Application of R-transaminase and mutant thereof in synthesis of chiral beta-amino aryl ether compound

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of R-transaminase and a mutant thereof in synthesis of chiral beta-amino aryl ether compounds. The amino acid sequence of the R-transaminase mutant is obtained by mutation of one site or simultaneous mutation of two or more different sites in the 134 site, the 150 site, the 167 site and the 169 site in the amino acid sequence shown in R-transaminase SEQ ID NO.1 or at least one amino acid sequence with 80% homologous consistency with the amino acid sequence shown in the SEQ ID NO.1. The R-transaminase and the R-transaminase mutant are used as biocatalysts in synthesis of chiral beta-amino aryl ether compounds, show excellent catalytic activity and strict stereoselectivity, and have practical industrial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to application of an R-aminotransferase and a mutant thereof in the synthesis of chiral β-aminoaryl ether compounds. Background Art

[0002] Mexiletine, an important β-aminoaryl ether drug, is used to treat symptoms such as arrhythmias, allodynia, and myotonia. Currently, the marketed mexiletine is in its racemic form. However, R-mexiletine is more active than its S isomer in terms of arrhythmia and cardiac sodium channel binding. Furthermore, the racemic form may have side effects in the treatment of neuromuscular disorders, limiting its application (Journal of Medicinal Chemistry, 2003, 46(24): 5238-5248). Therefore, developing a method for efficiently synthesizing optically pure mexiletine has important scientific significance and application value. In recent years, a variety of methods for synthesizing such compounds have been developed, such as by establishing chiral pools, chemical or enzymatic resolution, and multi-step stereoselective synthesis (Tetrahedron: Asymmetry (2000), 11(17), 3619-3634; Tetrahedron (2008), 64(49), 11110-11114; Tetrahedron Letters, 2015, 56(28): 4195-4199). However, these methods are often plagued by problems such as high pressure conditions, the use of complex chiral ligand catalysts, or heavy metal contamination.

[0003] Transaminases are pyridoxal phosphate (PLP)-dependent enzymes that catalyze the transfer of amino groups from amine donors to prochiral ketones to generate the corresponding chiral amines. As an important class of biocatalysts, they have attracted widespread attention in industry and academia. In particular, with the development of protein engineering, transaminases have shown great potential in the green synthesis of chiral drugs, and a variety of biocatalytic preparation processes for blockbuster drugs based on transaminases have been reported, such as sitagliptin, sacubitril, and remdesivir (Science, 2010, 329(5989):305-309; ACS Catalysis, 2021, 11(6):3762-3770; Organic Process Research & Development, 2022, 26(7):1971-1977). R-transaminases can synthesize R-mexiletine in one step using 1-(2,6-dimethylphenoxy)ethanone as a substrate without the need for an additional cofactor regeneration system. The synthesis of mexiletine using R-aminotransferase as a biocatalyst not only offers the advantages of high stereoselectivity but also, compared to chemical catalysis, can be performed in aqueous solution under mild reaction conditions. It also eliminates the need for the addition of metals and complex ligand catalysts, facilitating subsequent product purification. However, the catalytic activity of transaminases is currently known to be relatively low. Therefore, the development of more efficient enzyme catalysts is needed to reduce reaction costs and promote industrial application. Summary of the Invention

[0004] The present invention aims to solve the problem of low catalytic efficiency of existing R-transaminase in synthesizing β-aminoaryl ether compounds. Through enzyme engineering strategy, an excellent transaminase mutant is obtained to efficiently synthesize chiral β-aminoaryl ether compounds.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] The amino acid sequence of the R-aminotransferase provided by the present invention is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] MTLADSGTDFSTSNLVAVEPGAIREDTPPGSVIQYSDYELDTSSPYAGGAAWIEGEYVPASE

[0009] ARISIFDTGFGSDLTYTVAHVWHGNIFRLADHIERLLDGARKLRLASPYDETEIAEIAKRC

[0010] VGLSQLREAYVNITLTRGYGKRKGEKDLSKLTSQIYVYAIPYLWAFPPYEQIFGTSAVVPRH

[0011] VQRAGRNTIDPTIKNYQWGDLTAASFEAKDRGARTGILLDADGCVAEGPGFNVVVVKDG

[0012] ALASPSRNALPGITRKTVFEIAHARGISAELRDVTSRELYDADELMAVTTAGGVTPITSLDGAAVGDGEPGPITVAIRDRFWALMDEPSDLIDTIRYDVDR。

[0013] A gene encoding the foregoing R-transaminase, the nucleotide sequence of which is as shown in SEQ ID NO.2.

