product. The reaction temperature is 50-120℃, and the reaction conditions are more intense and demanding.

[0004] Based on the analysis of the above schemes, it can be found that the one-step production of ectoine using L-2,4-diaminobutyric acid hydrochloride as a raw material and triethyl orthoacetate has the advantages of fewer process steps, smaller equipment investment, mild reaction conditions, and high conversion rate, making it a current research hotspot. On the other hand, L-2,4-diaminobutyric acid dihydrochloride, a key intermediate in the preparation of ectoine, is an important pharmaceutical intermediate with wide applications, stable chemical properties, readily available raw materials, mild reaction conditions, and controllable costs.

[0005] Taking invention patents CN201510992842.4 and CN201910683022.5 as examples, the preparation method of L-2,4-diaminobutyric acid salt is summarized, including the following steps: S1: L-glutamine is protected with Boc under alkaline conditions to obtain an aqueous solution of N-Boc-L-glutamine; S2: Saturated sodium hypochlorite solution is added dropwise to the N-Boc-L-glutamine aqueous solution obtained in step S1 to carry out a degradation reaction, and a crude solution of L-2-N-Boc-4-aminobutyric acid is obtained; S3: The crude solution of L-2-N-Boc-4-aminobutyric acid obtained in step S2 is concentrated for later use. In invention patent CN201510992842.4, it is extracted with ethyl acetate five times, the ethyl acetate is combined, washed three times with saturated brine, and then the ethyl acetate is evaporated to separate solid tert-butoxycarbonyl-L- Glutamine, ethyl acetate, tetrahydrofuran, and water were added and cooled to 0°C. Iodobenzene diacetate was added, and the reaction was carried out at room temperature for 12 hours. After the reaction was complete, the aqueous layer was separated, and the organic layer was washed three times with water. The aqueous layers were combined, and water was distilled under reduced pressure with anhydrous ethanol and stirred to crystallize. The mixture was filtered, washed twice with ethanol, dried under vacuum, and dried at 60°C to obtain solid L-2-N-tert-butoxycarbonyl-4-aminobutyric acid. The solid was heated under reflux in water to remove protection, yielding free L-2,4-diaminobutyric acid, with a total yield of 21.8%. This preparation process suffers from high energy consumption, low yield, and cumbersome post-processing. Another invention patent, CN201910683022.5, concentrates the crude L-2-N-Boc-4-aminobutyric acid solution obtained in step S2 and then adjusts the pH with 2N hydrochloric acid. Desalination was then performed using cation exchange resin, with dilute ammonia as the eluent. The eluent was then concentrated, and concentrated hydrochloric acid was added to adjust the pH. Anhydrous ethanol was then added, followed by crystallization, filtration, and drying to obtain L-2,4-diaminobutyrate. This desalination process using ion exchange resin generates a large amount of acidic wastewater. The eluent is then concentrated after elution with ammonia, and the pH is adjusted with hydrochloric acid before adding anhydrous ethanol for crystallization. The highest yield is 85%, but this process also faces the challenges of high equipment costs, high energy consumption, low yield, and the generation of wastewater containing large amounts of inorganic salts. Furthermore, methanol is generated as a byproduct in the final cyclization step to form the target product. Current processes often use methanol and ethanol as reaction solvents, which, from a reaction kinetics perspective, is unfavorable for the forward reaction. The low boiling points of the reaction solvents are also unsuitable for byproduct removal, reducing the reaction rate. Additionally, excessively long reaction times are detrimental to maintaining the stereochemical structure. Summary of the Invention

[0006] To address the aforementioned technical deficiencies, the present invention aims to provide a low-cost, efficient, and environmentally friendly method for preparing ectoine.

