Silanization purification method for ursodesoxycholic acid prepared by biological enzyme method

Abstract

The invention discloses a silanization purification method for ursodesoxycholic acid prepared by a biological enzyme method, and belongs to the field of biological pharmacy. According to the method, the whole-cell reaction liquid is used as a purification object, insoluble macromolecular impurities are removed through preliminary impurity removal and high-concentration acetone-water dissolution, the target content of UDCA is 80% or above successfully, and the product is pure white; the method comprises the following steps: carrying out a reaction on a UDCA silanization derivative and-OH by using a silanization reagent, separating the UDCA silanization derivative from other silanization derivatives, and hydrolyzing the UDCA silanization derivative to realize purification of UDCA. The content of CDCA in the final product is effectively controlled to be 0.5% or below, the purity of the UDCA crude product is improved from 53% to 98.5% or above, and the requirements of UDCA bulk drugs are met. The method is simple in process and low in cost, the obtained product is high in purity, the chenodeoxycholic acid impurity content is low, and the chenodeoxycholic acid can be used as a raw material of a pharmaceutical preparation.

Description

Technical Field

[0001] This invention relates to a silanization purification method for the preparation of ursodeoxycholic acid by a biological enzymatic process, which belongs to the field of biopharmaceuticals. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is a type of animal bile acid, chemically named (3α,7β)-dihydroxy-5β-cholanic acid. It was first isolated and purified from bear bile by Japanese researchers, hence its name. Later, it was marketed by the Japanese pharmaceutical company Tanabe. Clinically, ursodeoxycholic acid is mainly used to treat hepatobiliary diseases such as cholestasis, fatty liver, hepatitis, and liver damage. Ursodeoxycholic acid is a non-toxic bile acid, thus preventing toxic damage to hepatobiliary cells.

[0003] Chenodeoxycholic acid (CDCA) is the 7-isomer of ursodeoxycholic acid (UDCA), the starting material for its synthesis, and also a major impurity component in UDCA tablets. Both chemical and biosynthetic methods involve oxidizing CDCA to the intermediate 7-ketolithocholic acid (7-KLCA), followed by reducing 7-ketolithocholic acid to UDCA. During this reaction, CDCA is rarely completely reacted, leaving 3%–10% impurities. Additionally, small amounts of 7-ketolithocholic acid remain in the product. Both are chemically similar to UDCA and are difficult to remove. Therefore, finding suitable methods to remove the raw material impurity CDCA and the intermediate impurity 7-KLCA to improve the quality of the UDCA raw material is a key challenge in the research of enzymatic synthesis of UDCA.

[0004]

[0005] Patent EP119040A1 involves inoculating a culture medium with *Fusarium equisetifolium* to convert lithocholic acid to ursodeoxycholic acid. After the reaction, sodium hydroxide is added to adjust the pH to 2-7, followed by extraction with n-butanol. The extract is then pyrolyzed with 15% sodium hydroxide for 5 hours, extracted with ethyl acetate, concentrated, and loaded onto a silica gel column. Ursodeoxycholic acid is eluted with chloroform-acetone-acetic acid (100:100:1), concentrated, and crystallized with a small amount of ethyl acetate. This process is complex, has low production capacity, and is not suitable for industrial production.

[0006] Another patent uses ethyl acetate to selectively dissolve and remove chenodeoxycholic acid from a mixture of ursodeoxycholic acid and chenodeoxycholic acid, but the selectivity is not strong, the required amount to be dissolved is large, and the yield is not high.

[0007] Patent CN113666982A purifies monomers from ursodeoxycholic acid (UDCA) in biological reactions. First, calcium chloride and diatomaceous earth are used to adsorb and remove enzymes and proteins. Then, triethylamine is added to prepare the triethylamine salt of ursodeoxycholic acid, followed by hydrolysis to obtain high-purity ursodeoxycholic acid. However, this method suffers from low recovery rates, difficulty in completely removing soluble impurities from enzymes and proteins, and a low UDCA content in the final product, failing to meet the requirements of the European Pharmacopoeia.

