Lipase mutant and application thereof in preparation of nebivolol intermediate

Abstract

The invention discloses a lipase mutant and an application of the lipase mutant in preparation of a nebivolol intermediate I. The lipase mutant disclosed by the invention can be used for selectively catalyzing and hydrolyzing a compound II to obtain a nebivolol intermediate I, namely (R)-(+)-6-fluoro-chroman-2-carboxylic acid. According to the method, the conversion substrate concentration can reach 120 g / L, the conversion rate is larger than or equal to 49%, the ee value is larger than or equal to 99%, and the method is easy to operate, high in reaction efficiency and high in stereoselectivity.

Description

Technical field:

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to a lipase mutant and application thereof in preparing a nebivolol intermediate. Background technology:

[0002] Nebivolol (Formula A) was first developed by Johnson & Johnson of the United States and was first launched in Germany and the Netherlands in 1997 for the treatment of essential hypertension. The tablet trade name is Nebilet. The drug has the advantages of significant efficacy, convenient medication, and few adverse reactions. In addition, because nebivolol has the advantages of both cardioprotection and vasodilation, it is predicted to be one of the "top ten blockbusters" with good market prospects in the future.

[0003]

[0004] (R)-(+)-6-Fluoro-chroman-2-carboxylic acid (structural formula shown in I, CAS number: 129101-37-7) is a key chiral intermediate in the synthesis of nebivolol, and its resolution effect and efficiency directly affect the product quality and synthesis efficiency of nebivolol.

[0005] The document Tetrahedron: Asymmetry, 26(17), 2015, 912-917. discloses a method for preparing a key intermediate I by chemical method, as shown in Scheme 1. This route uses 2-hydroxybutyrate as the starting material, first demethylates it under the action of aluminum chloride, and then reacts it with Ph 3 Under the action of P and the like, a cyclization reaction occurs to obtain chromanone, and finally the target product I is obtained through reduction and hydrolysis reaction. The total yield is no more than 41%. The route is lengthy and the synthesis efficiency is low.

[0006]

[0007] Patent CN114150036A discloses a method for preparing a key intermediate I by a two-step enzyme-catalyzed reaction, as shown in Scheme 2. This route uses racemic 6-fluorochroman-2-carboxylic acid methyl ester as a substrate, firstly selectively catalyzes the hydrolysis of (S)-6-fluoro-chroman-2-carboxylic acid methyl ester under the action of freeze-dried bacteria (lycEstS), and after the reaction is completed, freeze-dried bacteria (lycEstR) is added to selectively catalyze the hydrolysis of (R)-6-fluoro-chroman-2-carboxylic acid methyl ester, and the ee value of the final product compound I is 99.07%, and the conversion rate is 49.3%.

[0008]

[0009] The preparation route of compound I disclosed in patent CN103228640B is shown in Scheme 3. This route uses esterase from the microorganism Ophiostoma novoulmi to resolve racemic 6-fluoro-chroman-2-carboxylic acid ethyl ester, and compound I is obtained after separation and purification, with an ee value of 90.72% and a conversion rate of 50.28%.

[0010]

[0011] In the prior art, the preparation method of nebivolol intermediate I has low synthesis efficiency, low product ee value and high cost. Therefore, we need to develop a lipase with high catalytic activity and high stereoselectivity, and use it in the preparation of nebivolol intermediate I. Summary of the invention:

[0012] The object of the present invention is to provide a lipase mutant and its application in the preparation of nebivolol intermediate I in view of the deficiencies of the prior art.

[0013] On the one hand, the present invention provides a new lipase mutant, which is obtained by site mutation based on the amino acid sequence of the wild-type lipase shown in SEQ ID NO.1 as a reference sequence.

[0014] Furthermore, the wild-type lipase is derived from Acinetobacter sp., the wild-type template NCBI accession number is AIK29633.1, and the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0015] Furthermore, the 145th glutamic acid (Glu) of the wild-type lipase was mutated to histidine (His) to obtain the lipase mutant Mut1 (E145H).

[0016] Furthermore, the amino acid sequence and nucleotide sequence of the lipase mutant Mut1 are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0017] Furthermore, the phenylalanine (Phe) at position 82 of the wild-type lipase was mutated to proline (Pro), the glutamate (Glu) at position 145 was mutated to histidine (His), the glutamine (Gln) at position 157 was mutated to glutamate (Glu), and the glycine (Gly) at position 250 was mutated to serine (Ser) to obtain the lipase mutant Mut2 (F82P / E145H / Q157E / G250S).

[0018] Furthermore, the amino acid sequence and nucleotide sequence of the lipase mutant Mut2 are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0019] Furthermore, the lipase mutant is expressed in genetically engineered bacteria.