[0007] In order to overcome the defects of the prior art, the present invention provides a method for preparing ectoine that is easy to obtain, produces high-purity products, is simple to operate, environmentally friendly, has low preparation cost, and is suitable for industrial production. Its key features include using L-glutamine as a raw material, first protecting it with Boc, then removing the carbonyl group through Hoffmann degradation without separation, and directly performing a second amino protection. This significantly reduces the polarity of the pre-protected compound, thereby increasing the efficiency of organic solvent extraction and significantly reducing the amount of extractant and the number of extractions. After extraction, hydrogen chloride gas is directly introduced into the organic phase to directly deprotect and precipitate L-2,4-diaminobutyric acid dihydrochloride solid. This key process significantly reduces the polarity of the intermediate, allowing it to enter the organic phase, avoiding complex and cumbersome dehydration and desalination processes and the generation of large amounts of acidic wastewater. The organic solvent can be recycled and reused, featuring high yield, simple post-processing, and environmental friendliness, which is the core innovation of this invention. The L-2,4-diaminobutyric acid dihydrochloride solid then reacts with trimethyl orthoacetate in the reaction solvent to undergo a cyclization reaction to generate crude ectoine. The crude ectoine is recrystallized in a crystallization solvent to obtain high-purity ectoine. The reaction consists of five steps, and the equations are as follows:

[0008] A method for preparing high-purity ectoine involves first protecting the α-amino group of L-glutamine with an amino protecting agent under alkaline conditions, then adding a decarbonylating agent to induce a Hofmann degradation reaction to remove the carbonyl group of the amide group. The 4-amino group is then protected with Boc anhydride or chloroformate to obtain L-2,4-bis((tert-butoxycarbonyl)amino)butyric acid. After extraction and separation, hydrogen chloride gas is passed through the organic phase to complete the removal of bis-Boc protection and salt formation to obtain the key intermediate L-2,4-diaminobutyric acid dihydrochloride. This intermediate then undergoes a cyclization reaction with trimethyl orthoacetate in a reaction solvent. During the incubation process, the generated byproduct methanol is removed, promoting rapid reaction and yielding the target product, ectoine.

[0009] The above-mentioned method for preparing high-purity ectoine is as follows: S1: Under alkaline conditions, Boc anhydride is added dropwise to L-glutamine to react and obtain an aqueous solution of N-Boc-L-glutamine. S2: Under stirring conditions, the N-Boc-L-glutamine aqueous solution obtained in step S1 is added dropwise to an alkaline solution containing a decarbonylating agent to carry out the Hoffmann degradation decarbonylation reaction. After the reaction is completed, the reaction solution is cooled to room temperature, and an appropriate amount of sodium bisulfite is added to quench excess sodium hypochlorite to obtain an L-2-N-Boc-4-aminobutyric acid solution. S3: The amino protecting agent solution is added dropwise to the solution obtained in S2 to react and obtain L-2,4-bis((tert-butoxycarbonyl)amino)butyric acid solution. After desolvation under reduced pressure, an extractant is added and the organic phases are combined to obtain L-2,4-bisBocaminobutyric acid organic solution. S4: Under stirring conditions, hydrogen chloride gas is slowly introduced into the organic solution of L-2,4-bis((tert-butoxycarbonyl)amino)butyric acid to carry out the reaction. After the deprotection of Boc is completed, the solid is directly precipitated as a salt. After filtration and drying, L-2,4-diaminobutyric acid dihydrochloride solid is obtained. S5: Under stirring conditions, L-2,4-diaminobutyric acid dihydrochloride and trimethyl orthoacetate undergo a cyclization reaction in a solvent. During the reaction, methanol, a byproduct, is removed to promote rapid reaction. After the reaction is completed, the product is cooled to precipitate, filtered, and dried to obtain crude ectoine. Then, high-purity ectoine product is obtained by recrystallization.

[0010] In the above-mentioned method for preparing high-purity ectoine, the amino protecting agent is one or more of Boc anhydride, tert-butyl chloroformate, isopropyl chloroformate, ethyl chloroformate, and methyl chloroformate.

[0011] In the above-mentioned method for preparing high-purity ectoine, the decarbonylating agent is sodium hypochlorite.

[0012] In the above-mentioned method for preparing high-purity ectoine, in step S1, sodium hydroxide is added under alkaline conditions, and the molar ratio of L-glutamine:Boc anhydride:sodium hydroxide is 1:1.1-1.5:1-2, preferably 1:1.2:1.1. The reaction temperature is 0~30℃, preferably 10℃; The protected reaction solvent includes one or more of the following: water, dichloroethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetonitrile, methanol, and ethanol, preferably a mixture of 1,4-dioxane and water.

