Method for preparing S-(-)-1-(2-naphthyl) ethylamine by enzyme method

Abstract

The invention discloses a method for preparing S-(-)-1-(2-naphthyl) ethylamine by an enzyme method. According to the method, 2-acetylnaphthalene is used as a substrate and is converted into S-(-)-1-(2-naphthyl) ethylamine under the catalytic action of transaminase, the conversion rate is as high as 96%, and the ee value of the product is as high as 99%. The method disclosed by the invention is simple to operate and high in atom economy, and can be used for industrial production.

Description

Technical Field:

[0001] The present invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing S-(-)-1-(2-naphthyl)ethylamine by enzymatic method. Background Art:

[0002] S-(-)-1-(2-naphthyl)ethylamine, with the structure shown in Formula I and CAS number 3082-62-0, is an important pharmaceutical intermediate and can be used to prepare human immunoglobulin E synthesis inhibitors, such as for the treatment of allergic asthma, rhinitis, etc. In the future, this compound can be used to prepare more drugs.

[0003]

[0004] The preparation route of S-(-)-1-(2-naphthyl)ethylamine disclosed in Patent CN1070847C is shown in Scheme 1. This route uses racemic 1-amino-1-(β-naphthyl)ethane as the substrate and an esterase derived from Pseudomonas spec. DSM8246 as the resolving agent to obtain Compound I with an ee value of 89.5%. In this route, the material loss in the resolution step is relatively large and the yield is low.

[0005]

[0006] The preparation route of S-(-)-1-(2-naphthyl)ethylamine disclosed in Patent CN102863304B is shown in Scheme 2. This route uses 2-acetylnaphthalene as the substrate, first reacts with 2-hydroxybenzylamine and a chiral base at 80 °C for 72 hours to obtain an intermediate aldehyde imine, and then hydrolyzes it under acidic conditions for 24 hours to obtain Compound I, with a yield of 73% and an ee value of 81%.

[0007]

[0008] The preparation route of S-(-)-1-(2-naphthyl)ethylamine reported in the literature Catal. Sci. Technol., 2012, 2, 1556-1559. is shown in Scheme 3. This route uses 2-acetylnaphthalene as the substrate and is converted into Compound I under the catalytic action of PIG5 enzyme, with a conversion rate of 90% and an ee value of 99%.

[0009]

[0010] Therefore, we need to develop a preparation method that is simple to operate, has a high yield, good selectivity, is safe, environmentally friendly and suitable for industrialization. Summary of the Invention:

[0011] The object of the present invention is to provide a method for preparing S-(-)-1-(2-naphthyl)ethylamine that is simple to operate and easy to industrialize in view of the deficiencies of the prior art.

[0012] The technical solution adopted by the present invention is as shown in Scheme 4:

[0013]

[0014] The present invention provides a method for preparing S-(-)-1-(2-naphthyl)ethylamine, which specifically comprises the following steps: using 2-acetylnaphthalene as a substrate and converting it into S-(-)-1-(2-naphthyl)ethylamine under the catalytic action of a transaminase.

[0015] Furthermore, the transaminase is derived from Naegleria gruberi, and the NCBI accession number of its amino acid sequence is XP_002681137.1, and the NCBI accession number of its nucleotide sequence is XM_002681091.1.

[0016] Furthermore, the amino acid sequence and nucleotide sequence of the transaminase are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0017] Furthermore, the transaminase participates in the catalytic reaction in the form of transaminase enzyme powder, transaminase enzyme solution, transaminase homogenate, transaminase lyophilized powder, cells containing transaminase, etc., and transaminase cells are preferred.

[0018] Furthermore, the transaminase expression receptor strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis, and Escherichia coli is preferred.

[0019] Furthermore, an amino donor needs to be added to the reaction, and the amino donor is selected from (±)-phenylethylamine, (+)-phenylethylamine, (-)-phenylethylamine and isopropylamine, and (±)-phenylethylamine is preferred.

[0020] The beneficial effect of the present invention is that the present invention provides a method for preparing S-(-)-1-(2-naphthyl)ethylamine. A new transaminase is used in this method, which can selectively convert 2-acetylnaphthalene into S-(-)-1-(2-naphthyl)ethylamine. The substrate conversion rate is as high as 96%, and the ee value of the product is as high as 99%. This method is simple to operate, has high atom economy and good selectivity, and can be used for industrial production. Description of the Drawings

[0021] Figure 1 Protein expression diagram of transaminase in Example 3 Detailed Description

[0022] The technical content of the present invention will be further elaborated below in conjunction with specific embodiments, with the aim of better understanding the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0023] Example 1 Construction of Transaminase Enzyme Library

[0024] Select the transaminase sequence from Naegleria fowleri as the template, search on the NCBI website, search for relevant invertase genes within the range of 35%-80% identity to construct a screening enzyme library, and finally obtain 55 transaminases. Gene cloning was performed on these transaminases respectively, and the above-mentioned transaminases were constructed onto pET28a using NcoI and XhoI as restriction sites. Each glycerol tube containing the transaminase strain was spread on a Kan + resistant plate and cultured upside down at 37°C. The next day, single colonies were picked into a 96-well deep well plate containing LB medium and cultured at 37°C for 12 h. Then, the seed liquid was transferred to a 96-well deep well plate containing 2YT medium and cultured with shaking for 5 h. After that, 0.1 mM IPTG was added for induction, and at the same time, the temperature was lowered to 25°C. After 16 h of induction expression, the cultured 96-well plate was placed in a centrifuge and centrifuged at 4000 rpm for 15 min. The supernatant was removed, and the 96-well plate containing the cells was frozen at -40°C for standby.

