Industrial preparation method of levocarnitine

Abstract

The present invention relates to the technical field of pharmaceutical and chemical synthesis, and particularly to an industrial preparation method of levocarnitine. The method comprises the following steps: adding an Escherichia coli engineered bacterium cell containing carbonyl reductase and glucose dehydrogenase, anhydrous glucose, and coenzyme into a buffer salt solution, dropping ethyl 4-chloroacetoacetate for reaction, and obtaining ethyl (R)-4-chloro-3-hydroxybutyrate through extraction, concentration under reduced pressure, and high-vacuum distillation; dropping ethyl (R)-4-chloro-3-hydroxybutyrate into an aqueous solution of trimethylamine in the presence of an inorganic base, keeping the temperature for reaction, adjusting the pH with an acid first and then with a base after the reaction ends; and obtaining the levocarnitine compound through electrodialysis for desalting. The present invention uses a whole-cell catalysis method, which has stronger adaptability; does not use a cosolvent, is green and environmentally friendly, has little influence on enzyme activity, and has higher optical purity of the product. By changing the addition mode of the intermediate, the reaction is more complete and the yield is higher. The electrodialysis for desalting has good effect, high efficiency, and is more suitable for industrialization.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical and chemical synthesis, and particularly relates to an industrial preparation method of levocarnitine. Background Art

[0002] Levocarnitine, also known as L-carnitine, chemical name: (R)-3-carboxy-2-hydroxy-N,N,N-trimethyl-1-ammonium hydroxide inner salt, molecular formula: C 7 H 15 NO 3 , molecular weight: 161.20. Levocarnitine is essential for human metabolism, is an important component of food, and is considered a "vitamin-like" nutrient. There are two sources of levocarnitine in the human body. One is ingested from the diet, which is most abundant in meat and dairy products, and extremely low or absent in vegetables, grains, and fruits. The other is endogenous synthesis. Under the action of a series of liver enzymes, with Vc, VB 6 , niacin, and iron as cofactors, levocarnitine is synthesized from lysine and methionine, and this synthesis is limited. There are two main reasons for normal people to supplement levocarnitine: First, many individuals are in a state of deficiency or marginal deficiency, manifested by low blood and tissue carnitine levels. Second, it is the current view of nutritional science to achieve the "optimal health level". Levocarnitine has many clinical applications. For example, it has therapeutic or adjuvant therapeutic effects on levocarnitine deficiency, cardiovascular diseases, hyperlipidemia, dialysis nephropathy patients, cirrhosis, and diabetes patients, etc. The United States Pharmacopoeia has included levocarnitine raw materials, tablets, and injections for the prevention and treatment of levocarnitine deficiency. At present, it has been specified as a legal multi-purpose nutrient by Switzerland, France, the United States, and the World Health Organization, and the Ministry of Health of China has also included levocarnitine in the list of nutritional fortifiers. With the continuous deepening of the understanding of this product, a craze for levocarnitine health products has emerged at home and abroad, and health products such as weight loss, lipid-lowering, and improving exercise energy have been developed.

[0003] The currently reported methods for synthesizing levocarnitine are as follows: 1) extraction method, 2) chemical synthesis method, and 3) biosynthesis method. The extraction method has a low yield and high cost and has been phased out. Currently, most manufacturers use the chemical synthesis method to prepare levocarnitine. Starting from epichlorohydrin, after kinetic resolution, dextrorotatory epichlorohydrin is obtained, which reacts with trimethylamine hydrochloride to produce a quaternary ammonium salt, then reacts with sodium cyanide, and finally undergoes hydrolysis to obtain levocarnitine. Due to the long process route, large equipment investment, use of highly toxic sodium cyanide, strong danger, environmental unfriendliness, and difficult treatment of three wastes, the manufacturing cost is relatively high. The biosynthesis method is currently mostly in the laboratory research stage, and there are few reports on industrial applications. Usually starting from ethyl 4-chloroacetoacetate, asymmetric reduction is carried out using carbonyl reductase. Additionally, glucose dehydrogenase, coenzyme, and glucose need to be added, and a cosolvent is used to carry out the reaction in a buffer solution. Although this route uses a relatively advanced enzyme-catalyzed method, it requires the simultaneous addition of two separated and purified enzymes and coenzymes, and a cosolvent is also needed. The cosolvent is an organic solvent, which not only pollutes the environment but also has a toxic effect on the enzyme, affecting the enzyme activity and thus the reaction conversion rate. In the purification process of levocarnitine, a resin column is used, which has low efficiency and cumbersome operation and is not conducive to continuous industrial operation.

