Preparation method of sitagliptin intermediate

Abstract

The invention discloses a preparation method of a sitagliptin intermediate, which comprises the following steps of: catalyzing sitagliptin precursor ketone 1-(3-trifluoromethyl)-5, 6-dihydro-8H-[1, 2, 4] triazole-[4, 3-a] pyrazine-7 (8H)-yl)-4-(2, 4, 5-trifluorophenyl)-1, 3-butanedione by transaminase in the presence of a solvent system to obtain (R)-3-amino-1-[3-(trifluoromethyl)-5, 6, 7, 8-tetramethyl-1, 3-pentanedione; the invention relates to a preparation method of 2, 4, 6, 7, 8-tetrahydro-1, 2, 4-triazolo [4, 3-a] pyrazine-7-yl]-4-(2, 4, 5-trifluorophenyl) butyl-1-one. The product obtained by the preparation method is high in conversion rate, separation and purification of the product are facilitated, the preparation process is simplified, and the cost is reduced.

Description

Technical Field

[0001] The present application relates to the technical field of chemical synthesis, and in particular to a method for preparing a sitagliptin intermediate. Background Art

[0002] Sitagliptin phosphate, the ingredient is (3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one. The key step in the synthesis of sitagliptin phosphate is the construction of chiral amino intermediates. The chiral amino intermediates generated by transaminase catalysis have high stereoselectivity, and the ee value of the transamination product can reach 99% or more. It can directly meet the pharmacopoeia requirements without recrystallization, and the reaction conditions are mild, environmentally friendly, and can greatly reduce industrial costs.

[0003] In the catalytic process of the transaminase catalytic synthesis of sitagliptin chiral amine found at present, the conversion rate is unstable due to the reaction conditions, and the organic solvent used, such as DMSO, is difficult to recycle, and the product recovery is difficult, so that the product yield is not high. These catalytic processes cannot meet the high conversion rate of the product and the cost requirements of industrial production. Therefore, the development of a suitable enzymatic reaction process is the key to reduce the enzyme catalytic synthesis of sitagliptin chiral amine. Summary of the invention

[0004] The purpose of the present application is to provide a method for preparing a sitagliptin intermediate, which can solve the above-mentioned technical problems.

[0005] The present invention provides a method for preparing a sitagliptin intermediate, comprising the following steps:

[0006] Sitagliptin precursor ketone 1-(3-trifluoromethyl)-5,6-dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)-1,3-butanedione is catalyzed by transaminase in the presence of a solvent system to obtain (R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one.

[0007] In some embodiments, the solvent system includes at least one of methyl tert-butyl ether, ethyl acetate, or isopropanol.

[0008] In some embodiments, the mass percentage of water in the solvent system is ≤10%.

[0009] In some embodiments, the mass percentage of water in the solvent system is 0.01% to 10%.

[0010] In some embodiments, the sitagliptin precursor ketone is catalyzed by transaminase in the presence of isopropylamine to obtain the sitagliptin intermediate.

[0011] In some embodiments, the sitagliptin precursor ketone is catalyzed by transaminase in the presence of pyridoxal phosphate to obtain the sitagliptin intermediate.

[0012] In some embodiments, the concentration of the sitagliptin proketone is 0.1-0.8 mol / L.

[0013] In some embodiments, the concentration of isopropylamine is 0.2-5 mol / L.

[0014] In some embodiments, the concentration of pyridoxal phosphate is 5-100 mmol / L.

[0015] In some embodiments, the mass ratio of the sitagliptin precursor ketone to the transaminase is 1:(2-5).

[0016] In some embodiments, the reaction temperature is 15-65°C.

[0017] In some embodiments, the transaminase is at least one of whole cells, enzyme powder, ammonium sulfate enzyme powder and immobilized enzyme.

[0018] In some embodiments, the nucleotide sequence encoding the transaminase is as shown in SEQ ID NO: 1 to SEQ ID NO: 5, or is a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to SEQ ID NO: 1 to SEQ ID NO: 5.

[0019] In some embodiments, the amino acid sequence of the transaminase is as shown in SEQ ID NO: 6 to SEQ ID NO: 10, or is an amino acid that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to SEQ ID NO: 6 to SEQ ID NO: 10.

