Transaminase mutant and application thereof in preparation of atozepam intermediate

Abstract

The invention discloses a transaminase mutant and application of the transaminase mutant in preparation of an atozepam intermediate compound I. The invention discloses a novel transaminase mutant which can convert 50g / L of a compound III into an atozepam intermediate compound I, the conversion rate is greater than 99%, the dr value is greater than 65: 1, and the novel transaminase mutant is more suitable for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a transaminase mutant and its application in the preparation of atorgipam intermediates. Background Technology

[0002] Atogepant is a calcitonin gene-related peptide (CGRP) receptor antagonist developed by AbbVie Biopharmaceuticals in the United States. It was approved by the U.S. FDA on September 28, 2021, for the preventive treatment of episodic migraine in adults.

[0003]

[0004] ((5S,6R)-6-methyl-2-oxo-5-(2,3,6-trifluorophenyl)piperidin-3-yl)tert-butyl carbamate (compound I, CAS 1456803-42-1) is an important intermediate for atorgipam. How to prepare compound I simply and efficiently has a significant impact on the preparation of atorgipam.

[0005] US8754096B2 discloses a method for chemically synthesizing compound I, as shown in Scheme 2. This method uses compound II as a substrate, requires two-step reaction, and needs to be chirally separated by high performance liquid chromatography to obtain the target compound. This results in material waste, poor atom economy, and is not suitable for industrial production.

[0006]

[0007] Patent CN104168768B discloses an enzymatic method for preparing compound I, as shown in Scheme 1. This method uses compound III as a substrate and converts it to compound I at 55°C using 50% wt transaminase. The dr ratio at the C5 and C6 positions is >60:1, and the yield is 70.5%. This transaminase is derived from *Arthrobacter* sp., with a substrate concentration of approximately 30 g / L.

[0008]

[0009] Enzymatic preparation of atorgipan I has high catalytic efficiency and atom economy, but there is a need to develop a transaminase mutant with higher substrate concentration, stronger chiral selectivity, and better suitability for industrial application. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of existing technologies by providing a transaminase mutant and its application in the preparation of atorgipam intermediate I.

[0011] On the one hand, the present invention provides a transaminase mutant, which is obtained by site-directed mutagenesis using the amino acid sequence of the wild-type transaminase shown in SEQ ID NO.1 as a reference sequence.

[0012] Furthermore, the wild-type transaminase is derived from Aspergillus fumigatus, and the wild-type template has the NCBI accession number XP_748821.1. The amino acid sequence and nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0013] Furthermore, the amino acid sequence of the transaminase mutant differs from the wild-type amino acid sequence SEQ ID NO.1 by one or more residues selected from the following: L36F; E49F; M52T; H53Q; D55W; L56A; D59T; V60T; Q76K; I78L; M85S; K97R; N98K; F113L; E115N; V116I; S128R; K129Q; Y134F; N136F; I146M; W147T; V148L; N153K; L155R; E159H; L181F; W183V; L190N; M194A; T199E; N209H; R235E.

[0014] Furthermore, the amino acid residue differences of the transaminase mutant are preferably selected from a combination of the following residue differences:

[0015] (a)Mut1: I146M / W147T;

[0016] (b)Mut2: W183V / N209H;

[0017] (c)Mut3: I146M / W147T / W183V;

[0018] (d)Mut4: L36F / D59T / F113L / E115N / I146M / E159H / L181F / W183V;

[0019] (e)Mut5: E49F / M52T / D55W / L56A / I146M / V148L / N153K / L190N;

[0020] (f)Mut6: H53Q / V60T / I78L / N136F / L155R / W183V / T199E / R235E;

[0021] (g)Mut7: Q76K / W147T / W183V / M85S / V116I / K129Q / M194A;

[0022] (h)Mut8: K97R / N98K / S128R / Y134F / I146M / W147T / W183V.

[0023] Furthermore, the amino acid sequence of the transaminase mutant is shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17.

[0024] Furthermore, the gene sequences of the transaminase mutants are shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18.

[0025] Furthermore, the transaminase mutant is expressed in genetically engineered bacteria.

[0026] Furthermore, the transaminase mutant expression strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis, with Escherichia coli being preferred.

[0027] On the other hand, this invention provides a method for preparing atorgipam intermediate I using a transaminase mutant. This method uses compound III as a substrate, which is converted to compound I under the catalysis of the transaminase mutant, as shown in Scheme 3.

[0028]

[0029] Furthermore, the transaminase mutants are preferably Mut1 to Mut8.

[0030] Furthermore, the transaminase mutant participates in the catalytic reaction in the form of transaminase powder, transaminase solution, transaminase homogenate, transaminase clear solution, transaminase lyophilized powder, cells containing transaminase, etc., preferably transaminase clear solution.

[0031] Furthermore, coenzymes can be added to the reaction system to promote the reaction. When using cells containing transaminase, the cells contain a small amount of coenzyme, so coenzyme may not need to be added. In some cases, the transaminase powder also contains a small amount of coenzyme, so coenzyme may also not need to be added. If coenzyme is added to the reaction system, the coenzyme is selected from PLP.