[0004] Therefore, developing an environmentally friendly, easy-to-operate, and high-conversion industrial process to simultaneously solve the problems in the prior art that the cosolvent affects the conversion rate, and the low production efficiency and cumbersome operation are not conducive to industrialization has good economic benefits and application prospects. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides an industrial preparation method for levocarnitine. This method solves the problem that the existing biosynthesis method of levocarnitine requires the simultaneous use of two enzymes and a cosolvent, which affects the enzyme activity and reaction conversion rate; it also solves the problem of low desalting efficiency and cumbersome operation when using a resin column device. The product yield and purity of this method are higher than those of the current mainstream methods.

[0006] The preparation method of the levocarnitine compound of the present invention comprises the following steps:

[0007] (1) Add Escherichia coli engineering bacteria cells (bacterial sludge) containing carbonyl reductase and glucose dehydrogenase, anhydrous glucose, and coenzyme to a buffer salt solution, slowly dropwise add ethyl 4-chloroacetoacetate over about 2 h, and simultaneously dropwise add an alkali solution to maintain the pH value within a certain range, control the temperature at 30 ± 5 °C, and carry out a catalytic reduction reaction. After the reaction is completed, through extraction, vacuum concentration, and high-vacuum distillation, ethyl (R)-4-chloro-3-hydroxybutyrate is obtained;

[0008] (2) Ethyl (R)-4-chloro-3-hydroxybutyrate was added dropwise to an aqueous solution of trimethylamine containing an inorganic base over about 2 h, and after the addition, the mixture was kept warm for reaction for 1 - 2 h. After the reaction was completed, the pH was adjusted to 5 - 6 with a hydrochloric acid solution first, and then to 7 - 8 with an aqueous ammonia solution; finally, the salt was removed by electrodialysis to obtain the levocarnitine compound.

[0009] Among them:

[0010] The buffer salt solution described in step (1) is a phosphate buffer solution or a Tris-HCl buffer solution; its pH value is 6 - 8.

[0011] The pH value of the catalytic reduction reaction described in step (1) is 6 - 8.

[0012] The preparation method of the Escherichia coli engineered bacteria cells described in step (1) is as follows: 4 μL of a 50 mg / mL kanamycin aqueous solution was added to a test tube containing 4 mL of seed medium in a sterile operating bench, and then 10 μL of the Escherichia coli engineered bacteria preserved with glycerol was inoculated, and the mixture was shaken and cultured in a shaker at 37 °C and 200 rpm for 10 h; the seed liquid was transferred to a 500 mL Erlenmeyer flask containing 100 mL of seed medium and continued to be cultured in a shaker at 37 °C and 200 rpm for 3 h; the seed liquid was transferred into a 100 L seed tank and continued to be cultured at 37 °C and 200 rpm for 3 h; the seed liquid was transferred to a fermenter, the temperature and stirring speed were set at 37 °C and 200 rpm respectively, the aeration rate was adjusted to 60 L / min, and the dissolved oxygen was calibrated to 100%; the seed liquid was transferred into the fermenter under the protection of a flame, and the fermentation started; as the cells grew, the dissolved oxygen (DO) gradually decreased, and when the DO dropped to about 10%, the stirring speed was gradually increased; during the fermentation process, ammonia water was added dropwise to control the pH at about 7.0; after culturing for 4 h, the carbon and nitrogen sources were supplemented; when culturing for 5 h, the temperature of the fermenter was set at 25 °C, and the temperature began to decrease gradually, and the temperature dropped to 25 °C in about 30 min; after culturing for 6 h, an IPTG aqueous solution was added to a final concentration of 0.2 mM to induce the expression of the target protein, and after inducing for 10 h, the fermentation was ended; the Escherichia coli engineered bacteria cells were collected by high-speed centrifugation.