[0020] The beneficial effect of the present application is that the present application provides a method for synthesizing a sitagliptin intermediate, comprising the following steps: the sitagliptin precursor ketone 1-(3-trifluoromethyl)-5,6-dihydro-8H-[1,2,4]triazol-[4,3-a]pyrazine-7(8H)-yl)-4-(2,4,5-trifluorophenyl)-1,3-butanedione is catalyzed by transaminase in the presence of a solvent system to obtain (R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazine-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one. The preparation method of the present application has a high conversion rate of the product obtained, is conducive to the separation and purification of the product, simplifies the preparation process, and reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 This is the HPLC chromatogram of the conversion rate detection of Example 1 of the present application;

[0023] Figure 2 This is the HPLC chromatogram of the conversion rate detection of Example 7 of the present application;

[0024] Figure 3 This is the HPLC chromatogram for conversion rate detection of Example 20 of the present application;

[0025] Figure 4 This is an HPLC chromatogram for detecting the conversion rate of the immobilized enzyme reaction system prepared using Sequence 1 in Example 24 of the present application. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application. In addition, in the description of the present application, the term "including" means "including but not limited to". The terms first, second, third, etc. are used only as markings, and no numerical requirements or order are imposed. Various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a hard limit to the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered range, such as 1, 2, 3, 4, 5 and 6, which are applicable regardless of the range. Additionally, whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.

[0027] In order to solve the problems of low conversion rate of sitagliptin intermediate products, low product separation and purification rate, low solvent recovery rate, high production cost, etc., the embodiment of the present application provides a method for preparing sitagliptin intermediates, comprising the following steps: preparing sitagliptin precursor ketone 1-(3-trifluoromethyl)-5,6-dihydro-8H-[1,2,4]triazole-[4,3-a]pyrazine-7(8H)-yl)-4-(2,4,5-trifluorophenyl)-1,3-butanedione ( CAS: 764667-65-4) in the presence of a solvent system, catalyzed by transaminase to obtain (R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one ( CAS: 486460-32-6). The preparation method of the sitagliptin intermediate of the present application can at least solve one of the above technical problems.

[0028] In some embodiments, the solvent system includes at least one of methyl tert-butyl ether, ethyl acetate, or isopropanol.

[0029] In some embodiments, the solvent system comprises methyl tert-butyl ether (MTBE). The present application adopts MTBE as a solvent because MTBE has a low boiling point, which is conducive to the separation and purification of the product.

[0030] In some embodiments, the mass percentage of water in the solvent system is ≤10%.

[0031] In some embodiments, the mass percentage of water in the solvent system is 0..01% to 10%, such as the mass percentage (%) of water in the solvent system is 0.01, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or any range consisting of any two values. The applicant unexpectedly discovered that controlling the water content in the solvent system for the synthesis of the sitagliptin intermediate can increase the conversion rate of the product and simplify the purification step. The applicant also found that the solvent system is universal in the synthesis process of the sitagliptin intermediate and is suitable for different transaminase systems, avoiding the situation where the product conversion rate fluctuates due to changes in the reactants.

[0032] In some embodiments, the applicant first dehydrates the reactants added to the reaction system to control the water content in the entire system.

[0033] In some embodiments, the sitagliptin precursor ketone is catalyzed by transaminase in the presence of isopropylamine to obtain a sitagliptin intermediate. In some embodiments, the sitagliptin precursor ketone is catalyzed by transaminase in the presence of pyridoxal phosphate to obtain a sitagliptin intermediate. Wherein, pyridoxal phosphate is used as a coenzyme and isopropylamine is used as a cosubstrate.

[0034] In some embodiments, the concentration of sitagliptin proketone is 0.1-0.8 mol / L, such as the concentration (mol / L) of sitagliptin proketone is any value of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or a range consisting of any two values.

[0035] In some embodiments, the concentration of isopropylamine is 0.2-5 mol / L, such as the concentration (mol / L) of isopropylamine is any value of 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or a range consisting of any two values.

[0036] In some embodiments, the concentration of pyridoxal phosphate is 5 to 100 mmol / L, such as the concentration of pyridoxal phosphate (mmol / L) is any value of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or a range consisting of any two values.