[0008] Patent CN104804055A uses a silanization reagent to purify 7-ketolithocholic acid, a chemical oxidation intermediate of CDCA, thereby reducing by-reaction impurities in the final product. However, this reaction is a chemical reaction, and the impurities are relatively simple and easy to separate. Nucleic acids, proteins, and ions from the buffer solution in the whole-cell reaction solution also all enter the reaction product, increasing the difficulty of subsequent separation and purification. Summary of the Invention

[0009] Based on the problems mentioned above, and to overcome the shortcomings of existing technologies, this invention proposes a process for purifying whole-cell enzymatically prepared ursodeoxycholic acid (UDCA) using a silanization method. By combining the characteristics of impurities in whole-cell enzymatic fermentation with the dissolution, crystallization, and silanization reactions, the aim is to prepare high-purity UDCA.

[0010] Although the whole-cell enzymatic conversion of CDCA to UDCA reduces the cost of enzyme purification and the product conversion reaction, the nucleic acids, proteins, and ions in the buffer solution from the whole-cell fermentation broth all enter the reaction product, increasing the difficulty of subsequent separation and purification. Furthermore, because UDCA, CDCA, and 7-KLCA have similar properties, simple solvent extraction alone cannot achieve effective separation of the three. While column chromatography can achieve partial separation, it cannot completely remove CDCA, and its industrial production cost is high.

[0011] The reaction product after preliminary impurity removal still contains impurities such as soluble proteins, nucleic acids, and inorganic salt ions. If silanization is carried out directly at this stage, the process will be unstable. Sometimes, the silanized derivatives of UDCA cannot be crystallized and separated from UDCA, and sometimes the product recovery rate is very low, only 20%–30%, which cannot meet the process requirements. In this case, by dissolving the product in high-concentration acetone-water and heating under reflux to remove some insoluble macromolecular impurities, followed by precipitation with purified water, UDCA with a target content of over 80% can be successfully obtained. The product is white, and almost all macromolecular impurities have been removed. This method uses simple solvents, all of which are recyclable, and the mother liquor can be reused in the next batch of product, achieving a target overall recovery rate of over 90%. Further purification of UDCA followed by reaction with a silanizing reagent can significantly improve reaction efficiency and the target recovery rate. After further purification, the main impurities of UDCA are CDCA and 7K. These three substances have similar polarities, making it difficult to separate them using conventional methods such as column chromatography. Therefore, a silanizing agent is introduced to react with -OH groups. By controlling the type, amount, reaction time, and temperature of the silanizing agent, the UDCA silanized derivatives can be separated from other silanized derivatives. The UDCA silanized derivatives are then hydrolyzed to achieve UDCA purification. Finally, regarding the UDCA recrystallization process, traditional methods typically use ethyl acetate. However, UDCA has very low solubility in ethyl acetate; even with heating, more than 40 times the volume of ethyl acetate is required for dissolution. The high temperature leads to ethyl acetate volatilization and loss, and also seriously affects workshop safety and the production environment. Through extensive experimental comparisons, this invention has found that when using 30% to 80% acetone, preferably 70% acetone, UDCA precipitates in a better crystal form with a high yield (≥90%). Furthermore, only one or two crystallization cycles are required to obtain UDCA products with a purity greater than 99.0%, which greatly improves the workshop production environment and saves solvent costs.

[0012] This invention utilizes 7α-hydroxysteroid dehydrogenase cells and 7β-hydroxysteroid dehydrogenase cells constructed in patent CN109402212A for whole-cell transformation to produce ursodeoxycholic acid, specifically including the following steps: (1) Take 7α-steroid dehydrogenase cells and 7β-steroid dehydrogenase cells, add 3 times the volume of purified water and stir well, add to chenodeoxycholic acid CDCA, then add coenzyme II-NADP, stir well, add 5% sodium hydroxide to adjust the pH to 7-8, react at 37℃ for 18-24h to obtain whole cell reaction solution; The mass ratio of 7α-steroid dehydrogenase cells to CDCA was 0.5:1; the mass ratio of 7β-steroid dehydrogenase cells to CDCA was 3.5:1; and the mass ratio of coenzyme II-NADP to CDCA was 0.1:1.