[0013] The coenzyme described in step (1) is NADP, that is, β-nicotinamide adenine dinucleotide phosphate-sodium salt; its molecular weight is 765.39.

[0014] The material ratio m(ethyl 4-chloroacetoacetate):m(Escherichia coli engineered bacteria cells):m(anhydrous glucose):m(coenzyme) described in step (1) is 1:(0.2 - 0.4):(1.1 - 2.2):(0.5 - 2)×10 -3 , where m represents mass.

[0015] The alkali solution described in step (1) is one of potassium hydroxide, sodium hydroxide, and sodium carbonate solutions.

[0016] The inorganic base described in step (2) is one or both of potassium hydroxide and sodium hydroxide, preferably sodium hydroxide.

[0017] The temperature of the heat preservation reaction described in step (2) is -10 to 10 °C.

[0018] The molar ratio of ethyl (R)-4-chloro-3-hydroxybutyrate to aqueous trimethylamine solution described in step (2) is 1:(1 - 4).

[0019] The molar ratio of ethyl (R)-4-chloro-3-hydroxybutyrate to inorganic base described in step (2) is 1:(1 - 3).

[0020] The standard that the conductivity of the electrodialysis solution should reach after desalination by electrodialysis described in step (2) is 50 - 300 μs / cm.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The present invention uses the whole-cell catalysis method for carbonyl asymmetric reduction, developing a brand-new industrialization process; the whole-cell catalysis method adopted has the characteristics of stronger cell adaptability to the environment, and does not require enzyme separation, saving time, so the cost is lower; no cosolvent is used, which is green and environmentally friendly, has little toxicity to cells, does not affect enzyme activity, and has a higher conversion rate. The molar conversion rate of the optical intermediate prepared by the whole-cell catalysis method can reach more than 99%, and the optical purity can reach more than 99.5%.

[0023] (2) The present invention uses a one-pot method to prepare levocarnitine, with water as the reaction solvent and mild reaction conditions; by changing the addition method of the intermediate, the reaction is more complete, the product purity is high, and the yield can reach more than 95%.

[0024] (3) In the desalination process of the present invention, an electrodialysis device is used, which has higher efficiency, more convenient operation and is more suitable for industrialization compared with the traditional resin column method. Specific Embodiments

[0025] The following further illustrates the present invention with reference to embodiments, but the protection scope of the present invention is not limited thereto.

[0026] All raw materials used in the embodiments are commercially available unless otherwise specified.

[0027] The coenzyme described in the embodiments is NADP.

[0028] Example 1

[0029] (1) Prepare 2000 L of phosphate buffer solution with a pH value of 6.0 in a 5000 L reaction tank. Add 40 kg of Escherichia coli engineered bacteria cells (bacterial sludge), 220 kg of anhydrous glucose, and 100 g of coenzyme. Dropwise add 200 kg of ethyl 4-chloroacetoacetate over about 2 h. At the same time, dropwise add 1 M sodium hydroxide solution to maintain the pH value at 6.0 ± 0.1, control the temperature at 30 °C, and carry out the catalytic reduction reaction. After 6 h, take a sample and detect the reaction conversion rate by gas phase to be 99.91%. After confirming the reaction is complete, add ethyl acetate (450 kg * 3) for extraction, separate the liquid, and collect the organic phase. First, carry out concentration under reduced pressure at 40 °C to remove the ethyl acetate solvent. Then, carry out high-vacuum distillation under reduced pressure at 95 °C, collect the distillate, and obtain 188.7 kg of colorless (R)-ethyl 4-chloro-3-hydroxybutyrate product with a yield of 93.20% and an ee value of 99.6%.