[0020] Furthermore, the lipase mutant expression strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis, preferably Escherichia coli.

[0021] On the other hand, the present invention provides a method for preparing nebivolol intermediate I using a lipase mutant. The method uses racemic compound II as a substrate, and selectively catalyzes (R)-6-fluoro-chroman-2-carboxylic acid methyl ester to undergo a hydrolysis reaction under the catalytic action of lipase to generate an optically pure nebivolol intermediate I, i.e., the target compound (R)-(+)-6-fluoro-chroman-2-carboxylic acid, and the route is shown in Scheme 4.

[0022]

[0023] Furthermore, the lipase is selected from the lipase mutants Mut1 and Mut2.

[0024] Furthermore, the lipase participates in the catalytic reaction in the form of lipase powder, lipase solution, lipase homogenate solution, lipase freeze-dried powder, cells containing lipase, etc., preferably lipase cells.

[0025] Furthermore, the substrate concentration is 10 to 150 g / L, preferably 100 to 120 g / L.

[0026] Furthermore, the reaction needs to be controlled at a pH of 7.0 to 8.0, preferably at a pH of 7.5.

[0027] Furthermore, the reaction temperature is 25°C to 40°C, preferably 35°C to 40°C.

[0028] The beneficial effect of the present invention is that the present invention provides a new lipase mutant, which can selectively catalyze the hydrolysis of racemic compound II to obtain nebivolol intermediate I. The lipase conversion substrate concentration can reach 120g / L, the conversion rate is ≥49%, the ee value of the target product (R)-(+)-6-fluoro-chroman-2-carboxylic acid is ≥99%, and the method is simple to operate, has high reaction efficiency and high stereoselectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Gene electrophoresis of lipase in Example 2

[0030] Figure 2 Electrophoresis of lipase expression in Example 2

[0031] Figure 3 Chiral HPLC analysis of the product in Example 5 DETAILED DESCRIPTION

[0032] 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.

[0033] Example 1 Screening of lipase library

[0034] 0.5g of 6-fluorochroman-2-carboxylic acid methyl ester compound II was dissolved in 500μL methanol to prepare a mother solution, and added to a lipase enzyme plate (Shangke Biopharmaceuticals (Shanghai) Co., Ltd.) containing 81uL deionized water. After the addition was completed, it was placed in a shaker at 37°C for 24 hours of shaking reaction. After the reaction was completed, the sample was extracted with n-butanol and analyzed by HPLC. The results showed that the lipase (AsLip) from Acinetobacter sp. had the activity of splitting 6-fluorochroman-2-carboxylic acid methyl ester. When the substrate concentration was 20g / L, the conversion rate was 23.5%, and the ee value of compound I was 78.4%. The enzyme was later subjected to directed evolution.

[0035] Example 2 Inducible expression of wild-type lipase AsLip

[0036] The AsLip monoclonal strain was isolated in the presence of Kan + 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.

[0037] Then, the monoclonal cells were transferred into 5 mL of LB medium and cultured at 37°C overnight. The cultured seed solution was transferred into a Kan medium at a 1.5% inoculum. + The cells were cultured in resistant 2YT medium at 37°C until the biomass OD 600 When the value reached about 0.6, IPTG induction was performed, the temperature was lowered to 25°C, and the cells were collected after 13 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.

[0038] Example 3 Directed evolution of lipase

[0039] The molecular simulation software Discovery Studio was used to dock the substrate and protein to find the distance between the active center and the catalytic residue Ser. The amino acids were selected as evolutionary targets. After analysis, the key amino acid sites were confirmed to be Phe82, Glu145, Gln157, Lys238 and Gly250. Mutations were performed on these five sites, and the corresponding saturation mutation primers were designed using the software. The specific primer information is shown in Table 1:

[0040] Table 1 Nucleic acid sequences of saturation mutation primers

[0041] Primer name Sequence information F82X-F CGCCTGAGCTATNNKATGGGCCGTACCC F82X-R TGTAGTCTGGGGTMNNCATCCAGCCATA E145X-F GGGCCGTACCCCAGACNNKAAAGCTGCTTC E145X-R ACCGAAAGCAGCTTTMNNGTCTGGGGTACGG Q157X-F TGCGAAAGTGGTTGCCNNKTCCCGGCTGACC Q157X-R GTCAGCGCGGAMNNGGCAACCACTTTCGCAC K238X-F GGTTTTGCCCGTATGNNKCCGCTGCAAGCC K238X-R CAGGCTTGCAGCGGMNNCATACGGGCAAAACC G250X-F TATGTATCCGCTGCAANNKTGTGTGCGCCTG G250X-R GGCGCACACAMNNTTGCAGCGGATACATACGGG