[0037] In some embodiments, the mass ratio of sitagliptin proketone to transaminase is 1:(2-5), such as the mass ratio of sitagliptin proketone to transaminase is any value of 1:2, 1:3, 1:4, 1:5 or a range consisting of any two values.

[0038] In some embodiments, the reaction temperature is 15-65°C, such as the reaction temperature (°C) is any value of 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or a range consisting of any two values.

[0039] In some embodiments, the transaminase is at least one of whole cells, enzyme powder, ammonium sulfatase powder, and immobilized enzyme.

[0040] In some embodiments, the nucleotide sequence encoding the transaminase is as shown in SEQ ID NO: 1 to SEQ ID NO: 5, or is a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to SEQ ID NO: 1 to SEQ ID NO: 5.

[0041] In some embodiments, the amino acid sequence of the transaminase is as shown in SEQ ID NO: 6 to SEQ ID NO: 10, or is an amino acid that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to SEQ ID NO: 6 to SEQ ID NO: 10.

[0042] As used in this application, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0043] The preparation of the transaminase of the present application uses conventional means to express the transaminase gene. Different forms of transaminase can be obtained in the following ways: A: collect whole cells by centrifugation and spray dry to obtain dehydrated whole cells; B: whole cells are ultrasonically broken and freeze-dried to obtain enzyme powder; C: whole cells are ultrasonically broken and precipitated with 80% ammonium sulfate to obtain ammonium sulfate enzyme powder; D: after cells are ultrasonically broken, the enzyme is immobilized using an immobilized enzyme carrier to obtain an immobilized enzyme.

[0044] In some embodiments, the transaminase of the present application is prepared by the following method:

[0045] Constructing genetically engineered bacteria containing transaminase encoding genes as shown in SEQ ID NO: 1 to SEQ ID NO: 5: Construct genetically engineered bacteria containing SEQ ID NO: 1 to SEQ ID NO: 5 respectively. The following is a detailed description of constructing a genetically engineered bacteria containing a transaminase encoding gene as shown in SEQ ID NO: 1 as an example:

[0046] Construction of recombinant expression vector pUC19-WT: pUC-19 plasmid without foreign gene inserted was selected as the vector. pUC-19 plasmid has restriction sites BamH I and Nde I. The construction of pUC19-WT includes the following steps:

[0047] Artificially synthesizing a nucleotide sequence as shown in SEQ ID NO: 1, and then using BamH I and Nde I restriction endonucleases to double-digest the synthesized nucleotide sequence, and after detecting the complete digestion by 1% agarose gel electrophoresis, recovering the target gene fragment by gel, wherein the operation of recovering the target gene fragment after double digestion is carried out according to the operating instructions of the gel recovery kit;

[0048] The pUC-19 plasmid was double-digested with BamH I and Nde I restriction endonucleases, and the vector skeleton was recovered by gel electrophoresis after the complete digestion was detected by 1% agarose gel electrophoresis. The operation of recovering the vector skeleton after double-digestion was carried out according to the operating instructions of the gel recovery kit;

[0049] The obtained target gene fragment was mixed with the obtained vector backbone, and connected at 16°C overnight under the action of T4 ligase. The ligation product was then transformed into DH5a competent cells, and single clones were picked for sequencing verification. The recombinant plasmid with correct sequencing was extracted to obtain a recombinant expression vector containing the transaminase encoding gene of SEQ ID NO: 1. The ligation system was 20 μL, specifically: 2 μL of 10×T4 ligase buffer (Buffer), 5 μL of target gene fragment, 5 μL of vector backbone, 2 μL of T4 ligase and 6 μL of double distilled water (ddH2O).