[0013] (2) Add 6 to 10 times the volume of organic reagent to the above CDCA whole cell enzyme reaction solution, stir evenly, let stand at 5-20℃ for 4 hours, centrifuge to take the supernatant, concentrate and dry the supernatant to obtain the reaction product. (3) Add 10 times the volume of 70%~80% acetone solution (solvent is water) to the ursodeoxycholic acid drying reaction product of step (1), reflux and heat at 50-60℃ for 0.5~2h, stir and cool to 10~20℃, filter and take the filtrate. (4) Slowly add 5 to 10 times the volume of purified water to the filtrate obtained in step (3) while stirring. After the addition is complete, concentrate to 40% to 50% of the volume, stop the recovery, filter while hot, dry the filter cake, and obtain crude ursodeoxycholic acid. The crude product contains about 80% ursodeoxycholic acid. (5) Dissolve the crude ursodeoxycholic acid in an organic reagent and suspend it. Heat the temperature to 30-70°C and maintain it for 0.5-1h. Slowly add the silanizing reagent while stirring. Maintain the temperature and react for 1-4 hours to carry out the silanization reaction. (6) After the silanization reaction is complete, cool the solution to -5~0℃ and keep it for 24 hours until crystallization is complete. Filter the solution, wash it with a small amount of solvent, and collect the precipitate. (7) Add an appropriate volume of inorganic acid to the precipitate in step (6) while stirring, and reflux at 30~50℃ for 15~30 min. (8) After hydrolysis, cool the reaction solution in (7) to 4~10℃, filter, and repeatedly rinse the filter cake with purified water to remove residual acid and collect the filter cake; (9) Take the filter cake from step (8), recrystallize it with acetone-water, and dry it to obtain purified ursodeoxycholic acid. The purified ursodeoxycholic acid contains less than 0.5% CDCA and 98.5% UDCA, meeting the requirements for UDCA raw materials.

[0014] In one embodiment, the settling time in step (2) is preferably 20°C.

[0015] In one embodiment, the organic reagent in step (2) includes, but is not limited to, methanol, ethanol, and acetone.

[0016] In one embodiment, the organic reagent in step (5) is acetonitrile, acetone, N,N-dimethylformamide, etc.

[0017] In one embodiment, the amount (mass) of the silanizing agent added in step (5) is 3 to 5 times the volume of crude ursodeoxycholic acid.

[0018] In one embodiment, the solvent in step (6) is acetonitrile, acetone, or N,N-dimethylformamide.

[0019] In one embodiment, the inorganic acid mentioned in step (7) is sulfuric acid or hydrochloric acid, with a concentration of 5% to 10%.

[0020] In one embodiment, the mixed solvent in step (9) is 35%~80% acetone, and the amount added is 10 times the volume.

[0021] The beneficial effects of this invention are as follows: This invention uses whole-cell transformation fluid as the purification target. After preliminary impurity removal, insoluble macromolecular impurities are removed by dissolving in high-concentration acetone-water, successfully obtaining UDCA with a target content of over 80%, and the product is white, with virtually all macromolecular impurities removed. Then, a silanizing reagent is used to react with -OH groups to separate the UDCA silanized derivatives from other silanized derivatives. Finally, the UDCA silanized derivatives are hydrolyzed to achieve UDCA purification. The CDCA content in the final product is effectively controlled below 0.5%, increasing the purity of crude UDCA from 53% to over 98.5%, meeting the requirements for UDCA raw materials. Detailed Implementation

[0022] The following examples are provided to help better understand the present invention, but do not limit the invention.

[0023] The 7α-steroid dehydrogenase cells and 7β-steroid dehydrogenase cells used in the following examples can be constructed with reference to patent CN109402212A.