[0042] The mutant library was constructed for each selected target site using the whole plasmid amplification technology. 190 single clones were selected for each site and placed in LB containing Kan + ) medium in a 96-well plate, and then cultured at 37°C for 16 hours. The seed solution was then transferred to a 96-deep-well plate containing 2YT medium for culture. After shaking culture for 14 hours, the bacteria were collected by centrifugation, the supernatant was removed, and the collected bacteria were frozen at -20°C. After 4 hours, the bacteria were taken out, and the substrate 6-fluorochroman-2-carboxylic acid methyl ester was added to each well for screening reaction. The primary screening mutants were determined according to the reduction of substrate by TLC spot plate analysis. By a similar method, superposition screening of other sites was performed, and finally mutants Mut1 (E145H) and Mut2 (F82P / E145H / Q157E / G250S) 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.

[0043] Example 4 Lipase mutant catalytic activity test

[0044] The mutants Mut1 and Mut2 were expressed in 400 mL of 2YT medium to obtain the corresponding mutant cells. After freezing at -20°C, 0.4 g of the cells were weighed and put into a reactor. At the same time, 0.4 g of substrate 6-fluorochroman-2-carboxylic acid methyl ester, 2 mL of methanol, and 18 mL of 100 mM phosphate buffer were added. The pH value was adjusted to 7.5 with ammonia water, and an oscillation reaction was carried out at 37°C. The reaction was terminated after 24 hours. The samples were extracted with n-butanol and sent for HPLC detection and analysis. The results are shown in Table 2. The catalytic activity of Mut1 and Mut2 was significantly improved compared with the wild type WT.

[0045] Table 2 Catalytic activity test of mutant Mut 1 / 2 and wild type WT

[0046] sample Catalytic substrate concentration (g / L) Conversion rate (%) ee value (%) WT 20 23.5 78.4 Mut1(E145H) 20 43.2 94.1 Mut2(F82P / E145H / Q157E / G250S) 20 49.7 99.2

[0047] Example 5 Lipase mutants catalyze high concentration substrates

[0048] In order to reduce the substrate inhibition, 4.8g of substrate was added to the reaction system in 4 times, with an interval of 2 hours between each addition. That is, 0.4g of cells, 4mL of methanol, 36mL of 100mM phosphate buffer, and 1.2g of substrate 6-fluorochroman-2-carboxylic acid methyl ester were added to the reactor in sequence, and the pH value was adjusted to 7.5 with ammonia water. The reaction was carried out at 37°C with an oscillation reaction, and the substrate was added once every 2 hours for a total of 3 times. After the reaction was completed, the sample was extracted with n-butanol and sent for HPLC detection and analysis. The conversion results are shown in Table 3, and the chiral HPLC analysis spectrum of the product is shown in Figure 3 .

[0049] Table 3 Mutant Mut 1 / 2 and wild type WT transform high concentration substrate

[0050] sample Catalytic substrate concentration (g / L) Conversion rate (%) ee value (%) WT 120 10.7 70.0 Mut1(E145H) 120 43.1 94.0 Mut2(F82P / E145H / Q157E / G250S) 120 49.8 99.3

Claims

1. A lipase mutant, It is characterized in that Taking the wild-type lipase amino acid sequence shown in SEQ ID NO.1 as the reference sequence, single-point or multiple-point mutations occur at positions 82, 145, 157, and 250, wherein the phenylalanine at position 82 mutates to proline, the glutamic acid at position 145 mutates to histidine, the glutamine at position 157 mutates to glutamic acid, and the glycine at position 250 mutates to serine.

2. The lipase mutant according to claim 1, It is characterized in that The amino acid sequences of the lipase mutant are shown in SEQ ID NO.3 and SEQ ID NO.

5.

3. The lipase mutant as claimed in claim 1, It is characterized in that The nucleotide sequences of the lipase mutant are shown in SEQ ID NO.4 and SEQ ID NO.

6.

4. The lipase mutant according to claim 1, It is characterized in that The lipase mutant is expressed in genetically engineered bacteria.

5. A method for preparing nebivolol intermediate I, It is characterized in that The racemic compound II undergoes a selective hydrolysis reaction under the action of lipase to be converted into compound I, wherein the lipase is selected from the lipase mutant in claim 1, 6. The preparation method according to claim 5, It is characterized in that The lipase participates in the catalytic reaction in the form of enzyme powder, enzyme liquid, homogenized liquid, freeze-dried powder, cells containing the enzyme, and the like.

7. The preparation method according to claim 5, It is characterized in that The substrate concentration is 10-150 g / L.

8. The preparation method according to claim 5, It is characterized in that The reaction temperature is 25°C to 40°C.

9. The preparation method according to claim 5, It is characterized in that The reaction needs to be controlled at pH 7.0-8.0.

Images