An improved method for preparing sitagliptin by transaminase catalysis

Abstract

The present invention discloses an improved method for preparing sitagliptin by transaminase catalysis, which is characterized in that: 1) feeding and reacting, using a low-concentration cosolvent-water buffer medium system for the transaminase-catalyzed reaction, and directly putting the crushed sitagliptin precursor ketone into the reaction system without dissolving it with a solvent; 2) reaction process control, using a pH automatic control liquid adding machine to automatically regulate the pH during the enzyme reaction process; 3) reaction treatment, after the reaction ends, the enzyme reaction solution is acidified and then the product is separated by a nano-ceramic membrane, the membrane filtrate is alkalized to precipitate the product, and then the product sitagliptin is prepared by recrystallization. The present invention greatly reduces the solvent consumption, effectively improves the preparation efficiency of sitagliptin, is easy to operate in industrial production, and reduces the production cost.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals. More specifically, the present invention relates to an improved method for preparing sitagliptin by transaminase catalysis. Background Art

[0002] Sitagliptin, also known as sitagliptin, with the chemical name of (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)-butan-2-amine, is a novel hypoglycemic drug developed by Merck and approved by the FDA for the treatment of type II diabetes. Sitagliptin can increase insulin secretion in a glucose-dependent manner, has a moderate hypoglycemic effect, does not cause hypoglycemia, and has no side effects such as weight gain, nausea, and vomiting. The trade name of sitagliptin is Januvia. The chemical structural formula of sitagliptin is as follows:

[0003]

[0004] Many synthetic processes of sitagliptin have been reported [Zhejiang Chemical Industry, 2017, 48(9): 15-19]. The main method is asymmetric synthesis. Industrially, the method developed by Merck is mainly used. Its first-generation synthesis method uses methyl 4-(2,4,5-trifluorophenyl)-3-oxobutyrate as a raw material and undergoes multiple steps of reaction catalyzed by a chiral ruthenium catalyst to obtain sitagliptin; the second-generation synthesis method uses 2,4,5-trifluorophenylacetic acid as a raw material to synthesize an enamine intermediate, and then uses a chiral rhodium catalyst to catalyze an asymmetric hydrogenation reaction to obtain sitagliptin; the third-generation synthesis method uses 2,4,5-trifluorophenylacetic acid as a raw material to synthesize 1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)-1,3-butanedione (sitagliptin precursor ketone), and then uses transaminase as a biocatalyst to catalyze a transamination reaction to obtain sitagliptin. The reaction formula is as follows:

[0005]

[0006] The method for synthesizing sitagliptin by transaminase was initially jointly developed by Merck and Codexis. Subsequently, many similar methods have been developed at home and abroad. Due to the poor water solubility of the raw materials, a large amount of co-solvent is often used for dissolution before being added to the reaction. For example, in the original transaminase-catalyzed synthesis process [Science, 2010, 329(5989): 305-309], the dosage of co-solvent DMSO is 50% (V / V). In CN109777813 B and [Chemical Engineering Design Communications, 2019, 45(12): 215-216] as well as [Journal of Shenyang Pharmaceutical University, 2014, 31(11): 867-869, 890], etc., the dosage of co-solvent DMSO is 20-50% (V / V). In CN116240189A and CN112048485B, etc., the dosage of co-solvent methanol is 30-50% (V / V). WO2023181046 uses a large amount of isopropyl acetate as the co-solvent. Using a large amount of organic solvents in the enzyme reaction system will cause many disadvantages. First, a large amount of solvents are likely to cause enzyme inactivation. Second, solvent recovery is difficult. Third, the separation process of the product is lost due to solvent dissolution. Fourth, the increase in solvent dosage and waste solvents leads to an increase in cost. Fifth, after the raw materials are dissolved in the solvent and added to the aqueous reaction medium in large quantities, if they are not reacted in time, they are likely to precipitate into an oily state and form lumps, affecting the reaction effect. In terms of the reaction process control of transaminase for synthesizing sitagliptin, since the co-substrate isopropylamine is consumed to generate acetone, the pH of the reaction process will decrease, and the pH needs to be continuously adjusted. The specific method of pH adjustment is not mentioned in the relevant literature including the original research process. Finally, in terms of the treatment and separation of the enzyme reaction solution to obtain the product, all relevant literatures use the extraction method. Due to enzyme protein emulsification and the use of a large amount of co-solvents, the extraction separation effect is not good, and the solvent dosage is relatively large.