[0014] ATGACCCTGGCGGATAGCGGCACCGATTTTAGCACGAGCAACCTGGTGGCGGTGGAAC

[0015] CGGGCGCGATTCGCGAAGATACCCCGCCGGGCAGCGTGATTCAGTATAGCGATTATGAA

[0016] CTGGATACGAGCAGCCCGTATGCGGGCGGCGCGGCGTGGATTGAAGGCGAATATGTGC

[0017] CGGCGAGCGAAGCGCGCATTAGCATTTTTGATACCGGCTTTGGCCATAGCGATCTGACC

[0018] TATACCGTGGCGCATGTGTGGCATGGCAACATTTTTCGCCTGGCGGATCATATTGAACGC

[0019] CTGTTAGATGGCGCCCGCAAACTGCGCCTGGCGAGCCCGTATGATGAAACCGAAATTG

[0020] CGGAAATTGCGAAACGCTGCGTGGGCCTGAGTCAGCTGCGCGAAGCGTATGTGAACAT

[0021] TACCCTGACCCGCGGCTATGGCAAACGCAAAGGCGAAAAAGATCTGAGCAAACTGAC

[0022] GAGTCAGATTTATGTGTATGCGATTCCGTATCTGTGGGCGTTTCCGCCGTATGAACAGAT

[0023] TTTTGGCACGAGCGCGGTGGTGCCGCGCCATGTGCAGCGCGCGGGCCGCAACACCATT

[0024] GATCCGACGATTAAAAACTATCAGTGGGGCGATCTGACCGCGGCGAGCTTTGAAGCGA

[0025] AAGATCCGGCGCGCGCACCGGCATTCTGCTGGATGCGGATGGCTGCGTGGCGGAAGG

[0026] CCCGGGCTTTAACGTGGTTGTGGTGAAAGATGGCGCCTGGCGAGCCCGAGCCGCAAC

[0027] GCGCTGCCGGGCATTACCCGCAAAACCGTGTTTGAAATTGCGCATGCGCGCGGCATTAG

[0028] CGCGGAACTGCGCGATGTGACGAGCCGCGAACTGTATGATGCGGATGAGCTGATGGCG

[0029] GTGACCACCGCGGGCGGTGGTGACCCCAATTACGAGCCTGGATGGTGCGGCCGTGGGCG

[0030] ATGGCGAACCGGGCCCGATTACCGTGGCGATTCGCGATCGCTTTTGGGCGCTGATGGATGAACCGAGCGATCTGATTGATACCATTCGCTATGATGTGGATCGC.

[0031] Furthermore, the present invention provides an R-transaminase mutant, whose amino acid sequence is obtained by mutating one of positions 134, 150, 167, and 169 in the amino acid sequence of R-transaminase shown in SEQ ID NO.1 or an amino acid sequence with at least 80% homology to the amino acid sequence shown in SEQ ID NO.1, or mutating two or more different positions simultaneously.

[0032] Furthermore, in the mutant, positions 134, 150, 167, and 169 are mutated identically or differently to A, T, V, I, F, and S.

[0033] Furthermore, the 134th position mutated to F, the 150th position mutated to A or V or I, the 167th position mutated to A or S or T, and the 169th position mutated to V or I.

[0034] Furthermore, the mutant is a mutant in which position 134 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to F, or position 150 is mutated to V, or position 150 is mutated to I, or position 169 is mutated to V, or position 169 is mutated to I, or position 134 is mutated to F and position 150 is mutated to V, or position 134 is mutated to F and position 150 is mutated to I, or position 150 is mutated to A and position 169 is mutated to I, or position 150 is mutated to V and position 169 is mutated to I, or position 150 mutates to I and position 169 mutates to I, or position 150 mutates to S and position 169 mutates to I, or position 134 mutates to F and position 169 mutates to I, or position 167 mutates to T and position 169 mutates to I, or position 150 mutates to A and position 169 mutates to I and position 134 mutates to F, or position 150 mutates to V and position 169 mutates to I and position 134 mutates to F.

[0035] The present invention provides the use of the aforementioned R-transaminase and R-transaminase mutants in synthesizing chiral β-aminoaryl ether compounds.