[0050] Induced expression and post-treatment of genetically engineered bacteria containing transaminase encoding genes: The genetically engineered bacteria containing the transaminase encoding gene of SEQ ID NO: 1 are inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 r / min until OD600 is 0.6-0.8 to obtain a seed bacterial liquid. The seed bacterial liquid is inoculated into a fresh auto-induction culture medium containing 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 30°C for 18 hours to obtain a culture solution. The culture solution is centrifuged at 25°C and 8000 r / min for 10 minutes, the supernatant is discarded to collect the precipitate, the precipitate is washed several times with PB buffer at pH 7.0, and the wet bacteria are collected for later use. The preparation method of the autoinduction medium is as follows: 120g of yeast powder, 60g of tryptone, 0.75g of magnesium sulfate (MgSO4), 16.5g of ammonium sulfate ((NH4)2SO4), 32.5g of potassium dihydrogen phosphate (KH2PO4), 35.5g of disodium hydrogen phosphate (Na2HPO4), 2.5g of glucose and 10g of α-lactose are weighed respectively, and then all the weighed components are added to the grinder, and fully ground into powder to obtain the powdered autoinduction medium. The powdered autoinduction medium is dissolved in 1L of deionized water, the pH is adjusted to 7.0 after being fully mixed, and then sterilized at 121°C for 30min.

[0051] The wet bacterial cells are resuspended in ultrapure water to obtain a bacterial solution with a bacterial concentration of 20%. The obtained bacterial cells that can express transaminase can be spray-dried to obtain dehydrated whole cells, or the bacterial solution can be treated by ultrasonic or high-pressure homogenization. After the bacterial solution is treated, the dehydrated enzyme powder can be obtained by freeze-drying or ammonium sulfate precipitation, or the enzyme solution can be adsorbed on an immobilized enzyme carrier to obtain an immobilized enzyme, which is the transaminase encoded by SEQ ID NO: 1. Similarly, the transaminase encoded by SEQ ID NO: 2 to SEQ ID NO: 5 can be obtained respectively by the above method.

[0052] In the present application, the addition method of transaminase can be: directly adding the dehydrated whole cells of transaminase to the premixed system; or, adding the freeze-dried enzyme powder obtained by fermentation and culturing the transformant containing the coding gene of transaminase; or, adding the dehydrated enzyme powder obtained by ammonium sulfate precipitation of the cell lysis liquid after the wet bacteria are crushed to the premixed system; or, adding the product of the cell lysis liquid after being treated with an immobilized carrier to the premixed system.

[0053] Example 1: 10 g of dehydrated whole cell enzyme expressing the transaminase encoded by SEQ ID NO: 1 and 1.2 g of isopropylamine were added to a 40 mL reaction system (40 mL of solvent, 4 g of substrate, 50 mg of PLP and 2 g of water). Final assay concentration: 95.6 g / L substrate compound (sitagliptin precursor ketone), 5 mM PLP (pyridoxal phosphate) and 100% solvent, 0.5 M isopropylamine. The solvent used is detailed in Table 1. The reaction bottle was sealed in a 45°C water bath and stirred for 24 hours. After the reaction was completed, the components of the catalytic reaction solution were analyzed by liquid chromatography (HPLC).

[0054] The test method for product conversion rate is: use HPLC to detect the concentration of the product and calculate the conversion rate:

[0055] Preparation of injection solution: dilute 20 μL of the reaction solution with 980 μL of methanol, mix well, centrifuge, and take the supernatant for injection;

[0056] Conversion rate LC conditions: Instrument used: Shimadzu LC20ADXR;

[0057] Chromatographic column: Diamonsil 150*4.6mm 5μm;

[0058] Mobile phase: 1‰ TFA: acetonitrile;

[0059] Flow rate: 1 mL / min;

[0060] Detection wavelength: 268nm.

[0061] Product conversion rate (%): product production / (substrate residue+product production)*100.

[0062] Examples 2-3: The reaction system is the same as that of Example 1, except that different solvents are used, as shown in Table 1 for details.

[0063] Table 1 Solvent systems and product conversion rate test results used in Examples 1 to 3

[0064] Solvents Conversion rate (%) Example 1 MTBE 20％ Example 2 Butyl acetate 5％ Example 3 Isopropyl alcohol 6％

[0065] Examples 1 to 3 of the present application screened the solvent system for the reaction, and the results are shown in Table 1. From the results in Table 1, it can be seen that the product conversion rate is high in the MTBE system containing a trace amount of water, and the MTBE boiling point is low, which is conducive to the separation and purification of the product.