[0007] This application aims to improve the existing method by adopting the strategy of low co-solvent concentration and adding the raw materials in one-time after crushing. After the raw materials are crushed and added to the reaction system, a small amount of co-solvent is added, reducing the impact of the solvent on the reaction and eliminating the solvent recovery process, reducing the inactivation effect of the solvent on the enzyme, reducing the loss of product separation caused by the solvent, making the raw materials easier to disperse without forming lumps, and making the reaction simple and easy to handle; by using a pH automatic control liquid adding machine to automatically regulate the reaction pH and improve the reaction efficiency; using a nano-ceramic membrane for product separation, simplifying the separation operation and reducing the solvent dosage. After the improvement, the solvent dosage is significantly reduced, the operation steps are simplified, the production cost is reduced, showing good potential for industrial application. Summary of the Invention

[0008] In view of the technical deficiencies in the method for synthesizing sitagliptin by transaminase disclosed in the existing public literature, the present invention provides an improved method for synthesizing sitagliptin by transaminase, and the method comprises the following steps:

[0009]

[0010] Step 1) Feeding and reaction: The transaminase-catalyzed reaction is carried out in a low-concentration co-solvent-water buffer medium system. After the raw material sitagliptin precursor ketone is pulverized, it is directly added to the reaction system without being dissolved in a solvent. First, prepare an isopropylamine-water buffer solution, then add PLP (pyridoxal phosphate), a small amount of co-solvent, and the raw material powder. Turn on the temperature to the set temperature, and finally add the transaminase to start the reaction.

[0011] Step 2) Reaction process control: During the enzyme reaction process, the pH is automatically regulated using a pH automatic control liquid addition machine. First, prepare an isopropylamine-water solution for the pH automatic control liquid addition machine to adjust the reaction pH to the set pH value.

[0012] Step 3) Reaction treatment: After the reaction ends, the enzyme reaction solution is acidified and then separated by a nanoceramic membrane to obtain the product. The membrane filtrate is alkalized to precipitate the product, and then the product sitagliptin is prepared by recrystallization.

[0013] Furthermore, in Step 1), the R-transaminase is a transaminase capable of converting sitagliptin precursor ketone prepared according to the methods in the published literature, such as the mutant ATA-117 transaminase (code ATA-117M, gene sequence NCBI ID: JA717225.1) derived from Arthrobacter sp. KNK168 in [Science, 2010, 329(5989):305-309], the transaminase mutant ATA-64 (code ATA-64, amino acid sequence SEQ ID NO: 3 and gene sequence SEQ ID NO: 4) derived from Arthrobacter sp. KNK168 in CN116240189A, and the amino acid sequence SEQ ID NO: 11 (code ATA-M11, gene sequence SEQ ID NO: 12) of the transaminase mutant derived from Aspergillus fumigatus in CN112048485B. Preferably, it is the mutant ATA-117 transaminase derived from Arthrobacter sp. KNK168.

[0014] Furthermore, in Step 1), the co-solvent is DMSO, methanol, ethanol, isopropanol, tert-butanol, acetonitrile, dioxane, ethylene glycol, THF, isopropyl acetate, etc. Preferably, it is ethanol.

[0015] Furthermore, in Step 1), the concentration of the co-solvent ethanol is ≤20% V / V. Preferably, the ethanol concentration is 5% V / V.

[0016] Furthermore, in Step 1), the water buffer is Tris-hydrochloric acid, triethanolamine-hydrochloric acid, phosphate, boric acid-borax, sodium carbonate-sodium bicarbonate, glycine-sodium hydroxide, etc. Preferably, it is glycine-sodium hydroxide.

[0017] Further, in step 1), the raw material powder is prepared by pulverizing sitagliptin precursor ketone with a pulverizer and then passing through an 80-mesh sieve.

[0018] Further, in step 2), the concentration of the isopropylamine aqueous solution used for adjusting the pH by the pH automatic control liquid adding machine is not limited, and preferably a 4M isopropylamine aqueous solution is used.

[0019] Further, in step 3), the pore diameter of the ceramic membrane is ≤ 5 nm, and preferably the pore diameter is 2 nm.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] a) By adopting the strategy of low cosolvent concentration and adding the raw materials in one go after pulverization, the pulverized raw materials are added into the reaction system, then a small amount of cosolvent is added, the influence of the solvent on the reaction is reduced, the solvent recovery process is omitted, the inactivation effect of the solvent on the enzyme is reduced, the separation loss of the product caused by the solvent is reduced, the raw materials are more easily dispersed and do not form lumps, and the reaction is simple and easy to handle.