[0025] Example 2 Screening of Transaminases

[0026] The ATA cell scanning reaction system is shown in Table 1. Add (±)-phenylethylamine and PBS to the reaction kettle in sequence, adjust the pH value to 9.1 with 6M HCl, then add the substrate 2-acetylnaphthalene (100 g / L in DMSO), coenzyme PLP, and the frozen cells in sequence, and perform an oscillating reaction at 30°C. After 24 h of reaction, the reaction is terminated, samples are taken and sent for HPLC detection and analysis. The results show that the transaminase (NG-ATA) from Naegleria gruberi has the best conversion rate and chiral result.

[0027] Table 1 ATA cell scanning reaction system

[0028]

[0029] Example 3 Expression and Catalysis of NG-ATA

[0030] Transfer the NG-ATA strain into an LB test tube with Kan + resistance and culture it overnight at 37°C. Then, transfer the seed liquid to a 2YT medium with Kan + resistance according to an inoculation amount of 1.5%, and culture the bacteria at 37°C. Wait for the biomass OD 600When the value reaches about 0.8, IPTG induction is carried out. The temperature needs to be reduced to 25 °C. After expressing for 16 h, the cells are collected, and the cells are broken by ultrasound for electrophoresis analysis. The results are shown in Figure 1 , the size of the protein is consistent with the expectation, and there is good soluble expression.

[0031] Catalytic tests were carried out on NG-ATA cells. The reaction system is shown in Table 2. (±)-phenethylamine, 2-acetylnaphthalene, PBS, and purified water were sequentially added to the system, and then the pH was adjusted to 9.1 with 6 M hydrochloric acid. Then methanol, PLP, and cells were added. After reacting with shaking at 30 °C for 24 h, samples were taken and sent for HPLC detection and analysis. The substrate conversion rate was 70.3%, and the ee value of the product was 99.8%.

[0032] Table 2 Reaction system of NG-ATA cells

[0033]

[0034] Optimization of reaction conditions in Example 4 - Selection of amino donors

[0035] The amino donors in the transformation process were optimized. The amino donors include isopropylamine, (±)-phenethylamine, (+)-phenethylamine, and (-)-phenethylamine. The reaction system refers to Table 3. Substrate, amino donor, PBS, and purified water were sequentially added to the reaction kettle, and the pH was adjusted to 9.1 with 6 M hydrochloric acid. Then methanol, PLP, and cells were added. After reacting with shaking at 30 °C for 24 h, samples were taken and sent for HPLC detection, and the reaction results were analyzed. The specific data are shown in Table 4. The screening results show that when the amino donor is (±)-phenethylamine, the ee value and conversion rate results are the best.

[0036] Table 3 Parameters of reaction systems with different amino donors

[0037]

[0038] Table 4 Optimization results of different amino donors

[0039]

[0040] Optimization of reaction conditions in Example 5 - Selection of cosolvents

[0041] Optimize the cosolvent in the conversion process. The cosolvents include tetrahydrofuran, methanol, ethyl acetate, and methyl tert-butyl ether. Refer to Table 5 for the reaction system. Add the substrate, (±)-phenethylamine, PBS, and purified water into the reaction kettle in sequence. Adjust the pH to 9.1 with 6M hydrochloric acid. Then add the cosolvent (select another group without cosolvent as a control), PLP, and cells. After oscillating and reacting at 30°C for 24 hours, take samples and send them for HPLC detection. Analyze the reaction results. The specific data are shown in Table 6. The screening results show that when no cosolvent is added, the ee value and conversion rate results are the best.

[0042] Table 5 Parameters of the reaction system with different cosolvents

[0043]

[0044]

[0045] Table 6 Optimization results with different cosolvents

[0046]

Claims

1. A method for enzymatically preparing S-(-)-1-(2-naphthyl)ethylamine, characterized in that, This method uses 2-acetylnaphthalene as a substrate, which is converted into S-(-)-1-(2-naphthyl)ethylamine under the catalysis of a transaminase, and the amino acid sequence of the transaminase is as shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the transaminase is as shown in SEQ ID NO.

2.

3. The preparation method according to claim 1, characterized in that, The transaminase is derived from Naegleria gruberi.

4. The preparation method according to claim 1, characterized in that, The transaminase participates in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenate, lyophilized powder, cells containing the enzyme, etc.

5. The preparation method according to claim 1, characterized in that, The transaminase expression strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis.

6. The preparation method according to claim 1, characterized in that, An amino donor needs to be added in the reaction, and the amino donor is selected from (±)-phenylethylamine, (+)-phenylethylamine, (-)-phenylethylamine and isopropylamine.

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