[0066] Example 4: Add 10 g of enzyme and 1.2 g of isopropylamine to a 40 mL reaction system (40 mL of MTBE, 4 g of substrate, 50 mg of PLP). Final concentration: 95.6 g / L substrate compound, 100% MTBE, 0.5 M isopropylamine, water content is a variable, and the water content added to the reaction system is shown in Table 2. The reaction bottle is sealed in a 45°C water bath and stirred for 24 hours. After the reaction is completed, the components of the catalytic reaction liquid are analyzed by liquid chromatography (HPLC).

[0067] Examples 5 to 13: The reaction system and conditions are the same as those of Example 4, except that the water content in the system changes, as shown in Table 2.

[0068] Table 2 Solvent systems and product conversion rate test results used in Examples 4 to 13

[0069] Water content (%) Conversion rate (%) Example 4 0 0.1％ Example 5 0.01 5.9％ Example 6 0.05 30％ Example 7 0.1 96.6％ Example 8 0.5 96.7％ Example 9 1 96.5％ Example 10 2.5 90％ Embodiment 11 5 20％ Example 12 10 6％ Embodiment 13 15 0.5％

[0070] From the results in Table 2, it can be found that the solvent system of the present application contains a small amount of water, which can improve the conversion rate of the product. The mass percentage of water content in the system is 0.05-5%. More preferably, the water content is in the range of 0.1-2.5%, which can achieve a better conversion rate.

[0071] Example 14: Add 10 g of enzyme and 1.2 g of isopropylamine to 40 mL of reaction system (40 mL of MTBE, 4 g of substrate, appropriate amount of PLP, 2 g of water). Final concentration: 95.6 g / L substrate compound, 100% MTBE, 0.5 M isopropylamine, PLP variable, see Table 3 for details. Seal the reaction bottle and place it in a 45°C water bath, stir and react for 24 hours. After the reaction is completed, the components of the catalytic reaction liquid are analyzed by liquid chromatography (HPLC).

[0072] Example 15 to Example 19: The reaction system is the same as Example 14, except that the amount of PLP used is different, see Table 3 for details.

[0073] Table 3 Reaction system and product conversion rate test results used in Examples 14 to 19

[0074]

[0075]

[0076] It can be seen from the data in Table 3 that the PLP dosage of the present application is controlled within the range of 0.1-20 mM, and the entire reaction system has a high conversion rate, especially within the range of 0.5-20 mM PLP, the conversion rate of the entire reaction system is higher.

[0077] Example 20: Add 10 g of enzyme and an appropriate amount of isopropylamine to a 40 mL reaction system (MTBE 40 mL, substrate 4 g, PLP 25 mg, water 2 g), see Table 4 for details. Final assay concentration: 95.6 g / L substrate compound, 5 mM PLP and 100% solvent, see Table 4 for the content of isopropylamine. Seal the reaction bottle in a 45°C water bath and stir the reaction for 24 hours. After the reaction is completed, the components of the catalytic reaction liquid are analyzed by liquid chromatography (HPLC).

[0078] Example 21 to Example 23: The reaction system is the same as that of Example 20, except that the amount of isopropylamine used is different, see Table 4 for details.

[0079] Table 4 Reaction system and product conversion rate test results used in Examples 20 to 23

[0080] Isopropylamine dosage (g) Product conversion rate (%) Embodiment 20 0.6 86.7 Embodiment 21 1.2 96.6 Embodiment 22 1.8 96.5 Embodiment 23 2.4 96.3

[0081] It can be seen from the data in Table 4 that the amount of isopropylamine in the present application is controlled within the range of 0.25 to 1 mol / L, and the entire reaction system has a high conversion rate, especially when the amount of isopropylamine is within the range of 0.5 to 1 mol / L, the conversion rate of the entire reaction system is higher.

[0082] Example 24: 4 g of substrate, 29.6 g (40 mL) of methyl tert-butyl ether (MTBE), 2 g of water, and 25 mg of pyridoxal phosphate (PLP) were added to a reaction bottle equipped with a magnetic rotor and sealed with a hollow stopper;

[0083] Place the reaction bottle in a 45°C water bath, stir and heat, add 5 g of enzyme and 1.2 g of isopropylamine, and seal with a hollow stopper;

[0084] The reaction was sealed and kept warm for 24 hours, and then samples were taken;

[0085] Reaction conditions: 45°C, sealed for 24 hours;

[0086] The enzyme forms are shown in Table 5.