[0013] In the above-mentioned method for preparing high-purity ectoine, in step S2, Based on L-glutamine, the molar ratio of L-glutamine:sodium hypochlorite:sodium hydroxide:sodium bisulfite is 1:1.1~2:3~5:0.1~0.5, preferably 1:1.5:4:0.2; The reaction temperature for the Hoffmann degradation decarbonylation reaction is -5 to 60℃, preferably 55±2℃.

[0014] The pH value of the Hoffmann degradation decarbonylation reaction is controlled at 9-13, preferably at 11±0.5.

[0015] In the above-mentioned method for preparing high-purity ectoine, in step S3, the molar ratio of L-glutamine to Boc anhydride or chloroformate is 1:1.1~1.5, preferably 1:1.1, based on L-glutamine. The reaction temperature is 5~30℃, preferably 10±2℃; The pH value of the reaction is controlled at 7~9, preferably 8±0.5; The amino protecting agent is one or more of Boc anhydride, tert-butyl chloroformate, isopropyl chloroformate, ethyl chloroformate, and methyl chloroformate, with Boc anhydride being preferred. The extractant used is one or more of dichloromethane, ethyl acetate, methyl acetate, butyl acetate, butanone, and methyl isobutyl ketone, with ethyl acetate being preferred.

[0016] In the above-mentioned method for preparing high-purity ectoine, in step S4, the molar ratio of L-glutamine to hydrogen chloride is 1:4~8, preferably 1:4.5, based on L-glutamine. The reaction is carried out at a temperature controlled between 5 and 30°C, preferably 10 ± 2°C.

[0017] In the above-mentioned method for preparing high-purity ectoine, in step S5, the molar ratio of L-2,4-diaminobutyric acid dihydrochloride to triethyl orthoacetate is 1:2~5, preferably 1:4; The solvent for the reaction is one or more of methanol, ethanol, isopropanol, tert-butanol, water, toluene, and xylene, with isopropanol being preferred.

[0018] The recrystallization solvent is one or more of methanol, ethanol, isopropanol, tert-butanol, and water, with methanol being preferred.

[0019] The beneficial effects of this invention are: This invention uses inexpensive and readily available L-glutamine with its own chiral structure as a raw material. First, after Boc protection, it undergoes a Hoffmann degradation decarbonylation reaction without separation. These two steps are mild and easily controlled. The reaction solution is then directly protected with the decarbonylated amino group without separation. All three steps are performed without separation, and the mild reaction conditions maintain the chiral structure while avoiding product loss during separation, significantly improving preparation efficiency. Furthermore, the protection of the diamino group of L-2,4-diaminobutyric acid greatly enhances the lipid solubility of the L-2,4-diamino acid, avoiding the use of ion exchange resins for desalting in aqueous extraction processes. Organic solvent extraction completely solves the problem of increased extraction efficiency due to the high polarity of the pre-protected compound, significantly reducing the amount of extractant and the number of extractions, requiring only three conventional organic solvent extractions. The combined organic phases are then directly... By introducing hydrogen chloride gas, high-quality L-2,4-diaminobutyric acid dihydrochloride solid is directly precipitated after deprotection, with a total yield of up to 95%. This key process significantly improves the yield and post-processing efficiency, avoiding complex and cumbersome dehydration and desalination processes and the generation of large amounts of acidic wastewater. The extractant can be recycled. This process features high yield, simple post-processing, and environmental friendliness, which is the core innovation of this invention. Simultaneously, the L-2,4-diaminobutyric acid dihydrochloride solid is then reacted with trimethyl orthoacetate. In the optimization of the reaction solvent, isopropanol is used as the reaction solvent, which increases the reflux temperature and accelerates the separation of the reaction byproduct methanol, thereby increasing the reaction rate. On the other hand, the use of isopropanol with its branched structure is beneficial to the preservation of the chiral structure of ectoine. After recrystallization and purification of the crude product, the purity of ectoine can reach 99.8% (HPLC detection), with an optical rotation D20 +179° (c = 1.0 in CH3OH). Attached Figure Description

[0020] Figure 1 This is the H-NMR spectrum of ectoine prepared in Example 1.