[0036] Furthermore, R-aminotransferase and its mutants are used as biocatalysts to catalyze the asymmetric amination of phenoxyacetone compounds to prepare chiral β-aminoaryl ether compounds.

[0037] The reaction formula is:

[0038]

[0039] The phenoxyacetone compound is selected from any one of the following:

[0040]

[0041] Furthermore, the concentration of the phenoxyacetone compound in the reaction system is 1-500 mM; the final concentration of the amine donor is 1-2000 mM; the final concentration of the cofactor is 0.1-10 mM; the pH value of the reaction system is 6.0-10.0, and the temperature of the reaction system is 20-45°C; the cosolvent concentration is 0.1-30% (v / v) of the reaction system; and the buffer concentration is 0.05-0.2 M. Preferably, the concentration of the phenoxyacetone compound in the reaction system is 1-300 mM; and the final concentration of the amine donor is 10-1000 mM.

[0042] Furthermore, the biocatalyst is in the form of free cells, free enzymes, immobilized cells or immobilized enzymes.

[0043] Furthermore, the cofactor is pyridoxal phosphate (PLP); the amine donor is alanine, phenylethylamine or isopropylamine, preferably isopropylamine; the cosolvent is selected from one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, and acetone, preferably methanol or dimethyl sulfoxide; and the buffer is Tris-HCl buffer or phosphate buffer.

[0044] Beneficial technical achievements of the present invention:

[0045] The present invention utilizes enzyme engineering technology to modify the R-aminotransferase PaTA, resulting in a series of superior mutants capable of efficiently synthesizing chiral β-aminoaryl ether compounds. These superior mutants enable efficient enzymatic synthesis of R-mexiletine (R-1d) and its aryl-substituted derivative (R-12d). Compared to existing enzymatic synthesis processes, this method offers high substrate loading and space-time yields, further reducing production costs and possessing practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the SDS-PAGE electrophoresis diagram of PaTA.

[0047] Figure 2 The reaction time-conversion curves of compounds 1c and 12c are shown.

[0048] Figure 3 Gas phase diagram showing the conversion rate of substrate 1c catalyzed by PaTA mutants.

[0049] Figure 4 Gas phase diagram showing the conversion rate of substrate 12c catalyzed by PaTA mutants.

[0050] Figure 5 Liquid phase diagram showing the ee value of the PaTA mutant catalyzing the conversion of substrate 1c to product R-1d.

[0051] Figure 6 Liquid phase diagram showing the ee value of the PaTA mutant catalyzing the conversion of substrate 12c to product R-12d. DETAILED DESCRIPTION

[0052] The following further illustrates the use of an R-aminotransferase and its mutants in the synthesis of chiral β-aminoaryl ether compounds, using the accompanying drawings and specific examples. Unless otherwise specified, the experimental protocols used in the present invention are well known to those skilled in the art. Furthermore, the examples should be construed as illustrative rather than limiting.

[0053] Example 1 Acquisition of R-transaminase PaTA gene

[0054] The amino acid sequence of PaTA (NCBI sequence number: WP_093355841.1) was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / genome / ). A gene synthesis company was commissioned to synthesize the codon-optimized target DNA sequence.

[0055] Example 2 Obtaining the R-transaminase PaTA mutant gene

[0056] Using the plasmid containing the wild-type gene of R-aminotransferase PaTA as a template, the mutant gene was obtained by overlap extension PCR. The primers involved in the mutant are shown in Table 1:

[0057] Table 1 List of primers involved

[0058]

[0059]

[0060] The PCR amplification system is shown in Table 2:

[0061] Table 2 PCR amplification system

[0062]

[0063] Amplification program: 94°C: 10 min, 35 cycles of (94°C: 30 s, 50°C: 30 s, 72°C: 90 s), 72°C, 10 min.

[0064] Two fragments were generated from the first round of PCR. After verifying the correct band size by agarose gel electrophoresis, the fragments were recovered using a standard DNA gel recovery kit and used as templates for the second round of PCR. The second round of PCR amplified the mutant gene using the terminal primers PaTA-Nde IF and PaTA-Xho IR according to the system described in Table 2. After the second round of PCR, the product was recovered using a DNA purification kit to obtain a DNA product containing the mutation site.