[0024] Example 1 (1) Take 7α-steroid dehydrogenase cells and 7β-steroid dehydrogenase cells, add 3 times the volume of purified water and stir well, add to chenodeoxycholic acid CDCA, then add coenzyme II-NADP, stir well, add 5% sodium hydroxide to adjust the pH to 7-8, react at 37℃ for 18-24h to obtain whole cell reaction solution; The mass ratio of 7α-steroid dehydrogenase cells to CDCA was 0.5:1; the mass ratio of 7β-steroid dehydrogenase cells to CDCA was 3.5:1; and the mass ratio of coenzyme II-NADP to CDCA was 0.1:1.

[0025] (2) Take 100g of CDCA whole cell reaction solution, add 6 times 95% ethanol and stir evenly. Let stand at 20℃ for 4h, centrifuge, concentrate and dry the supernatant to obtain 110.2g of reaction product.

[0026] (3) Add 15 times the amount of 70% acetone to the above reaction product, reflux and stir at 55°C for 0.5h, cool to 15°C and filter to obtain the filtrate.

[0027] (4) Add 10 times the volume of purified water to the filtrate while stirring. After the addition is complete, concentrate to 40%~50% of the volume, stop the recovery, filter while hot, dry the filter cake, and obtain 57.3g of crude ursodeoxycholic acid with a content of 80.8%.

[0028] (5) Take the above crude ursodeoxycholic acid, add 10 times the volume of acetone to suspend it, slowly heat it to 60°C, keep it for 1 hour, add 3 times the volume of hexamethyldisilazane (mass-volume ratio) of crude ursodeoxycholic acid dropwise, and stir the reaction at 60°C for 2 hours.

[0029] (6) After the reaction is complete, cool the reaction solution to -5~0℃ and keep it for 24 hours until crystallization is complete. Filter the solution, wash it with a small amount of ice acetone, and collect the precipitate.

[0030] (7) Add 8 times the volume of 5% hydrochloric acid solution to the above precipitate and reflux at 50°C for 30 min.

[0031] (8) After hydrolysis, cool the reaction solution to 4~10℃, filter, and wash repeatedly with purified water to remove residual acid, collect the filter cake, and dry it.

[0032] (9) Dissolve the above filter cake in 15 times 50% acetone water, recrystallize at 0~5℃ to obtain about 43.8g of ursodeoxycholic acid concentrate, with UDCA content of 99.2%, CDCA content of 0.08%, and 7-KLCA not detected.

[0033] Example 2 (1) Same as step (1) in Example 1.

[0034] (2) Take 50g of CDCA whole cell reaction solution, add 6 times 95% ethanol and stir evenly. Let stand at 20℃ for 4h, centrifuge, concentrate and dry the supernatant to obtain 55.6g of reaction product.

[0035] (3) Add 12 times the amount of 80% acetone to the above reaction product, reflux at 60°C and stir for 0.5 h, cool to 10°C and filter to obtain the filtrate.

[0036] (4) Add 10 times the volume of purified water to the filtrate while stirring. After the addition is complete, concentrate to 40%~50% of the volume, stop the recovery, filter while hot, dry the filter cake, and obtain 29.4g of crude ursodeoxycholic acid with a content of about 79.6%.

[0037] (5) Take the above crude ursodeoxycholic acid, add 10 times the volume of N,N-dimethylformamide (DMF) to dissolve it, slowly heat it to 55°C, keep it for 0.5h, add 5 times the volume of hexamethyldisilazane (mass-volume ratio) of crude ursodeoxycholic acid dropwise, and stir the reaction at 55°C for 3h.

[0038] (6) After the reaction is complete, cool the reaction solution to -5~0℃ and keep it for 24 hours until crystallization is complete. Filter the solution, wash it with a small amount of DMF, and collect the precipitate.

[0039] (7) Add 10 times the volume of 5% hydrochloric acid solution to the above precipitate and reflux at 50°C for 30 min.

[0040] (8) After hydrolysis, cool the reaction solution to 4~10℃, filter, and wash repeatedly with purified water to remove residual acid, collect the filter cake, and dry it.

