Imine reductase mutant and application of imine reductase mutant in preparation of aboxitinib intermediate

Abstract

The invention discloses an imine reductase mutant and an application of the imine reductase mutant in preparation of an aboxitinib intermediate I. The imine reductase mutant disclosed by the invention can convert a compound II into an aboxitinib intermediate I, the yield reaches 96%, the ee value of the product reaches 92%, and the method disclosed by the invention is simple to operate and high in yield and can be used for industrial production.

Description

Technical field:

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to an imine reductase mutant and an application thereof in preparing an abrocitinib intermediate. Background technology:

[0002] Abrocitinib is a new generation of oral Janus kinase 1 (JAK1) selective inhibitor developed by Pfizer, with the trade name Cibinqo, and its structure is shown in Formula A. The drug is suitable for adult patients with refractory, moderate to severe atopic dermatitis who have poor response to other systemic treatments such as hormones or biological agents or are not suitable for the above treatments.

[0003]

[0004] Pfizer disclosed a method for preparing abrocitinib in patent CN105008362B, as shown in Scheme 1. This route uses 3-oxocyclobutane carboxylic acid as a starting material, first reacts with diphenylphosphoryl azide (DPPA) and benzyl alcohol to generate compound II, then reacts with methylamine, and then undergoes reduction amination with lithium borohydride at 0°C to obtain a mixture of compound I with a cis-to-trans ratio of 4:1. The cis-compound I obtained by recrystallization then undergoes a substitution reaction with 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine, and finally undergoes deprotection and sulfonylation to obtain abrocitinib.

[0005]

[0006] Through route Scheme 1, it was found that (1S, 3S)-3-(methylamino)cyclobutyl)benzyl carbamate (Compound I, CAS No.: 1353501-22-0) is a key chiral intermediate for the synthesis of Abrocitinib. Therefore, the preparation research of Compound I is of great importance.

[0007] Patent US2022259209A1 discloses a method for preparing a key intermediate I, as shown in Scheme 2. This route also uses compound II as the starting material, but the reducing agent is replaced with sodium borohydride. After separation and purification, the yield of compound I is only 33%.

[0008]

[0009] In the prior art, the preparation method of abrocitinib intermediate I has low yield and high cost, and is not suitable for industrialization. Therefore, we need to develop a preparation method that is green, environmentally friendly, simple to operate, high in yield, and suitable for industrialization. Summary of the invention:

[0010] The object of the present invention is to provide an imine reductase mutant and its application in preparing abrocitinib intermediate I in view of the deficiencies in the prior art.

[0011] On the one hand, the present invention provides a novel imine reductase mutant, which is obtained by site mutation with the amino acid sequence of the wild-type imine reductase shown in SEQ ID NO.1 as the reference sequence.

[0012] Furthermore, the wild-type imine reductase is derived from Streptomyces sp. The NCBI accession number of the wild-type template is 4OQY_A, and the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0013] Furthermore, the valine (Val) at position 88 of the wild-type imine reductase was mutated to threonine (Thr) to obtain the imine reductase mutant Mut1 (V88T).

[0014] Furthermore, the amino acid sequence and nucleotide sequence of the imine reductase mutant Mut1 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0015] Furthermore, the valine (Val) at position 88 of the wild-type imine reductase was mutated to threonine (Thr), and the alanine (Ala) at position 162 was mutated to proline (Pro), to obtain the imine reductase mutant Mut2 (V88T / A162P).

[0016] Furthermore, the amino acid sequence and nucleotide sequence of the imine reductase mutant Mut2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0017] Furthermore, the imine reductase is expressed in genetically engineered bacteria.

[0018] Furthermore, the imine reductase expression strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis, preferably Escherichia coli.

[0019] On the other hand, the present invention provides a method for preparing abrocitinib intermediate I using an imine reductase mutant. The method uses compound II as a substrate and converts it into compound I under the catalysis of imine reductase, and the route is shown in Scheme 3.

[0020]

[0021] Furthermore, the imine reductase is selected from the imine reductase mutants Mut1 and Mut2.

[0022] Furthermore, the imine reductase participates in the catalytic reaction in the form of imine reductase enzyme powder, imine reductase enzyme solution, imine reductase homogenized solution, imine reductase lyophilized powder, cells containing imine reductase, etc., preferably imine reductase cells.

[0023] Furthermore, coenzymes can be added to the reaction system to promote the reaction. When cells containing imine reductase are used, a small amount of coenzymes are contained in the cells, and the coenzymes may not be added at this time. In some cases, the prepared imine reductase powder also contains a small amount of coenzymes, and the coenzymes may not be added at this time. However, coenzymes can also be added to the reaction system to further promote the reaction. When coenzymes are added to the reaction system to promote the reaction, the coenzymes are selected from NAD + , NADH, NADP + , NADPH or a combination thereof, preferably NADPH.