[0021] Figure 2 This is the high-performance liquid chromatography (HPLC) spectrum of ectoine prepared in Example 1.

[0022] Figure 3 This is the infrared spectrum of ectoine prepared in Example 1. Example 1

[0023] S1:

[0024] Add 120 kg of water to a 500 L reactor, start stirring, add 11 kg (0.26 kmol) of sodium hydroxide in batches until completely dissolved, and add 35 kg (0.24 kmol) of L-glutamine while maintaining the water temperature at 5-10℃ and stir until completely dissolved. Add 62.7 kg (0.29 kmol) of powdered Boc anhydride in batches to the reaction system, maintain the reaction temperature at 10±2℃, and react for 8-9 hours. Check that the reaction is complete, and set aside the S1 reaction solution for later use.

[0025] S2:

[0026] 382 kg of 7% sodium hypochlorite aqueous solution (0.36 kmol) was added to a 1000 L reactor. The temperature was controlled at -5 to 0 °C. Under stirring, 38.4 kg of sodium hydroxide (0.96 kmol) was slowly added in three batches, keeping the temperature no higher than 10 °C. The pH value was measured to be 11 ± 0.5. The S1 reaction solution was slowly added dropwise to the reactor. After the addition was completed, the temperature was raised to 55 ± 2 °C and stirred for 2 hours. Gas was generated, indicating that the reaction was complete. The temperature was then lowered to room temperature, and 5 kg (0.048 kmol) of sodium bisulfite was added. Stirring was continued for 1 hour to prepare the S2 reaction solution for later use.

[0027] S3:

[0028] Dissolve 68 kg (0.31 kmol) of Boc anhydride in 45 kg of 1,4-dioxane and add it dropwise to the S2 reaction solution at 20 ± 2 °C. React for 2-4 hours, then check for the end of the reaction. After desolvation under reduced pressure at 35-40 °C, cool to 5-10 °C and adjust the pH to 7 ± 0.5 with concentrated hydrochloric acid. Add 140 kg of ethyl acetate, stir, and allow to stand for separation. Extract the aqueous phase with 70 kg of ethyl acetate, and combine the organic phases to obtain the S3 ethyl acetate solution for later use.

[0029] S4: In a 500-liter reactor, the ethyl acetate solution of S3 was cooled to 10±2℃, stirring was started, and 40 kg (1.1 kmol) of dry HCl gas was slowly introduced. Solid precipitated. After the reaction was complete, the mixture was separated by centrifugation. The solid was washed with a small amount of ethyl acetate and dried at 50℃ to obtain 43.5 kg of L-2,4-diaminobutyric acid dihydrochloride crystalline powder, with a yield of 95%. [α]20 / D +15° (c=3.5% H2O 25℃) S5:

[0030] Add 100 kg of isopropanol to a 300 L reactor, start stirring, add 20 kg (0.105 kmol) of L-2,4-diaminobutyric acid dihydrochloride, heat to 80 °C, add 50.3 kg (0.42 kmol) of trimethyl orthoacetic acid in batches, react for 10-12 hours, distill off the methanol solution produced in the reaction, check that the reaction is complete, cool to room temperature, separate by centrifugation, wash the solid with a small amount of isopropanol, dry the solid at 50 °C for 4 hours, the crude product weighs about 14.1 kg, yield 90%; after recrystallization from methanol, the purity of the crude product is 99.8%.

[0031] Figure 1 This is the ¹H NMR spectrum of ectoin. ¹H-NMR (D₂O): 3.87-3.89 (¹H, t), 3.25-3.29 (¹H, m), 3.08-3.13 (¹H, m), 2.05 (³H, s), 1.88-1.99 (²H, m).

[0032] HPLC detection such as Figure 2 As shown, the high performance liquid chromatography (HPLC) conditions are as follows: Column: DIKMA Diamonsil 5μm C18(2), 250×4.6mm; Mobile phase: A methanol: B potassium dihydrogen phosphate aqueous solution (0.05mol / L, pH=3); Mobile phase ratio: A:B=25:75; Detection wavelength: 210nm; Mobile phase flow rate: 0.5ml / min; Injection volume: 10μl; Column temperature: 30℃; Detection time: 20min; Sample concentration: 15mg sample was diluted to 25ml with chromatographic methanol.