[0041] (9) Dissolve the above filter cake in 20 times the amount of 35% acetone water, recrystallize at 0~5℃ to obtain approximately 22.3g of purified ursodeoxycholic acid, with UDCA content of 99.1%, CDCA content of 0.05%, and 7-K not detected.

[0042] Example 3 (1) Same as step (1) in Example 1.

[0043] (2) Take 150g of CDCA whole cell reaction solution, add 6 times 95% ethanol and stir evenly. Let stand at 20℃ for 4h, centrifuge, concentrate and dry the supernatant to obtain 158.2g of reaction product.

[0044] (3) Add 10 times the amount of 80% acetone to the above reaction product, reflux and stir at 55°C for 0.5h, cool to 20°C and filter to obtain the filtrate.

[0045] (4) Add 10 times the volume of purified water to the filtrate while stirring. After the addition is complete, concentrate to 40%~50% of the volume, stop the recovery, filter while hot, dry the filter cake, and obtain 85.6g of crude ursodeoxycholic acid with a ursodeoxycholic acid content of about 82.6%.

[0046] (5) Take the above crude ursodeoxycholic acid, add 10 times the volume of acetonitrile to suspend it, slowly raise the temperature to 50°C, keep it for 0.5 h, add 4 times the volume of tetramethyldisilazane (mass-volume ratio) of crude ursodeoxycholic acid dropwise, and stir the reaction at 50°C for 4 h.

[0047] (6) After the reaction is complete, cool the reaction solution to -5~0℃ and keep it for 24 hours until crystallization is complete. Filter the solution, wash it with a small amount of DMF, and collect the precipitate.

[0048] (7) Add 10 times the volume of 5% hydrochloric acid solution to the above precipitate and reflux at 50°C for 30 min.

[0049] (8) After hydrolysis, cool the reaction solution to 4~10℃, filter, and wash repeatedly with purified water to remove residual acid, collect the filter cake, and dry it.

[0050] (9) Dissolve the above filter cake in 10 times the amount of 70% acetone, recrystallize at 0~5℃ to obtain approximately 65.9g of purified ursodeoxycholic acid, with UDCA content of 99.7%, CDCA content of 0.05%, and 7-K not detected.

[0051] Comparative Example 1 Referring to patent CN114438046A, 150g of whole-cell reaction solution of CDCA was taken. At this time, the entire reaction product was in a jelly-like state. Six times the amount of ethanol was added and stirred evenly. The mixture was allowed to stand at room temperature for 4 hours, centrifuged, and the ethanol was recovered from the supernatant under reduced pressure. When the mixture was cooled to room temperature, it turned into a jelly-like state that could not be stirred. It was then reheated to dissolve the jelly. While the concentrated solution was hot, hydrochloric acid was added dropwise to adjust the pH to about 2. The mixture was then filtered. At this time, the precipitate was very fine and difficult to filter. There were also brown viscous fragments mixed in. The filtered filter cake was dried at 70°C to obtain crude UDCA.

[0052] The prepared crude UDCA was added to a reaction flask, followed by 10 times the volume of DMF. After stirring and dissolving, 3 times the volume of crude ursodeoxycholic acid (HMDS) was added. The mixture was reacted at 40°C for 2 hours, then cooled to room temperature and maintained for 24 hours. No crystals precipitated. The reaction solution was then placed in an ice-water bath overnight, resulting in crystal precipitation. The crystals were filtered and washed with ice water. The filter cake was a mixture of pale yellow crystals and brownish-yellow debris.

[0053] After hydrolysis of the above-mentioned UDCA silanized derivative, it was dissolved in 10 times the amount of 75% ethanol and recrystallized to prepare 42.2g of ursodeoxycholic acid final product. The target recovery rate was only 42.4%, the target content was 85.5%, and the product had a yellow appearance with brownish-yellow debris mixed in.

[0054] Comparative Example 2 The specific implementation method is the same as in Example 1, except that the silanizing reagent is trimethyldichlorosilane. After suspending crude ursodeoxycholic acid in acetone, the temperature was slowly raised to 60°C and maintained for 1 hour. Trimethyldichlorosilane was then added dropwise for purification. The final purified ursodeoxycholic acid yielded approximately 36.5 g, with a UDCA content of 93.5%, a CDCA content of 1.7%, and 7-K not detected.