[0024] Furthermore, the concentration of the added coenzyme is 0.02 to 0.40 g / L, preferably 0.10 to 0.25 g / L.

[0025] Furthermore, the coenzymes used in this technical solution are all selected from the coenzyme products sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0026] Furthermore, the substrate concentration is 5 to 40 g / L, preferably 10 to 30 g / L.

[0027] Furthermore, the reaction needs to be controlled at a pH of 7.5 to 10.0, preferably at a pH of 8.0 to 8.5.

[0028] Furthermore, the reaction temperature is 20°C to 40°C, preferably 25°C to 35°C.

[0029] The beneficial effect of the present invention is that the present invention provides a new imine reductase mutant, which can efficiently catalyze the conversion of compound II into abrocitinib chiral intermediate compound I, with a yield of 96% and a product ee value of 92%. The method is simple to operate, has high atom economy, and can be used for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Gene electrophoresis of IRED in Example 2

[0031] Figure 2 Electrophoresis of IRED expression in Example 2

[0032] Figure 3 IRED saturation mutation full plasmid gene amplification diagram in Example 3 DETAILED DESCRIPTION

[0033] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0034] Example 1 Preliminary Screening of IREDs

[0035] The substrate compound II was dissolved in DMSO to prepare a 10g / L mother liquor, and added to an IRED enzyme plate (Shanghai Suntech Biopharmaceutical Co., Ltd.) containing 90uL of deionized water. After the addition was completed, it was placed in a shaker at 30°C for 20 hours of shaking reaction. After the reaction was completed, the sample was extracted with ethyl acetate and analyzed by HPLC. The results showed that the IRED enzyme (SS-IRED) derived from Streptomycessp detected 1.7% of the product, and the ee value was 91.5%. The enzyme was later subjected to directed evolution.

[0036] Example 2 Inducible expression of wild-type SS-IRED

[0037] The SS-IRED monoclonal strain was cultured in a + The cells were inverted and cultured overnight on the resistant LB plate. The monoclonal cells grown on the plate were selected and the gene of the monoclonal cells was amplified using the universal primers on the vector. The results of the electrophoresis amplification bands were shown in Figure 1 The size of the exogenous genes contained in all monoclones was consistent with the theory, so the downstream protein expression was further carried out.

[0038] Transfer the above monoclonal cells into 5 mL of LB medium and culture at 37°C overnight. Transfer the cultured seed solution to a 1.5% inoculum containing Kan + The cells were cultured in resistant 2YT medium at 37°C until the biomass OD 600 When the value reached about 0.8, IPTG induction was performed, the temperature was lowered to 25°C, and the cells were collected after 16 hours of expression. The cells were broken by ultrasound for electrophoresis analysis. The results are shown in Figure 2 The protein size was consistent with expectations and had good soluble expression.

[0039] Example 3 Construction of IRED saturation mutation library

[0040] Through substrate docking and protein structure analysis, it was confirmed that the amino acid sites with more critical catalytic activity in SS-IRED are A41, V88, A103 and A162. Semi-rational mutation design was performed on these four sites, and the corresponding saturation mutation primers were designed using software. The specific primer information is shown in Table 1. The underlined parts in the table are the amino acids for saturation mutation.

[0041] Table 1 Nucleic acid sequences of saturation mutation primers

[0042]

[0043] Using PCR technology, under the action of high-fidelity polymerase Primer STAR max DNA, annealing and extension at different temperatures were used to achieve amplification of the entire plasmid, and the amplified product was analyzed by electrophoresis. The results are shown in Figure 3 , each pair of primers can amplify the entire plasmid fragment.

[0044] The PCR amplification product was digested with FD DpnI restriction endonuclease to remove the wild-type template DNA sequence, transferred into BL21 (DE3) competent cells, and plated on a plate containing 100ug / mL Kan + The cells were cultured on resistant LB plates at 37°C overnight, and a single clone was picked for sequencing. The sequencing results showed that the mutation was successful.

[0045] Example 4 IRED saturation mutation library screening

[0046] The monoclonal cells obtained after saturation mutation were picked and cultured in 96-well plates, each well of which contained 400uL of LB (containing Kan + ) medium, shake culture at 37°C overnight, and then transfer 100 μL / well of the seed solution to a 96-well plate for culture, where each well contains 900 μL of 2YT (containing 0.1 mM IPTG + , 100ug / mL Kan + ) medium, shake culture at 25°C for 16 h, centrifuge at 4000 rpm for 10 min, remove the supernatant, and freeze the 96-well plate at -20°C for reaction screening.

