An imine reductase mutant and its application in preparing abubinitin intermediate

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological catalysis, and particularly relates to an imine reductase mutant and application thereof in preparation of an intermediate of Abrocitinib. BACKGROUND

[0002] Abrocitinib is a new generation of oral Janus kinase 1 (JAK1) selective inhibitor developed by Pfizer, and its trade name is Cibinqo. The structure is shown as formula A. The drug is suitable for adult patients with moderate to severe atopic dermatitis who are poorly responsive or unsuitable for other systemic treatments such as hormones or biological agents.

[0003]

[0004] Pfizer discloses a preparation method of Abrocitinib in patent CN105008362B, as shown in Scheme 1. The route takes 3-oxocyclobutyl carboxylic acid as a starting material, first reacts with azide phosphoric acid diphenyl ester (DPPA) and benzyl alcohol to generate compound II, then reacts with methylamine, and is reduced and aminated by lithium borohydride at 0℃ to obtain a compound I mixture with a cis:trans ratio of 4:1. The cis compound I obtained by recrystallization is subjected to substitution reaction with 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine, and finally subjected to deprotection and sulfonylation to obtain Abrocitinib.

[0005]

[0006] It is found by the route Scheme 1 that (1S,3S)-3-(methylamino)cyclobutyl) benzyl carbamate (compound I, CAS No.: 1353501-22-0) is a key chiral intermediate for synthesizing Abrocitinib, and therefore, it is crucial to study the preparation of compound I.

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

[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, it is necessary to develop a green and environmentally friendly, simple to operate, high-yield and suitable for industrialization preparation method. SUMMARY

[0010] The present application aims at solving the problems of the prior art and provides an imine reductase mutant and its application in preparing Abubinitin intermediate I.

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

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

[0013] Further, the wild-type imine reductase is derived from Streptomyces sp, and the wild-type template has the accession number 4OQY_A in NCBI, and the amino acid sequence and nucleotide sequence are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0014] Further, 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] Further, the wild-type imine reductase is derived from Streptomyces sp, and the wild-type template has the accession number 4OQY_A in NCBI, and the amino acid sequence and nucleotide sequence are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0016] Further, 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] Further, the imine reductase is expressed in a genetically engineered bacterium.

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

[0019] In another aspect, the present application provides a method for preparing Abubinitin intermediate I using an imine reductase mutant. The method uses compound II as the substrate, which is converted into compound I under the catalysis of imine reductase, and the route is shown in Scheme 3.

[0020]

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

[0022] Further, the imine reductase participates in catalyzing the reaction in the form of imine reductase enzyme powder, imine reductase enzyme liquid, imine reductase homogeneous liquid, imine reductase freeze-dried powder, cells containing imine reductase, etc., and the imine reductase is preferably imine reductase cells.

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

[0024] Further, the concentration of the added coenzyme is 0.02-0.40 g / L, and is preferably 0.10-0.25 g / L.

[0025] Further, the coenzyme used in the technical solution is selected from the coenzyme products sold by Shangke Biomedicine (Shanghai) Co., Ltd.

[0026] Further, the concentration of the substrate is 5-40 g / L, and is preferably 10-30 g / L.

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

[0028] Further, the reaction temperature is 20℃-40℃, and is preferably 25-35℃.

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

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

[0031] Figure 2 Expression electrophoretogram of IRED in Example 2

[0032] Figure 3 IRED saturation mutation whole plasmid gene amplification map in Example 3 DETAILED DESCRIPTION

[0033] The technical content of the present application is further described below in combination with specific embodiments, and the purpose is to better understand the content of the present application, but the protection scope of the present application is not limited thereto.

[0034] Example 1 Preliminary screening of IRED

[0035] The substrate compound II was dissolved in DMSO to prepare a 10 g / L stock solution, which was added to an IRED enzyme plate (Shanghai Shengke Biomedicine Co., Ltd.) containing 90 uL of deionized water. After the addition was completed, the sample was placed in a 30℃ shaker for 20 hours of shaking reaction. After the reaction was completed, the sample was extracted with ethyl acetate, and HPLC detection analysis was performed. The results showed that the IRED enzyme (SS-IRED) derived from Streptomyces sp. detected 1.7% of the product, with an ee value of 91.5%. The enzyme was later subjected to directed evolution modification.

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

[0037] The monoclonal strain of SS-IRED was cultured overnight on a LB plate containing Kan + resistance, and single colony cells growing on the plate were selected. The single colonies were subjected to gene amplification using universal primers on the vector. The electrophoresis band results are shown in Figure 1 . All single colonies contained exogenous genes of the same size as the theoretical value, so further protein expression was performed.

[0038] The above single colony cells were transferred to 5 mL of LB medium and cultured at 37℃ overnight. The cultured seed liquid was transferred to 2YT medium containing Kan + resistance at a 1.5% inoculation amount, and the cells were cultured at 37℃. When the biomass OD 600 value reached about 0.8, IPTG induction was performed, the temperature was reduced to 25℃, and the cells were collected after 16h of expression. Ultrasonic cell disruption was performed for electrophoresis analysis, and the results are shown in Figure 2 . The protein size was consistent with the expected value, and the soluble expression was good.