[0030] (2) Add 1288 kg of sodium hydroxide solution (88 kg of sodium hydroxide and 1200 kg of water) and 212.8 kg of 30% aqueous trimethylamine solution to a 3000 L reaction tank. Control the temperature at 0 - 10 °C, and slowly add 180 kg of (R)-ethyl 4-chloro-3-hydroxybutyrate to the reaction tank over about 2 h. After dropping, keep the reaction at a constant temperature for 1.5 h. After the reaction is completed, first adjust the pH to 5 - 6 with 1 M hydrochloric acid solution, and then adjust the pH to 7 - 8 with 5% ammonia water solution. Finally, remove the salt through an electrodialysis device. When the final conductivity of the electrodialysis solution is 200 μs / cm, stop the electrodialysis. The electrodialysis solution is distilled to dryness under reduced pressure at 65 °C to obtain 188.7 kg of white levocarnitine compound with a yield of 97.50%. (Isomer content: 0.1%; Chloride meets the regulations: not higher than 0.02%).

[0031] Example 2

[0032] (1) Prepare 2000 L of phosphate buffer solution with a pH value of 7.0 in a 5000 L reaction tank. Add 60 kg of Escherichia coli engineered bacteria cells (bacterial sludge), 330 kg of anhydrous glucose, and 300 g of coenzyme. Dropwise add 200 kg of ethyl 4-chloroacetoacetate over about 2 h. At the same time, dropwise add 1 M sodium hydroxide solution to maintain the pH value at 7.0 ± 0.1, control the temperature at 30 °C, and carry out the catalytic reduction reaction. Take a sample and detect the reaction conversion rate by gas phase to be 99.85%. After confirming the reaction is complete, add ethyl acetate (450 kg * 3) for extraction, separate the liquid, and collect the organic phase. First, carry out concentration under reduced pressure at 40 °C to remove the ethyl acetate solvent. Then, carry out high-vacuum distillation under reduced pressure at 95 °C, collect the distillate, and obtain 183.0 kg of colorless (R)-ethyl 4-chloro-3-hydroxybutyrate product with a yield of 90.41% and an ee value of 99.8%.

[0033] (2) Add 1310 kg of sodium hydroxide solution (110 kg of sodium hydroxide and 1200 kg of water) and 426.7 kg of 30% aqueous trimethylamine solution to a 3000 L reaction kettle. Control the temperature at -10 - 0 °C, and slowly add 180 kg of (R)-4-chloro-3-hydroxybutyric acid ethyl ester to the reaction kettle over about 2 h. After the addition, keep the reaction at a constant temperature for 1.5 h. After the reaction is completed, first adjust the pH to 5 - 6 with 1 M hydrochloric acid solution, and then adjust the pH to 7 - 8 with 5% ammonia water solution. Finally, desalt with an electrodialysis device, and stop the electrodialysis when the final conductivity of the electrodialysis solution is 150 μs / cm. Distill the electrodialysis solution under reduced pressure to dryness at 65 °C to obtain 183.8 kg of white L-carnitine compound, with a yield of 94.96%. (Isomer content: 0.07%; Chloride meets the requirements: not higher than 0.02%).

[0034] Example 3

[0035] (1) Add 2000 L of phosphate buffer solution with a pH value of 8.0, 80 kg of Escherichia coli engineered bacteria cells (bacterial sludge), 440 kg of anhydrous glucose, and 400 g of coenzyme to a 5000 L reaction kettle. Add 200 kg of ethyl 4-chloroacetoacetate dropwise over about 2 h, and at the same time add 1 M sodium hydroxide solution dropwise to maintain the pH value at 8.0 ± 0.1. Control the temperature at 30 °C and carry out the catalytic reduction reaction. Take a sample and detect the reaction conversion rate by gas phase to be 99.96%. After confirming the completion of the reaction, add ethyl acetate (450 kg * 3) for extraction, separate the liquid, and collect the organic phase. Concentrate under reduced pressure at 40 °C to remove the ethyl acetate solvent. Then, under high vacuum and reduced pressure distillation at 95 °C, collect the distillate to obtain 192.4 kg of colorless (R)-4-chloro-3-hydroxybutyric acid ethyl ester product, with a yield of 95.05% and an ee value of 99.7%.