[0047] The reaction was carried out in a deep 96-well plate. First, 90 mL of the reaction system was prepared. 3 g of glucose monohydrate, 73.75 mL of water, 10 mL of PBS-K (pH 8.0, 1 M), and 2.25 mL of methylamine aqueous solution (33%) were added to the bottle in sequence. After adjusting the pH value to 8.0 with 6 M HCl, 1 mL of NADP solution (20 g / L) and 3 g of glucose dehydrogenase homogenate (333 g / L) were added. Then, 100 g / L of substrate solution was prepared, that is, 1 g of substrate was weighed and dissolved in 10 ml of DMSO. The prepared 90 mL of reaction system and 100 g / L substrate solution were dispensed into the 96-well plate with a dispenser, and 450 μl of reaction system and 50 μl of substrate solution were added to each well. In the final system, the substrate concentration was 10 g / L, the glucose monohydrate concentration was 30 g / L, the methylamine concentration was 7.5 g / L, the NADP concentration was 0.2 g / L, and the glucose dehydrogenase homogenate concentration was 30 g / L. The reaction was shaken in a shaker at 37°C. TLC spot plate analysis was performed after 24 hours of reaction. The initial screening mutants were determined based on the amount of substrate reduction. A similar method was used to perform superposition screening of other sites. Finally, mutants Mut1 (V88T) and Mut2 (V88T / A162P) with significantly improved catalytic efficiency and product concentration were obtained. The amino acid sequences of mutants Mut1 and Mut2 are shown in SEQ ID NO.3 and SEQ ID NO.5.

[0048] Example 5 IRED catalytic activity test

[0049] In a 2.5 mL reaction system, 0.025 g substrate, 0.075 g glucose monohydrate, 2.09 mL water, 0.25 mL PBS-K (pH 8.0, 1 M), 0.05625 mL methylamine aqueous solution (33%) were added in sequence, and the pH value was adjusted to 8.0 with 6 M HCl, and finally 0.025 mL NADP solution (20 g / L), 0.075 g glucose dehydrogenase homogenate (333 g / L) and 0.05 g cells were added. The reaction was shaken at 37 ° C, and samples were taken for HPLC analysis after 22 hours of reaction. The results are shown in Table 2. The catalytic activity of Mut1 and Mut2 was significantly improved compared with the wild type WT.

[0050] Table 2 Catalytic activity test of mutant Mut 1 / 2 and wild type WT

[0051] sample Catalytic substrate concentration (g / L) Yield (%) ee value (%) WT 10 2.1 91.3 Mut1(V88T) 10 37.5 91.5 Mut2(V88T / A162P) 10 96.6 91.7

[0052] Example 6 IRED catalysis of high concentration substrate

[0053] In a 10 mL reaction system, 0.2 g substrate (20 g / L), 0.3 g glucose monohydrate, 5.6 mL water, 1 mL PBS-K (pH 8.0, 1 M), 0.4 mL methylamine aqueous solution (33%) were added in sequence, and the pH value was adjusted to 8.0 with 6 M HCl. Finally, 0.1 mL NADP solution (20 g / L), 0.3 g glucose dehydrogenase homogenate (333 g / L) and 0.2 g wild type or mutant broken cells were added. The transformation results are shown in Table 3.

[0054] Table 3 Mutant Mut 1 / 2 and wild type WT transform high concentration substrate

[0055] sample Catalytic substrate concentration (g / L) Yield (%) ee value (%) WT 20 1.7 91.5 Mut1(V88T) 20 21.5 91.6 Mut2(V88T / A162P) 20 68.2 92.0

Claims

1. An imine reductase mutant, characterized in that: The amino acid sequences of the imine reductase mutants Mut1 and Mut2 are shown in SEQ ID NO: 3 and SEQ ID NO: 5, respectively.

2. The imine reductase mutant according to claim 1, characterized in that The nucleotide gene sequences of the imine reductase mutants Mut1 and Mut2 are shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively.

3. The imine reductase mutant according to claim 1, characterized in that The imine reductase mutant is expressed in genetically engineered bacteria.

4. A method for preparing an abrocitinib intermediate I, characterized in that: Compound II is converted into compound I under the catalysis of imine reductase, and the imine reductase is selected from the imine reductase mutant in claim 1.

5. The preparation method according to claim 4, characterized in that: The imine reductase participates in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenized solution, freeze-dried powder, cells containing the enzyme, and the like.

6. The preparation method according to claim 4, characterized in that: The coenzyme is selected from NAD + , NADH, NADP + , NADPH or a combination thereof.

7. The preparation method according to claim 4, characterized in that: The substrate concentration is 5-40 g / L.

8. The preparation method according to claim 4, characterized in that: The reaction temperature is 20°C to 40°C.

9. The preparation method according to claim 4, characterized in that: The reaction needs to be controlled at pH 7.5-10.0.

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