[0022] b) By using a pH automatic control liquid adding machine to automatically regulate the reaction pH, the reaction efficiency is improved.

[0023] c) Using a nano-ceramic membrane for product separation simplifies the separation operation and reduces the solvent consumption. The nano-ceramic membrane can effectively retain enzyme proteins, impurity proteins, cell debris, and water-insoluble unreacted raw materials. The membrane filtrate is easy to purify and separate the product subsequently, and no extraction operation is required.

[0024] After improvement, the solvent consumption is greatly reduced, the operation steps are simplified, the production cost is reduced, and it shows good potential for industrial application. Specific Embodiments

[0025] To further understand the present invention, the following examples are used to illustrate in detail the improved method for synthesizing sitagliptin by transaminase provided by the present invention. It should be understood that these embodiments are only for further elaborating the features of the present invention, rather than limiting the scope of the present invention or the scope of the claims of the present invention.

[0026] Experimental equipment: 1) pH automatic control liquid adding machine: model CPH-2-X, Changsha Cromar Instrument Equipment Co., Ltd.; 2) Nano-ceramic membrane separation system: model JWCMF-0.1, Jiangsu Jiuwu High-tech Co., Ltd.

[0027] Analysis methods: 1) Conversion rate HPLC detection method: Chromatographic column: Agilent Eclipse XDB-C8 column (4.6 × 150 mm, 5 μm), mobile phase: 10 mM ammonium acetate - acetonitrile (volume ratio 50:50), flow rate: 1.0 mL / min, column temperature: 40 °C, detection wavelength 210 nm. 2) Chiral HPLC detection method: Chromatographic column: Daicel Chiralpak AD-H (4.6 × 150 mm, 5 µm), mobile phase: ethanol - n-hexane - diethylamine (volume ratio 60:40:0.1), flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength 268 nm.

[0028] Enzyme solution preparation method: Obtain the mutant ATA-117 transaminase gene sequence (NCBI ID: JA717225.1) from Arthrobacter sp. KNK168 from [Science, 2010, 329(5989):305 - 309], obtain the gene sequence SEQ ID NO: 4 of the transaminase mutant ATA-64 from Arthrobacter sp. KNK168 from CN116240189A, obtain the gene sequence SEQ ID NO: 12 of the transaminase mutant SEQ ID NO: 11 from Aspergillus fumigatus from CN112048485B, synthesize the enzyme gene, clone it onto the pET28a vector, then construct recombinant genetically engineered Escherichia coli. Inoculate the genetically engineered bacteria into LB liquid medium containing 50 μg / mL kanamycin, culture at 37 °C and 200 rpm for 12 h. Inoculate the seed liquid at 1% (v / v) into LB liquid medium containing 50 μg / mL kanamycin, culture at 37 °C and 200 rpm for 3 h, cool down to 30 °C, add the inducer IPTG to a concentration of 0.2 mM, induce and culture at 30 °C and 200 rpm for 20 h, collect the fermentation broth, centrifuge to obtain the cells, suspend the cells in a 0.1 M buffer solution with pH 9.0, the cell concentration is 20%, and then break the cells with an ultrasonic crusher to obtain the enzyme solution.

[0029] Raw material treatment method: The sitagliptin precursor ketone, self-made by the company, with a purity > 99%, is crushed with a high-speed multi-functional crusher, the crushing degree is 70 - 300 mesh, and after crushing, it is sieved through an 80-mesh sieve.

[0030] Enzymatic catalysis small-scale reaction method: 1) Reaction conditions: feeding concentration 100 g / L, enzyme-substrate ratio 0.5 / 1 (bacterial cells / raw materials), reaction medium 0.1 M buffer and cosolvent, concentration of co-substrate isopropylamine 1 M, concentration of coenzyme PLP 1.0 mM, pH 10.0, temperature 50 °C, magnetic stirring reaction. 2) Reaction operation process for 10 g of feed: Weigh 5 g of bacterial cells, add 25 mL of buffer, and prepare enzyme solution according to the aforementioned enzyme solution preparation method; weigh 5.9 g of isopropylamine, add it to 20 mL of buffer, adjust to pH 10.0 with concentrated hydrochloric acid, and make up the volume to 35 mL with buffer; take a 250 mL reaction flask, add a magnetic stirring bar, the prepared isopropylamine buffer solution, 20 mg of PLP, 10 g of crushed and sieved raw materials, 40 mL of cosolvent and buffer in total, turn on the temperature and magnetic stirring, and finally add the prepared enzyme solution. The total reaction system is 100 mL. When the reaction starts, the pH automatic control liquid adding machine automatically regulates the pH to 10.0 with 4 M isopropylamine aqueous solution. After reacting for 24 h, add hydrochloric acid to adjust to pH 2.0 and stir for 0.5 h to terminate the reaction and hydrolyze the dimer, and then sample for analysis.