[0036] (2) Add 1266 kg of sodium hydroxide solution (66 kg of sodium hydroxide and 1200 kg of water) and 638.4 kg of 30% aqueous trimethylamine solution to a 3000 L reaction kettle. Control the temperature at -5 - 5 °C, and slowly add 180 kg of (R)-4-chloro-3-hydroxybutyric acid ethyl ester to the reaction kettle over about 2 h. After the addition, keep the reaction at a constant temperature for 1.5 h. After the reaction is completed, first adjust the pH to 5 - 6 with 1 M hydrochloric acid solution, and then adjust the pH to 7 - 8 with 5% ammonia water solution. Finally, desalt with an electrodialysis device, and stop the electrodialysis when the final conductivity of the electrodialysis solution is 100 μs / cm. Distill the electrodialysis solution under reduced pressure to dryness at 65 °C to obtain 186.1 kg of white L-carnitine compound, with a yield of 96.18%. (Isomer content: 0.085%; Chloride meets the requirements: not higher than 0.02%).

[0037] Comparative Example 1

[0038] (1) Add 20 g of ethyl 4-chloroacetoacetate, 160 g of methanol, 13 g of ketoreductase KRED-101, 5 g of coenzyme NADP, 10 g of glucose dehydrogenase, and 800 g of 6.5% glucose solution to a 2 L reaction flask. Adjust the pH value to 7.0, stir at 25 °C, and carry out the reduction reaction. After reacting for 6 h, take a sample and detect the conversion rate by gas phase, which is 97.56%. Extract with ethyl acetate, carry out reduced pressure distillation and high vacuum distillation to obtain 17.45 g of an oily product, yield: 86.2%, and the ee value is 96.3%. (Due to the toxic effect of the substrate and cosolvent on the enzyme, the substrate concentration is low, only 20 g / L; the product yield is low, and the optical purity is not high).

[0039] (2) Add 105.5 g of sodium hydroxide solution (5.5 g of sodium hydroxide and 100 g of water) and 53.2 g of 30% aqueous trimethylamine solution to the reaction flask. Slowly add 15 g of ethyl (R)-4-chloro-3-hydroxybutyrate to the reaction flask over about 2 h. After dropping, stir and react at 0 - 5 °C for 12 h, then raise the temperature to room temperature and react for 20 - 24 h. Dropwise add concentrated hydrochloric acid to adjust the pH value to 6, and purify with a 732-type cation exchange resin column. The purification process of the cation exchange resin column is as follows: Add the reaction solution to the ion exchange resin column, control the flow rate at the bottom of the column at 5 ml / min. After adding the material, add purified water to the resin column, start to control the flow rate at the bottom of the column at 5 ml / min, and end when the pH value of the discharged liquid at the bottom of the column is 6; then add dilute ammonia water with a mass fraction of 4% - 6%, control the flow rate at the bottom of the column at 2.5 ml / min, start to collect when the pH value of the discharged liquid at the bottom of the column is 7, and stop collecting when the pH value > 8. Combine the collected liquid, spin-dry the collected liquid to obtain 10.6 g of white L-carnitine, yield 73.2%. (Isomer content: 0.30%, chloride does not meet the regulations: significantly higher than 0.02%).

[0040] Comparative Example 2

[0041] (1) Add 20 g of ethyl 4-chloroacetoacetate, 160 g of methanol, 13 g of ketoreductase KRED-101, 5 g of coenzyme NADP, 10 g of glucose dehydrogenase, and 800 g of 6.5% glucose solution to a 2 L reaction flask. Adjust the pH value to 7.0, stir at 25 °C, and carry out the reduction reaction. After reacting for 6 h, take a sample and detect the conversion rate by gas phase, which is 97.56%. Extract with ethyl acetate, carry out reduced pressure distillation and high vacuum distillation to obtain 17.45 g of an oily product, yield: 86.2%, and the ee value is 96.2%. (Due to the toxic effect of the substrate and cosolvent on the enzyme, the substrate concentration is low, only 20 g / L; the product yield is low, and the optical purity is not high).