[0033] Figure 3 This is the infrared spectrum of ectoine, with the following data: IR (KBr, cm⁻¹): 3506.6, 3188.3, ​​3034.0, 2808.4, 1597.1, 1498.7, 1390.7, 1313.5, 1209.4, 1136.1, 902.7, 808.2, 651.9. Melting point: 323.1-324.3℃. Optical rotation D²⁰ +179° (c = 1.0 in CH₃OH). Example 2

[0034] S1:

[0035] Add 130 kg of water to a 500 L reactor and start stirring. Add 11.5 kg (0.29 kmol) of sodium hydroxide in batches until completely dissolved. While maintaining the water temperature at 20 ± 2 °C, add 35 kg (0.24 kmol) of L-glutamine and stir until completely dissolved. Dissolve 75 kg (0.34 kmol) of Boc anhydride in 50 kg of tetrahydrofuran and slowly and uniformly add it to the reactor while maintaining the temperature at 10-25 °C. React for 8-9 hours and check that the reaction is complete. Set aside the S1 reaction solution for later use.

[0036] S2:

[0037] Add 510 kg of 7% sodium hypochlorite aqueous solution (0.43 kmol) to a 1000 L reactor. Control the temperature at -5 to 0 °C and add 48 kg of sodium hydroxide (1.2 kmol) in three batches under stirring. Keep the temperature below 10 °C and measure the pH value as 11-13. Slowly add the S1 reaction solution dropwise into the reactor. After the dropwise addition is complete, raise the temperature to 58 ± 2 °C and stir for 2 hours. Check that the reaction is complete. Cool to room temperature and add 5 kg (0.05 kmol) of sodium bisulfite and stir for 1 hour to prepare the S2 reaction solution for later use.

[0038] S3:

[0039] Dissolve 78.6 kg (0.36 kmol) of Boc anhydride in 80 kg of tetrahydrofuran solvent, and add it dropwise to the S2 reaction solution at 10 ± 2 °C. React for 2-4 hours, then check for the end of the reaction. After desolvation under reduced pressure at 30-45 °C, cool to 5-10 °C and adjust the pH to 6-8 with concentrated hydrochloric acid. Add 200 kg of dichloromethane, stir, and allow to stand for separation. Extract the aqueous phase with 100 kg of dichloromethane, and combine the organic phases (S3 dichloromethane solution) for later use.

[0040] S4:

[0041] In a 500-liter reactor, the S3 dichloromethane solution was cooled to 20±2℃. While stirring, 44.5 kg (1.22 kmol) of dry HCl was slowly passed through, resulting in solid precipitation. The precipitate was separated by centrifugation, washed with a small amount of dichloromethane, and dried at 50℃ to obtain 41.2 kg of L-2,4-diaminobutyric acid dihydrochloride crystalline powder, with a yield of 90%. [α]20 / D +14.5° (c=3.5%H2O 25℃) S5:

[0042] Add 100 kg of anhydrous ethanol to a 300 L reactor, start stirring, add 21.5 kg (0.11 kmol) of L-2,4-diaminobutyric acid dihydrochloride, heat to reflux, and add 62.9 kg (0.52 kmol) of trimethyl orthoacetic acid in batches. React for 10-12 hours, check for complete reaction, cool to room temperature, filter by centrifugation, wash the solid with a small amount of anhydrous ethanol, dry the solid at 50 °C for 4-5 hours, the crude product weighs about 13.3 kg, yield 85%; after recrystallization from methanol, the purity of the crude product is 99.2% (HPLC detection), optical rotation D20 +170° (c = 1.0 in CH3OH). Example 3

[0043] S1:

[0044] Add 120 kg of water to a 500 L reactor, start stirring, add 11 kg (0.26 kmol) of sodium hydroxide in batches until completely dissolved, and add 35 kg (0.24 kmol) of L-glutamine while maintaining the water temperature at 5-10℃ and stir until completely dissolved. Add 62.7 kg (0.29 kmol) of powdered Boc anhydride in batches to the reaction system, maintain the reaction temperature at 15±2℃, and react for 5-6 hours. Check that the reaction is complete, and set aside the S1 reaction solution for later use.