[0031] Example 1 Transaminase reactions from different sources

[0032] The enzyme solution was prepared from bacterial cells with Tris-HCl buffer according to the aforementioned enzyme solution preparation method. The cosolvent was DMSO 30% (V / V), and other conditions were the same as the aforementioned enzymatic catalysis small-scale reaction method. The reaction results are shown in Table 1. All the transaminases investigated had high selectivity, and the product had >99.5% ee. However, the enzymatic catalytic activity of ATA-117M was the best, followed by ATA-64. The two had the same source and high similarity. The enzymatic activity of ATA-M11 from other sources was lower than the former two.

[0033] Table 1 Reaction results of transaminases from different sources

[0034] Transaminase Enzyme code Conversion rate ee% Arthrobacter sp. KNK168 mutant ATA-117 transaminase ATA-117M 94.35% 99.73% Arthrobacter sp. KNK168 transaminase mutant ATA-64 ATA-64 93.29% 99.74% Aspergillus fumigatus transaminase mutant SEQ ID NO: 11 ATA-M11 81.65% 99.59%

[0035] Example 2 Transaminase ATA-117M reactions with different types of cosolvents

[0036] The enzyme solution of ATA-117M from bacterial cells was prepared with Tris-HCl buffer according to the aforementioned enzyme solution preparation method. The cosolvent was 10% (V / V), and other conditions were the same as the aforementioned enzymatic catalysis small-scale reaction method. The reaction results are shown in Table 2. Among the cosolvents investigated, DMSO was the best, followed by methanol and ethanol.

[0037] Table 2 Reaction results of transaminase ATA-117M with different types of cosolvents

[0038] Cosolvent Conversion rate ee% None 76.95% 99.77% DMSO 91.22% 99.75% Methanol 88.95% 99.76% Ethanol 87.88% 99.75% Isopropanol 74.64% 99.73% tert-Butanol 77.50% 99.75% Acetonitrile 64.80% 99.70% Dioxane 69.85% 99.74% Ethylene glycol 85.44% 99.78% THF 75.86% 99.77% Isopropyl acetate 57.87% 99.71%

[0039] Example 3 Transaminase ATA-117M reactions with different concentrations of ethanol as cosolvent

[0040] The enzyme solution of the bacterial strain ATA-117M was prepared using Tris-HCl buffer according to the aforementioned method for preparing the enzyme solution. The cosolvent ethanol was 0 - 30% (V / V), and other conditions were the same as the aforementioned small-scale enzyme-catalyzed reaction method. The reaction results are shown in Table 3. The investigation result shows that 5% (V / V) ethanol as the cosolvent is the best. Both too high and too low concentrations are not conducive to the conversion reaction. Therefore, the amount of the cosolvent ethanol was determined to be 5% (V / V).

[0041] Table 3 Reaction results of transaminase ATA-117M with different concentrations of cosolvent ethanol

[0042] Ethanol dosage (V / V) Conversion rate ee% 30% 37.33% 99.73% 20% 69.95% 99.77% 10% 87.95% 99.72% 5% 90.26% 99.76% 0% 77.91% 99.78%

[0043] Example 4 Reaction of transaminase ATA-117M in different reaction media

[0044] The enzyme solution of the bacterial strain ATA-117M was prepared using different reaction media (buffers) according to the aforementioned method for preparing the enzyme solution. The cosolvent was 5% (V / V) ethanol, and other conditions were the same as the aforementioned small-scale enzyme-catalyzed method. The reaction results are shown in Table 4.