[0039] Example 3 Construction of IRED saturation mutation library

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

[0041] Table 1 Nucleic acid sequence of saturation mutation primer

[0042]

[0043] Using PCR technology, under the action of high-fidelity polymerase Primer STAR max DNA, using different temperature annealing and extension, the whole plasmid was amplified. The product at the end of amplification was analyzed by electrophoresis, and the results are shown in Figure 3 Each pair of primers can amplify the whole plasmid fragment.

[0044] The PCR amplification product was digested by FDDpnI restriction endonuclease to remove the wild-type template DNA sequence, and was transferred into BL21(DE3) competent cells and coated on LB plates containing 100ug / mL Kan + resistance, and cultured at 37℃ overnight. Single colonies were picked for sequencing. The sequencing results showed that the mutation was successful.

[0045] Example 4 Screening of IRED saturation mutation library

[0046] The single clone cells obtained after saturation mutation were picked into 96-well plates containing 400uL of LB (containing Kan + ) medium per well, and cultured at 37℃ overnight. Then 100μL of seed liquid was transferred into 96-deep well plates containing 900μL of 2YT (containing 0.1mM IPTG + , 100ug / mL Kan + ) medium per well, and cultured at 25℃ for 16h. After centrifugation at 4000rpm for 10min, the supernatant was discarded, and the 96-well plates were frozen at -20℃ for reaction screening.

[0047] The reaction was carried out in a deep 96-well plate, and 90 mL of reaction system was prepared first, 3 g of monohydrate glucose, 73.75 mL of water, 10 mL of PBS-K (pH 8.0, 1M), 2.25 mL of aqueous methylamine solution (33%), and 1 mL of NADP solution (20 g / L) and 3 g of glucose dehydrogenase homogenate (333 g / L) were sequentially added to the bottle after adjusting the pH value to 8.0 with 6M HCl, and then 100 g / L substrate solution was prepared, that is, 1 g of substrate was weighed and dissolved in 10 ml of DMSO. The prepared 90 mL reaction system and 100 g / L substrate solution were respectively dispensed into a 96-well plate using a row gun, and 450 μl of the reaction system and 50 μl of the substrate solution were added to each well. In the final system, the substrate concentration was 10 g / L, the monohydrate glucose 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 carried out in a shaking incubator at 37°C, and TLC spot plate analysis was carried out after 24 h of reaction. According to the amount of substrate reduction, the preliminary screening mutants were determined. Through similar methods, the superposition screening of other sites was carried out, and finally the mutants Mut1 (V88T) and Mut2 (V88T / A162P) with significantly improved catalytic efficiency and product concentration were obtained. The amino acid sequences of the 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 of substrate, 0.075 g of monohydrate glucose, 2.09 mL of water, 0.25 mL of PBS-K (pH 8.0, 1M), 0.05625 mL of aqueous methylamine solution (33%), and 0.025 mL of NADP solution (20 g / L), 0.075 g of glucose dehydrogenase homogenate (333 g / L), and 0.05 g of cells were sequentially added after adjusting the pH value to 8.0 with 6M HCl. The reaction was carried out at 37°C, and HPLC analysis was carried out after 22 h of reaction. The results are shown in Table 2. Mut1 and Mut2 have significantly improved catalytic activity compared with wild type WT.

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

[0051] Sample Catalytic substrate concentration (g / L) Yield (%) ee value (%) WT 10 2.1 91.3 Mut 1 (V88T) 10 37.5 91.5 Mut 2 (V88T / A162P) 10 96.6 91.7

[0052] Example 6 IRED catalyzing high concentration substrate

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

[0054] Table 3 Conversion of high concentration substrate by mutant Mut 1 / 2 and wild type WT

[0055] Sample Catalytic substrate concentration (g / L) Yield (%) ee value (%) WT 20 1.7 91.5 Mut 1 (V88T) 20 21.5 91.6 Mut 2 (V88T / A162P) 20 68.2 92.0

Claims

1. An imine reductase mutant, characterized in that, The amino acid sequence of the imine reductase mutant is shown in SEQ ID NO: 3 or SEQ ID NO: 5, respectively.

2. An imine reductase mutant, characterized in that, The gene nucleotide sequence of the imine reductase mutant is shown in SEQ ID NO: 4 or SEQ ID NO: 6, respectively.

3. A process for the preparation of Abubinitin intermediate I, characterized by, Compound II is converted into Abubinitin intermediate I under the catalysis of imine reductase selected from the imine reductase mutants in claim 1, and the reaction is shown as follows:

4. The production method according to claim 3, wherein The concentration of compound II is 5-40 g / L.

5. The production method according to claim 3, wherein The reaction temperature is 20-40℃.

6. The production method according to claim 3, wherein The reaction control pH is 7.5-10.0.

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