[0042] (2) Add 105.5 g of sodium hydroxide solution (5.5 g of sodium hydroxide and 100 g of water) and 53.2 g of 30% trimethylamine aqueous solution to the reaction kettle. Control the temperature at -10 - 0 °C, and slowly add 15 g of (R)-ethyl 4-chloro-3-hydroxybutyrate to the reaction kettle over about 2 h. After the addition, keep the reaction at a constant temperature for 1.5 h. After the reaction is completed, first adjust the pH to 5 - 6 with 1 M hydrochloric acid solution, and then adjust the pH to 7 - 8 with 5% ammonia aqueous solution. Finally, desalt with an electrodialysis device, and stop the electrodialysis when the final conductivity of the electrodialysis solution is 150 μs / cm. Distill the electrodialysis solution under reduced pressure to dryness at 65 °C to obtain 12.4 g of white L-carnitine compound, with a yield of 85.51%. (Isomer content: 0.29%; Chloride compliance: not higher than 0.02%).

Claims

1. An industrial preparation method of L-carnitine, characterized in that: It includes the following steps: (1) Add Escherichia coli engineered bacteria cells containing carbonyl reductase and glucose dehydrogenase, anhydrous glucose, and coenzyme to a buffer salt solution, dropwise add ethyl 4-chloroacetoacetate, and dropwise add an alkali solution to adjust the pH, and carry out a catalytic reduction reaction. After 6 hours, the reaction is completed. After extraction, concentration under reduced pressure, and high-vacuum distillation, ethyl (R)-4-chloro-3-hydroxybutyrate is obtained; (2) Dropwise add ethyl (R)-4-chloro-3-hydroxybutyrate to an aqueous solution of trimethylamine containing an inorganic base. After dropping, keep the reaction at a certain temperature for 1.5 hours. After the reaction is completed, first adjust the pH to 5-6 with a hydrochloric acid solution, and then adjust the pH to 7-8 with an ammonia water solution; finally, desalt by electrodialysis to obtain the L-carnitine compound; The material ratio in step (1) m(ethyl 4-chloroacetoacetate):m(Escherichia coli engineered bacteria cells):m(anhydrous glucose):m(coenzyme) is 1:(0.2-0.4):(1.1-2.2):(0.5-2)×10-3, where m represents mass; The standard of the conductivity of the electrodialysis solution after desalting by electrodialysis in step (2) is 50-300 μs / cm; Among them, the Escherichia coli engineered bacteria cells are the bacterial sludge of Escherichia coli engineered bacteria cells.

2. The industrial preparation method of L-carnitine according to claim 1, characterized in that: The buffer salt solution in step (1) is a phosphate buffer solution or a Tris-HCl buffer solution; its pH value is 6-8.

3. The industrial preparation method of L-carnitine according to claim 1, characterized in that: The pH value of the catalytic reduction reaction in step (1) is 6-8.

4. The industrial preparation method of L-carnitine according to claim 1, characterized in that: The coenzyme in step (1) is NADP.

5. The industrial preparation method of L-carnitine according to claim 1, characterized in that: The inorganic base in step (2) is one or two of potassium hydroxide and sodium hydroxide.

6. The industrial preparation method of L-carnitine according to claim 1, characterized in that: The temperature of the heat preservation reaction in step (2) is -10-10 °C.

7. The industrial preparation method of L-carnitine according to claim 1, characterized in that: The molar ratio of ethyl (R)-4-chloro-3-hydroxybutyrate to the aqueous solution of trimethylamine in step (2) is 1:(1-4).

8. The industrial preparation method of L-carnitine according to claim 1, characterized in that: The molar ratio of ethyl (R)-4-chloro-3-hydroxybutyrate to the inorganic base in step (2) is 1:(1-3).