[0045] S2:

[0046] 382 kg of 7% sodium hypochlorite aqueous solution (0.36 kmol) was added to a 1000 L reactor. The temperature was controlled at -5 to 0 °C. Under stirring, 38.4 kg of sodium hydroxide (0.96 kmol) was slowly added in three batches, keeping the temperature no higher than 10 °C. The pH value was measured to be 11 ± 0.5. The S1 reaction solution was slowly added dropwise to the reactor. After the addition was completed, the temperature was raised to 55 ± 2 °C and stirred for 2 hours. Gas was generated, indicating that the reaction was complete. The temperature was then lowered to room temperature, and 10 kg (0.096 mol) of sodium bisulfite was added. Stirring was continued for 1 hour to prepare the S2 reaction solution for later use.

[0047] S3:

[0048] Cool the S2 reaction solution in the 1000L reactor from the previous step to 5-10℃, and add 32.3kg (0.26kmol) of isopropyl chloroformate dropwise over 2-4 hours until the reaction is complete. Then, heat to 35-40℃ and maintain this temperature for 1-2 hours. Cool to 5-10℃, adjust the pH to 7±0.5 with concentrated hydrochloric acid, add 140kg of ethyl acetate, stir, and allow to stand for separation. Extract the aqueous phase with 70kg of ethyl acetate, and combine the organic phases to obtain the S3 ethyl acetate solution for later use.

[0049] S4: In a 500-liter reactor, the ethyl acetate solution of S3 was cooled to 10±2℃, stirring was started, and 40 kg (1.10 kmol) of dry HCl gas was slowly introduced. Solid precipitated. After the reaction was complete, the mixture was separated by centrifugation. The solid was washed with a small amount of ethyl acetate and dried at 50℃ to obtain 42.6 kg of L-2,4-diaminobutyric acid dihydrochloride crystalline powder, with a yield of 93%. [α]20 / D +15° (c=3.5% H2O 25℃) S5:

[0050] 100 kg of methanol was added to a 300 L reactor, and stirring was started. 20 kg (0.11 kmol) of L-2,4-diaminobutyric acid dihydrochloride was added, and the mixture was heated to reflux. 50.3 kg (0.42 kmol) of trimethyl orthoacetic acid was added in portions, and the reaction was allowed to proceed for 10-12 hours. The methanol solution produced during the reaction was distilled off, and the reaction was checked for completion. The mixture was cooled to room temperature, centrifuged, and the solid was washed with a small amount of methanol. The solid was then dried at 50 °C for 4 hours using a forced-air drying method. The crude product weighed approximately 12.67 kg, with a yield of 81%. After recrystallization from methanol, the crude product had a purity of 99.1% and an optical rotation D20 of +171° (c = 1.0 in CH3OH).

Claims

1. A method for preparing high-purity ectoine, characterized in that, Under alkaline conditions, the α-amino group of L-glutamine is first protected with an amino protecting agent, and then a decarbonylating agent is added to induce a Hofmann degradation reaction to remove the carbonyl group of the amide group. The 4-amino group is then protected with Boc anhydride or chloroformate to obtain L-2,4-bis((tert-butoxycarbonyl)amino)butyric acid. After extraction and separation, hydrogen chloride gas is passed through the organic phase to complete the deprotection of the bis-Boc group and the salt formation to obtain the key intermediate of L-2,4-diaminobutyric acid dihydrochloride. The intermediate then undergoes a cyclization reaction with trimethyl orthoacetate in the reaction solvent. During the heat treatment process, the generated byproduct methanol is removed, which promotes the rapid progress of the reaction and yields the target product, ectoin.