[0032] Furthermore, the concentration of added coenzyme is 0.02–0.40 g / L, preferably 0.10–0.20 g / L.

[0033] Furthermore, the coenzymes used in this technical solution are all selected from coenzyme products sold by Shangke Biomedical (Shanghai) Co., Ltd.

[0034] Furthermore, the concentration of compound III is 10–50 g / L, preferably 30–50 g / L.

[0035] Furthermore, the reaction needs to be controlled at pH 10.0–12.0, preferably pH 11.0–11.5.

[0036] Furthermore, the reaction temperature is 25℃~55℃, preferably 45℃~50℃.

[0037] The beneficial effect of this invention is that it provides a new transaminase mutant that can convert 50 g / L of compound III with a conversion rate of ≥99% and a dr value of 65:1, making it more suitable for industrial applications. Attached Figure Description

[0038] Figure 1 Electrophoresis diagram of wild-type transaminase expression in Example 1

[0039] Figure 2 Transformation map of substrate catalyzed by transaminase mutant in Example 5 Detailed Implementation

[0040] The technical content of the present invention will be further described below with reference to specific embodiments, in order to better understand the content of the present invention, but the scope of protection of the present invention is not limited thereto.

[0041] Example 1: Preliminary screening of transaminases

[0042] Deionized water, substrate (10 g / L), and 3 eq. of the amino donor of the substrate were added sequentially to a kit containing 86 transaminases (ATA)- (Shanghai Shangke Biomedical Co., Ltd.). The pH was adjusted appropriately with 6 M HCl, and then the reaction pH was controlled with 100 mM phosphate buffer. The reaction was then carried out at 40 °C with shaking for 24 hours. After the reaction, 1 mL of ethyl acetate was added to each well for extraction. After complete extraction, the extract was centrifuged at 12000 rpm for 3 min, filtered, and sent for analysis. The results showed that the transaminase derived from Aspergillus fumigatus (NCBI accession number XP_748821.1) could catalyze the substrate, but the conversion rate was only 45%. The dr ratio of the C5 and C6 positions of compound I was 47:1. Further directed evolutionary modification of this enzyme will be carried out to improve its catalytic efficiency.

[0043] Example 2: Culture and expression of wild-type transaminase

[0044] The wild-type transaminase strain from Aspergillus fumigatus was transformed into a strain containing Kan + In resistant LB liquid medium, cultured overnight at 37°C, when OD 600When the OD value reaches approximately 10, the cultured seed solution is transferred to 2YT medium at an inoculation rate of 1% for further culture. The culture temperature is maintained at 37℃ for the first 3 hours. 600 When the expression value reached 0.6–0.8, the temperature was lowered to 25°C, and 0.1 mM IPTG was added to induce expression. After 16 hours of expression, the bacterial cells were collected by centrifugation at 7500 rpm for 10 min. The collected bacterial cells were then subjected to low-temperature sonication to disrupt the protein expression after disruption. The results were analyzed by SDS-PAGE electrophoresis. (See attached figure.) Figure 1 The protein size was consistent with expectations, and it showed good soluble expression.

[0045] Example 3: Site-directed mutagenesis and saturation mutagenesis of wild-type transaminase

[0046] To improve the selectivity of wild-type transaminase derived from Aspergillus fumigatus, the enzyme's active site was targeted. Library construction and screening were performed using amino acids within a certain range. After analysis, 20 key sites were selected for saturation mutation library construction. Then, at least 200 single clones were screened for each site. Finally, mutants with improved chiral selectivity at sites I146, W147, and W183 were obtained. Then, mutations were added to the mutants to obtain mutants with significantly improved chiral selectivity. See Table 1 for details. Only some mutants with dr values ​​> 65:1 at C5 and C6 positions of compound I at a substrate concentration of 10 g / L are listed here.

[0047] Table 1. Mutants with a dr value > 65:1 at a substrate concentration of 10 g / L.

[0048]

[0049]

[0050] Through combined simulation structural analysis, site-directed and superimposed mutations were performed on multiple sites, and new mutants with significantly improved catalytic efficiency were finally obtained, as detailed in Table 2. Only some mutants of compound I with dr values ​​> 65:1 at C5 and C6 and substrate concentration ≥ 30 g / L are listed here.

[0051] Table 2 Mutants with substrate concentration ≥30 g / L and dr value >65:1

[0052] mutant Amino acid differences (compared to SEQ ID NO: 1) Conversion rate (%) Mut 4 L36F / D59T / F113L / E115N / I146M / E159H / L181F / W183V 85 Mut 5 E49F / M52T / D55W / L56A / I146M / V148L / N153K / L190N 87 Mut 6 H53Q / V60T / I78L / N136F / L155R / W183V / T199E / R235E 85 Mut 7 Q76K / W147T / W183V / M85S / V116I / K129Q / M194A 90 Mut 8 K97R / N98K / S128R / Y134F / I146M / W147T / W183V 91