[0055] Comparative Example 3 The specific implementation method is the same as in Example 1, except that the silanizing agent is dimethyldichlorosilane. After adding crude ursodeoxycholic acid to acetone for suspension, the temperature is slowly raised to 60°C and maintained for 1 hour. Dimethyldichlorosilane is then added dropwise for purification. The final purified ursodeoxycholic acid is about 32.7 g, with UDCA content of 95.6%, CDCA content of 2.3%, and 7-K not detected.

[0056] Comparative Example 4 The specific implementation method is the same as in Example 2, except that the final solution is dissolved in 10 times the amount of 85% acetone water and recrystallized at 0-5℃ to obtain approximately 17.6g of purified ursodeoxycholic acid with a UDCA content of 99.2%, a CDCA content of 0.06%, and 7-K not detected.

[0057] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for synthesizing ursodeoxycholic acid by whole cell transformation, characterized by, The specific steps are: (1) using the bacteria expressing 7α-hydroxysteroid dehydrogenase and the bacteria expressing 7β-hydroxysteroid dehydrogenase for whole cell transformation to obtain a whole cell reaction solution; (2) adding 6-10 times the volume of organic reagent to the whole cell reaction solution, stirring uniformly, standing at 5-20℃ for not less than 2h, centrifuging, and taking the supernatant, and concentrating and drying the supernatant to obtain a dried reaction product of ursodeoxycholic acid; (3) adding an acetone solution to the dried reaction product of ursodeoxycholic acid in step (2), refluxing at 50-60℃ for 0.5-2h, cooling, and filtering to obtain a filtrate; (4) adding purified water to the filtrate obtained in step (3), mixing, and concentrating to obtain a crude ursodeoxycholic acid product; (5) dissolving the crude ursodeoxycholic acid product obtained in step (4) in an organic reagent to perform a silylation reaction; (6) after the silylation reaction is completed, cooling the solution to below 0℃ until crystallization is complete, washing the crystals with a small amount of solvent, and collecting the precipitate; (7) adding an appropriate amount of inorganic acid to the precipitate in step (6) to perform hydrolysis; (8) after the hydrolysis is completed, cooling the reaction solution to 4-10℃, filtering to obtain a filter cake, and rinsing the filter cake with purified water to remove residual acid solution, and collecting the filter cake; (9) taking the filter cake in step (8), recrystallizing with acetone-water, drying, and obtaining a purified ursodeoxycholic acid product.

2. The method of claim 1, wherein, The organic reagent added in step (2) includes but is not limited to methanol, ethanol, and acetone.

3. The method according to claim 1 or 2, characterized in that, Step (3) is adding 5-20 times the volume of a 70%-80% concentration acetone solution to the dried reaction product of ursodeoxycholic acid in step (2).

4. The method according to any one of claims 1 to 3, characterized in that, The cooling temperature in step (3) is 10-20℃, and the filtration is suction filtration.

5. The method according to any one of claims 1 to 4, characterized in that, In step (4), 5-10 times the volume of purified water is added dropwise to the filtrate, and concentrated to 40%-50% volume.

6. The method according to any one of claims 1 to 5, characterized in that, The organic reagent in step (5) is dimethylformamide, ethyl acetate, methanol, acetonitrile, or acetone.

7. The method according to any one of claims 1 to 6, characterized in that, The silylation reagent in step (5) is one or more of tetramethyldisilazane, hexamethyldisilazane, or hexamethyldisilazane.

8. The method of any one of claims 1-7, wherein, The solvent in step (6) includes acetonitrile, N,N-dimethylformamide, or acetone.

9. The method of any one of claims 1-8, wherein, The inorganic acid in step (7) includes sulfuric acid or hydrochloric acid.

10. The method of any one of claims 1-9, wherein, In step (9), the acetone-water contains 35%-80% acetone.

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