[0065] Example 3 Construction of recombinant engineering plasmid

[0066] The PaTA and PaTA mutant gene fragments cloned in Examples 1 and 2 and the vector plasmid pET-28a(+) were double-digested with Nde I and Xho I, respectively (Table 3). After incubation at 37°C for 5 hours, the digested products were separated by agarose gel electrophoresis and recovered using a standard agarose gel DNA recovery kit to obtain DNA fragments and vector fragments with identical sticky ends. The digested target gene and vector plasmid were ligated using T4 DNA ligase to construct recombinant plasmids. The recombinant plasmids were transformed into competent E. coli Rosetta 2 (DE3), plated on Kana-resistant LB agar plates, and cultured overnight at 37°C.

[0067] Table 3 Enzyme digestion system

[0068]

[0069]

[0070] Example 4 Expression of transaminase PaTA protein

[0071] Different positive transformants verified on the LB agar plate of Example 3 were inoculated into 4 mL of LB liquid medium containing Kana resistance, and cultured at 37°C, 200 rpm for 6-8 h to obtain seed solutions of different mutants. 1 mL of seed solutions of different mutants were inoculated into 100 mL of LB liquid medium containing Kana resistance, and cultured at 37°C, 200 rpm until the culture solution OD reached 0. 600 When the concentration reaches 0.8-1.0, add IPTG solution with a final concentration of 0.1 mM and lower the temperature to 20-25°C to induce expression for 15-20 hours. Centrifuge at 3000 rpm for 10 minutes to collect the cells, wash with physiological saline, and centrifuge again to obtain the whole-cell biocatalyst.

[0072] Example 5 Gas Chromatography Conversion Rate Determination Method

[0073] Detection conditions were a SCION 456-GC system with an injection temperature of 240°C, a split ratio of 20:1, and an FID detector temperature of 240°C. The initial temperature was 60°C, and the temperature was increased to 240°C at a rate of 20°C / min. The gas phase retention times of substrates 1c-16c and products 1d-16d are shown in Table 4.

[0074] Table 4 Gas phase analysis retention time of substrates (1c-16c)

[0075]

[0076]

[0077] Example 6 Liquid Chromatography Method for Determining Product ee Value

[0078] A JASCO LC-1500 system was used, using a Daicel column and Boc derivatization. Detection conditions were: 220 nm, n-hexane:isopropanol = 90:10, and a flow rate of 0.7 mL / min. The R and S liquid chromatography retention times of products 1d-16d are shown in Table 5.

[0079] Table 5 Chiral analysis conditions and retention time of products

[0080]

[0081] Example 7 Transaminase PaTA and its mutants catalyze substrate 1c

[0082] Reaction system: 50 mg / mL wet cells of different mutants as described in Example 3, substrate 1c (100 mM), isopropylamine hydrochloride (500 mM), PLP (1 mM), methanol (10% v / v), and pH 9.0 Tris-HCl buffer (100 mM).

[0083] The reaction temperature was 25° C. After 40 min of reaction, the pH of the aqueous phase was adjusted to 12-13 with 10N NaOH solution, and the mixture was extracted with ethyl acetate. The reaction conversion rate was determined by gas chromatography. The conversion rate is shown in Table 6:

[0084] Table 6 Results of the conversion rate of substrate 1c catalyzed by PaTA and its mutants

[0085]

[0086] Example 8 Transaminase PaTA mutant catalyzes substrate 12c

[0087] Reaction system: 25 mg / mL wet cells of different mutants as described in Example 3, substrate 12c (100 mM), isopropylamine hydrochloride (500 mM), PLP (1 mM), methanol (10% v / v), and pH 9.0 Tris-HCl buffer (100 mM).

[0088] The reaction temperature was 25° C. After 40 min of reaction, the pH of the aqueous phase was adjusted to 12-13 with 10N NaOH solution, and the mixture was extracted with ethyl acetate. The reaction conversion rate was determined by gas chromatography. The conversion rate is shown in Table 7.

[0089] Table 7 Results of the conversion rate of substrate 12c catalyzed by PaTA and its mutants

[0090]

[0091] Example 9 Synthesis of Chiral β-Aminoaryl Ether Compounds Catalyzed by Transaminase PaTA Mutants

[0092] Reaction system: 50 mg / mL wet cells of different mutants as described in Example 3, substrates 1c-16c (100 mM), isopropylamine hydrochloride (500 mM), PLP (1 mM), methanol (10% v / v), and pH 9.0 Tris-HCl buffer (0.1 M). a represents 100 mg / mL wet cells; b represents 20 mM substrate, 100 mg / mL wet cells).