2. The method for preparing high-purity ectoine according to claim 1, characterized in that, The specific preparation method is as follows: S1: Under alkaline conditions, Boc anhydride is added dropwise to L-glutamine to react and obtain an aqueous solution of N-Boc-L-glutamine. S2: Under stirring conditions, the N-Boc-L-glutamine aqueous solution obtained in step S1 is added dropwise to an alkaline solution containing a decarbonylating agent to carry out the Hoffmann degradation decarbonylation reaction. After the reaction is completed, the reaction solution is cooled to room temperature, and an appropriate amount of sodium bisulfite is added to quench excess sodium hypochlorite to obtain an L-2-N-Boc-4-aminobutyric acid solution. S3: The amino protecting agent solution is added dropwise to the solution obtained in S2 to react and obtain L-2,4-bis((tert-butoxycarbonyl)amino)butyric acid solution. After desolvation under reduced pressure, an extractant is added and the organic phases are combined to obtain L-2,4-bisBocaminobutyric acid organic solution. S4: Under stirring conditions, hydrogen chloride gas is slowly introduced into the organic solution of L-2,4-bis((tert-butoxycarbonyl)amino)butyric acid to carry out the reaction. After the deprotection of Boc is completed, the solid is directly precipitated as a salt. After filtration and drying, L-2,4-diaminobutyric acid dihydrochloride solid is obtained. S5: Under stirring conditions, L-2,4-diaminobutyric acid dihydrochloride and trimethyl orthoacetate undergo a cyclization reaction in a solvent. During the reaction, methanol, a byproduct, is removed to promote rapid reaction. After the reaction is completed, the product is cooled to precipitate, filtered, and dried to obtain crude ectoine. Then, high-purity ectoine product is obtained by recrystallization.

3. The method for preparing high-purity ectoine according to claim 2, characterized in that, The amino protecting agent is one or more of Boc anhydride, tert-butyl chloroformate, isopropyl chloroformate, ethyl chloroformate, and methyl chloroformate.

4. The method for preparing high-purity ectoine according to claim 2, characterized in that, The decarbonylating agent is sodium hypochlorite.

5. The method for preparing high-purity ectoine according to claim 2, characterized in that, In S1, the alkaline conditions involve the addition of sodium hydroxide, and the molar ratio of L-glutamine:Boc anhydride:sodium hydroxide is 1:1.1-1.5:1-2, preferably 1:1.2:1.

1. The reaction temperature is 0~30℃, preferably 10℃; The protected reaction solvent includes one or more of the following: water, dichloroethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetonitrile, methanol, and ethanol, preferably a mixture of 1,4-dioxane and water.

6. The method for preparing high-purity ectoine according to claim 5, characterized in that, In S2, Based on L-glutamine, the molar ratio of L-glutamine:sodium hypochlorite:sodium hydroxide:sodium bisulfite is 1:1.1~2:3~5:0.1~0.5, preferably 1:1.5:4:0.2; The reaction temperature for the Hoffmann degradation decarbonylation reaction is -5 to 60℃, preferably 55±2℃; The pH value of the Hoffmann degradation decarbonylation reaction is controlled at 9-13, preferably at 11±0.

5.

7. The method for preparing high-purity ectoine according to claim 2, characterized in that, In S3, the molar ratio of L-glutamine to Boc anhydride or chloroformate, calculated as L-glutamine, is 1:1.1 to 1.5, preferably 1:1.1; The reaction temperature is 5~30℃, preferably 10±2℃; The pH value of the reaction is controlled at 7~9, preferably 8±0.5; The amino protecting agent is one or more of Boc anhydride, tert-butyl chloroformate, isopropyl chloroformate, ethyl chloroformate, and methyl chloroformate, with Boc anhydride being preferred. The extractant used is one or more of dichloromethane, ethyl acetate, methyl acetate, butyl acetate, butanone, and methyl isobutyl ketone, with ethyl acetate being preferred.

8. The method for preparing high-purity ectoine according to claim 2, characterized in that: In S4, the molar ratio of L-glutamine to hydrogen chloride is 1:4~8, preferably 1:4.5, based on L-glutamine. The reaction is carried out at a temperature controlled between 5 and 30°C, preferably 10 ± 2°C.

9. The method for preparing high-purity ectoine according to claim 2, characterized in that: In S5, the molar ratio of L-2,4-diaminobutyric acid dihydrochloride to triethyl orthoacetate is 1:2-5, preferably 1:4; The solvent for the reaction is one or more of methanol, ethanol, isopropanol, tert-butanol, water, toluene, and xylene, with isopropanol being preferred. The recrystallization solvent is one or more of methanol, ethanol, isopropanol, tert-butanol, and water, with methanol being preferred.

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