[0087] Table 5 Effect of different enzyme forms on conversion rate

[0088] Enzyme form Conversion rate (%) Sequence 1 Whole Cell 95.1 Sequence 1 Enzyme Powder 93.6 Sequence 1 ammonium sulfatase powder 91.5 Sequence 1 immobilized enzyme 98.3 Sequence 2 whole cell 50.1 Sequence 2 enzyme powder 46.1 Sequence 2 ammonium sulfatase powder 52.6 Sequence 2 immobilized enzyme 10.1 Sequence 3 whole cell 78.5 Sequence 3 enzyme powder 76.6 Sequence 3 ammonium sulfatase powder 74.3 Sequence 3 immobilized enzyme 70.2 Sequence 4 whole cell 16 Sequence 4 enzyme powder 18.5 Sequence 4 ammonium sulfatase powder 14.5 Sequence 4 immobilized enzyme 6.2 Sequence 5 whole cell 85 Sequence 5 enzyme powder 86.3 Sequence 5 ammonium sulfatase powder 82.5 Sequence 5 immobilized enzyme 90.9

[0089] Note: Sequence 1 represents the encoding nucleotide sequence of transaminase as shown in SEQ ID NO: 1, or the amino acid sequence as shown in SEQ ID NO: 6; Sequence 2 represents the encoding nucleotide sequence of transaminase as shown in SEQ ID NO: 2, or the amino acid sequence as shown in SEQ ID NO: 7; Sequence 3 represents the encoding nucleotide sequence of transaminase as shown in SEQ ID NO: 3, or the amino acid sequence as shown in SEQ ID NO: 8; Sequence 4 represents the encoding nucleotide sequence of transaminase as shown in SEQ ID NO: 4, or the amino acid sequence as shown in SEQ ID NO: 9; Sequence 5 represents the encoding nucleotide sequence of transaminase as shown in SEQ ID NO: 5, or the amino acid sequence as shown in SEQ ID NO: 10; whole cell, enzyme powder, ammonium sulfatase powder, and immobilized enzyme refer to the form in which transaminase is added.

[0090] It can be seen from the data in Table 5 that although there are differences in the catalytic efficiency of different transaminases, in the same enzymatic system, using the reaction system of the present application, the catalytic efficiency will not be affected by the change in the form of the enzyme, which will cause the conversion rate to fluctuate. The reaction system of the present application expands the application range of different enzyme forms.

[0091] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0092] The above is a detailed introduction to the preparation method of a sitagliptin intermediate provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method of the present application and its core idea. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A method for preparing a sitagliptin intermediate, characterized in that: The following steps are involved: Sitagliptin precursor ketone 1-(3-trifluoromethyl)-5,6-dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)-1,3-butanedione is catalyzed by transaminase in the presence of a solvent system to obtain (R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one.

2. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: The solvent system includes at least one of methyl tert-butyl ether, ethyl acetate or isopropanol.

3. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: In the solvent system, the mass percentage of water is ≤10%.

4. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: In the solvent system, the mass percentage of water is 0.01% to 10%.

5. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: The sitagliptin precursor ketone is catalyzed by transaminase in the presence of isopropylamine and pyridoxal phosphate to obtain the sitagliptin intermediate.

6. The method for preparing a sitagliptin intermediate according to claim 5, characterized in that: The concentration of the sitagliptin precursor ketone is 0.1-0.8 mol / L; the concentration of the isopropylamine is 0.2-5 mol / L; and the concentration of the pyridoxal phosphate is 5-100 mmol / L.

7. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: The mass ratio of the sitagliptin precursor ketone to the transaminase is 1:(2-5).

8. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: The reaction temperature is 15-65°C.

9. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: The transaminase is at least one of whole cells, enzyme powder, ammonium sulfate enzyme powder and immobilized enzyme.

10. The method for preparing a sitagliptin intermediate according to claim 1, characterized in that: The nucleotide sequence encoding the transaminase is shown in SEQ ID NO: 1 to SEQ ID NO: 5, or is a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to SEQ ID NO: 1 to SEQ ID NO: 5.

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