[0093] The reaction temperature was 25° C. After 40 min of reaction, the pH of the aqueous phase was adjusted to 12-13 with 10N NaOH solution, and the mixture was extracted with ethyl acetate. The reaction conversion rate was determined by gas chromatography. The conversion rate and ee value are shown in Table 8:

[0094] Table 8 Results of conversion rate and ee value of substrate 1c-16c catalyzed by PaTA and its mutants

[0095]

[0096]

[0097] Note: Mu 1 is a Y134F / K150V mutation, and Mu 2 is a K150V / A169I mutation.

[0098] Example 10 Synthesis of R-mexiletine (R-1d) catalyzed by transaminase PaTA mutant

[0099] The reaction system was sequentially added with substrate 1c (final concentration 150 mM), cofactor PLP (1 mM), isopropylamine hydrochloride (700 mM), mutant Mu 1 wet cell catalyst (150 g / L), methanol (10% v / v), and Tris-HCl buffer (0.1 M) at pH 9.0. The reaction was carried out on a thermostatic shaker at 25°C and 200 rpm for 1 hour. Upon completion of the reaction, the reaction conversion was greater than 95%, and the product ee value was greater than 99%.

[0100] Example 11 Synthesis of R-12d Catalyzed by Transaminase PaTA Mutant

[0101] The reaction system was sequentially added with substrate 12c (final concentration 200 mM), cofactor PLP (1 mM), isopropylamine hydrochloride (700 mM), mutant Mu 2 wet cell catalyst (150 g / L), methanol (10% v / v), and Tris-HCl buffer (0.1 M) at pH 9.0. The reaction was carried out on a thermostatic shaker at 25°C and 200 rpm for 1 hour. Upon completion of the reaction, the reaction conversion was greater than 98%, and the product ee value was greater than 99%.

Claims

1. An R-transaminase mutant, characterized in that The amino acid sequence is obtained by mutating one of positions 134, 150, 167, and 169 in the amino acid sequence of R-transaminase shown in SEQ ID NO. 1 or an amino acid sequence with at least 80% homology to the amino acid sequence shown in SEQ ID NO. 1, or by mutating two or more different positions simultaneously.

2. The R-transaminase mutant according to claim 1, characterized in that In the mutant, positions 134, 150, 167, and 169 are mutated identically or differently to A, T, V, I, F, and S.

3. The R-transaminase mutant according to claim 1, characterized in that The 134th position mutated to F, the 150th position mutated to A, V or I, the 167th position mutated to A, S or T, and the 169th position mutated to V or I.

4. Use of the R-aminotransferase according to claim 1 or the R-aminotransferase mutant according to any one of claims 1 to 3 in the synthesis of chiral β-aminoaryl ether compounds.

5. The use according to claim 4, characterized in that The use of the R-aminotransferase and the R-aminotransferase mutant as a biocatalyst in catalyzing the asymmetric amination of phenoxyacetone compounds to prepare chiral β-aminoaryl ether compounds; The reaction formula is:

6. The use according to claim 5, characterized in that The phenoxyacetone compound is selected from any one of the following:

7. The biocatalyst according to claim 5, characterized in that The biocatalyst is in the form of free cells, free enzymes, immobilized cells or immobilized enzymes.

8. The use according to claim 5, characterized in that The concentration of the phenoxyacetone compound in the reaction system is 1-500 mM; the final concentration of the amine donor is 1-2000 mM; the final concentration of the cofactor is 0.1-10 mM; the pH value of the reaction system is 6.0-10.0, and the temperature of the reaction system is 20-45° C.; the cosolvent content is 0.1-30% (v / v) of the reaction system; and the buffer concentration is 0.05-0.2 M. Preferably, the concentration of the phenoxyacetone compound in the reaction system is 1-300 mM; and the final concentration of the amine donor is 10-1000 mM.

9. The use according to claim 5, characterized in that: The cofactor is pyridoxal phosphate (PLP); the amine donor is alanine, phenylethylamine or isopropylamine, preferably isopropylamine; the cosolvent is selected from one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, and acetone, preferably methanol or dimethyl sulfoxide; and the buffer is Tris-HCl buffer or phosphate buffer.

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