[0045] Table 4 Reaction results of transaminase ATA-117M in different reaction media

[0046] Buffer solution Conversion rate ee% Tris-HCl 90.63% 99.76% Triethanolamine-HCl 88.75% 99.73% Phosphate 88.85% 99.77% Boric acid-borax 89.76% 99.78% Sodium carbonate-sodium bicarbonate 90.88% 99.72% Glycine-sodium hydroxide 90.30% 99.74%

[0047] Example 5 Preparation reaction of transaminase ATA-117M under optimized conditions

[0048] For the enzyme-catalyzed reaction with 100 g of transaminase ATA-117M on a magnified scale, under the optimized conditions of 5% (V / V) ethanol as the cosolvent and 0.1 M glycine-sodium hydroxide buffer, other conditions were the same as the aforementioned method for preparing the enzyme solution and the small-scale enzyme-catalyzed reaction method. The total reaction system was 1 L. After reacting for 24 h, acid hydrolysis treatment was carried out, and the conversion rate was measured to be 90.55% by sampling. The reaction solution was centrifuged, the precipitate was washed twice with 100 mL of water and centrifuged, the supernatant liquids were combined, extracted twice with 500 mL of isopropyl acetate to remove the residual raw materials, the aqueous phase was adjusted to pH 11.0, extracted twice with 500 mL of isopropyl acetate, the extraction phases were combined, washed with 200 mL of saturated sodium chloride solution, dehydrated with anhydrous sodium sulfate, and the product solid of 75.8 g was obtained by vacuum distillation, with a purity of 99.84% and an optical purity of 99.74% ee.

[0049] Example 6 Magnified preparation reaction of transaminase ATA-117M under optimized conditions

[0050] The transaminase ATA-117M was used for an enzyme-catalyzed reaction with a 1 kg scale-up feed. According to the optimized conditions, the cosolvent was 5% (V / V) ethanol and 0.1 M glycine-sodium hydroxide buffer solution. Other conditions were the same as those in the aforementioned enzyme solution preparation method and the small-scale enzyme-catalyzed reaction method. The total reaction system was 10 L. After reacting for 24 h, acid hydrolysis treatment was carried out, and the conversion rate was measured to be 90.47% by sampling. The 10.1 L reaction solution was filtered using a small ceramic membrane (membrane pore diameter 2 nm), and the membrane filtrate was collected. When the membrane filtrate was about 9 L, 2 L of 0.1 M hydrochloric acid aqueous solution was added to the membrane filtration mother liquor, and membrane filtration was continued. The combined membrane filtrates were collected to obtain 10.8 L, and the membrane filtration was ended. The mother liquor was discharged and discarded, and the membrane equipment was cleaned. The membrane filtrate was cooled to 30 °C, and 20% sodium hydroxide aqueous solution was slowly added with stirring until the pH reached 12 to precipitate the product. Stirring and heat preservation were carried out for 8 h, and 857 g of the crude product was obtained by filtration and drying. The crude product was dissolved in ethanol, filtered through diatomaceous earth, an appropriate amount of pure water was added to the filtrate, heated to dissolve clearly, cooled to crystallize, and 761 g of a white solid was obtained by filtration and drying. The purity was 99.87%, and the optical purity was 99.76% ee. The results of the scale-up reaction after process optimization reached the expectation.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An improved method for preparing sitagliptin by transaminase catalysis, the method comprising: Step 1) Feeding reaction, using a low concentration cosolvent-water buffer medium system for transaminase catalysis reaction, the raw material sitagliptin precursor ketone is crushed and directly added to the reaction system without solvent dissolution, first prepare isopropylamine-water buffer solution, then add PLP (pyridoxal phosphate), a small amount of cosolvent, raw material powder, turn on the temperature to the set temperature, and finally add transaminase to start the reaction; Step 2) reaction process control: the enzyme reaction process uses a pH automatic control liquid adding machine to automatically control the pH. First, an isopropylamine-water solution is prepared for the pH automatic control liquid adding machine to adjust the reaction pH to a set pH value; Step 3) reaction treatment, after the reaction is completed, the enzyme reaction solution is treated with acid and then separated by a nano-ceramic membrane, the membrane filtrate is alkali-adjusted to precipitate the product, and then recrystallized to prepare the product sitagliptin; In the step 1), the cosolvent is ethanol; the ethanol concentration is 10% V / V, and the raw material powder is prepared by grinding the sitagliptin precursor ketone in a grinder and then passing through an 80-mesh sieve; The R-aminotransferase is ATA-117M.

2. The method according to claim 1, characterized in that In the step 1), the aqueous buffer solution is Tris-hydrochloric acid, triethanolamine-hydrochloric acid, phosphate, boric acid-borax, sodium carbonate-sodium bicarbonate or glycine-sodium hydroxide.

3. The method according to claim 1, characterized in that In the step 2), the isopropylamine aqueous solution used for the pH automatic control liquid adding machine to adjust the pH is a 4M isopropylamine aqueous solution.

4. The method according to claim 1, characterized in that: In the step 3), the membrane pore diameter of the ceramic membrane is ≤5 nm.

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