[0053] Example 4: Temperature Optimization of Transaminase Mutants

[0054] To improve the conversion rate, the reaction temperature was optimized, set within the range of 25℃ to 55℃. Specifically, 2 mL of 0.1M sodium tetraborate buffer (12.5 mM) and 4 mL of DMSO solution were added sequentially to the reaction vessel. The temperature was lowered to 15±2℃, and 0.5 mL of isopropylamine was added. The pH of the reaction solution was adjusted to 11.0±0.1 by slowly adding 6M HCl, while controlling the temperature of the reaction solution to not exceed 20℃. Then, 0.3 g of substrate (30 g / L) and 1 mL of PL were added. Solution P (0.18 g / L) and 1.5 mL of enzyme solutions of different transaminase mutants (Mut4-Mut8) were sealed in the reaction apparatus and stirred at different temperatures. After 48 hours of reaction, 100 μL of the solution was dissolved in 900 μL of acetonitrile and centrifuged at 12000 rpm for 1 minute. The supernatant was collected for HPLC analysis. The reaction results were analyzed, and the specific data are shown in Table 3. The screening results showed that the conversion effect was better when the substrate concentration was 30 g / L and the reaction temperature was 50 °C.

[0055] Table 3. Optimization results of conversion rate (%) at different temperatures

[0056]

[0057]

[0058] Example 5: Optimization of the initial pH for the reaction of the transaminase mutant

[0059] To improve the conversion rate, the initial pH of the reaction was optimized, set within the range of 10.0–12.0. Specifically, 2 mL of 0.1 M sodium tetraborate buffer (12.5 mM) and 4 mL of DMSO solution were added sequentially to the reaction vessel. The temperature was lowered to 15 ± 2 °C, and 0.5 mL of isopropylamine was added. Then, 6 M HCl was slowly added dropwise to adjust the pH of the reaction solution to 12.0, 11.5, 11.0, 10.5, and 10.0, respectively. The reaction temperature was controlled to not exceed 20 °C to prevent the isopropylamine from volatilizing at high temperatures. Next, 0.5 g of substrate (50 g / L) and 1 mL of PLP were added. The reaction apparatus was sealed with 0.18 g / L solution and 1.5 mL of different transaminase mutant enzyme solutions (Mut4-Mut8). The temperature was initially raised to 50 °C and stirred for 48 hours. After 48 hours, 100 μL of the reaction solution was dissolved in 900 μL of acetonitrile and centrifuged at 12000 rpm for 1 minute. The supernatant was collected for HPLC analysis. The specific data are shown in Table 4. Mut8 mutant, by raising the initial pH of the reaction to 11.5, achieved a conversion rate of 99%. The chirality of compound I was also analyzed, and the results showed that the dr values ​​at C5 and C6 positions were >65:1. The substrate transformation chromatogram is attached. Figure 2 .

[0060] Table 4. Conversion rates (%) at different initial pH values ​​for the reactions

[0061]

Claims

1. A transaminase mutant, characterized in that, The amino acid sequence of the transaminase mutant differs from SEQ ID NO.1 from one or more residues selected from the following: L36F; E49F; M52T; H53Q; D55W; L56A; D59T; V60T; Q76K; I78L; M85S; K97R; N98K; F113L; E115N; V116I; S128R; K129Q; Y134F; N136F; I146M; W147T; V148L; N153K; L155R; E159H; L181F; W183V; L190N; M194A; T199E; N209H; R235E.

2. The transaminase mutant as described in claim 1, characterized in that, The amino acid residue differences of the transaminase mutant are selected from a combination of the following residue differences: (a)Mut1: I146M / W147T; (b)Mut2: W183V / N209H; (c)Mut3: I146M / W147T / W183V; (d)Mut4: L36F / D59T / F113L / E115N / I146M / E159H / L181F / W183V; (e)Mut5: E49F / M52T / D55W / L56A / I146M / V148L / N153K / L190N; (f)Mut6: H53Q / V60T / I78L / N136F / L155R / W183V / T199E / R235E; (g)Mut7: Q76K / W147T / W183V / M85S / V116I / K129Q / M194A; (h)Mut8: K97R / N98K / S128R / Y134F / I146M / W147T / W183V.

3. The transaminase mutant as described in claim 2, characterized in that, The amino acid sequences of the transaminase mutants are shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17.

4. The transaminase mutant as described in claim 2, characterized in that, The gene sequences of the transaminase mutants are shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18.

5. A method for preparing atorgipam intermediate I, characterized in that, Compound III is converted to compound I under the catalysis of a transaminase mutant, wherein the transaminase mutant is selected from the transaminase mutant of claim 1, and the reaction equation is shown below:

6. The preparation method according to claim 5, characterized in that, The transaminase mutant participates in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenized solution, clear solution, lyophilized powder, and cells containing the enzyme.

7. The preparation method according to claim 5, characterized in that, The concentration of compound III is 10–50 g / L.

8. The preparation method according to claim 5, characterized in that, The reaction temperature is 25℃~55℃.

9. The preparation method according to claim 5, characterized in that, The reaction requires the pH to be controlled between 10.0 and 12.0.

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