R-transaminase and application thereof in asymmetric synthesis of chiral N-heterocyclic amine compound

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to four R-transaminase and application thereof in asymmetric synthesis of chiral N-heterocyclic amine active drug intermediates. The amino acid sequences of the R-transaminase disclosed by the invention are shown as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7. Four kinds of R-transaminase are obtained through gene mining and screening, and recombinant escherichia coli is constructed and expressed by adopting a gene engineering technology. The obtained R-transaminase can be used for synthesizing chiral drug intermediates (R)-3-amino-N-Boc-pyrrole (1b) and (R)-3-amino-N-Boc-piperidine (2b) with high stereoselectivity, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and specifically relates to four recombinant R-transaminases and applications thereof in asymmetric synthesis of chiral N-heterocyclic amine active drug intermediates. Background Art

[0002] Optically pure N-heterocyclic amines are an important class of bioactive molecules with important application value in the pharmaceutical industry. For example, chiral N-heterocyclic amines (R)-3-amino-N-Boc-pyrrole (1b) and (R)-3-amino-N-Boc-piperidine (2b) can be used to prepare a variety of bioactive drugs, including hypoglycemic drugs dulagliptin, alogliptin, trelagliptin and linagliptin and antibiotic ceftriaxone. In the synthetic routes of 1b and 2b, the introduction of chiral amino groups is critical. The synthetic methods reported so far include chemical and biocatalytic methods. Among them, chemical synthesis methods usually face the defects of cumbersome steps and poor optical purity of products.

[0003] Transaminase, also known as aminotransferase, can specifically and selectively transfer the amino group to the ketone substrate in the presence of an amino donor to generate the corresponding chiral amine. Transaminase can be divided into α-transaminase and ω-transaminase according to the position of the amino group of its catalytic product. Among them, α-transaminase is more common, but it can only catalyze the transfer of α-amino group. ω-transaminase is less common. In addition to catalyzing the transfer of α-amino group, it can also catalyze the transfer of non-α-amino group. It has a wider substrate spectrum and strict stereoselectivity, and can catalyze the production of corresponding chiral amines from prochiral ketones under mild conditions.

[0004] Literature reports show that S-selective enzymes are more common among ω-transaminases, while R-selective enzymes are less common. Therefore, discovering new R-type transaminases with industrial application prospects is of great significance for the synthesis of chiral amines. Summary of the invention

[0005] The present invention aims to explore R-transaminase, expand the substrate spectrum of R-transaminase, and provide the application of R-transaminase in asymmetric synthesis of chiral N-heterocyclic amine compounds.

[0006] To achieve the above purpose, the present invention adopts the following technical solution:

[0007] The present invention provides the use of R-aminotransferase in asymmetric synthesis of chiral N-heterocyclic amine compounds. The amino acid sequence of the R-aminotransferase is shown in SEQ ID NOs. 1, 3, 5 and 7.

[0008] SEQ ID NO.1:

[0009] MTTSNDIDYSASNLVSVAPGAIREPTPSGSVIQYSEYELDESSPFAGGAAWVEG

[0010] EYVPAAEARISLFDTGFGHSDLTYTVAHVWHGNIFRLKDHIDRVFDGARELRL

[0011] QSPLSKAEVEDITKRCVSLSQLRESFVNITITRGYGARKGEKDLSKLTSQIYIYA

[0012] IPYLWAFPPEEQIFGTSAIVPRHVRRAGRNTVDPTVKNYQWGDLTAASFEAKD

[0013] RGARTAILLDSDNCVAEGPGFNVVMVKDGKLSSPSRNALPGITRLTVMELAA

[0014] DMGIEFTLRDITSRELYEADELIAVTTAGGITPITSLDGVPLGDGEPGPMTVAIR

[0015] DRFWALMDEPSPLVEAIEY

[0016] SEQ ID NO.3:

[0017] MTASIDHSMSNLVSVEPGAIREATPEGSVIQYSDYELDTSSEFAGGVAWIEGEY

[0018] VPASEARISIFDTGFGHSDLTYTVPHVWHGNIFRLGDHLDRLLDGARRLYLDP

[0019] GYTKEELADVVKKCVSLSQLRESFVNITITRGFGQRRGEKDLSKLTHQVYIYAI

[0020] PYLWAFPPDEQIFGTSVIVPRHVRRASRSTIDPTIKNYQWGDLTAASFEAKERG

[0021] ARSAILLDADNCVAEGPGFNVVLVKDGELFSPSRNALPGITRKTVFELADVMG

[0022] VPATLRDVTSRELYEADELMAVTTAGGVTPIIELDGEPQSGGKPGPLTTEIRDRF

[0023] WALMDEPSDLIEAIQY

[0024] SEQ ID NO.5:

[0025] MAITDSATTNLVSVEPGAIREDTPAGSVIQYSDYALDTSSRFAGGAAWIEGEYV

[0026] PASEARISIFDTGFGHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLPSPL

[0027] TKEELASIAKRCVALSQLRESYVNITLTRGFGARKGEKDLSKLTTQVYVYAIPY

[0028] LWAFPPHEQIFGTSAIVPRHVRRAGRNTIDPTVKNYQWGDLTAASFEAKDRGV

[0029] RTAILLDADGCVAEGPGFNVVIVKDGRLVSPSRNALPGITRRTVFEIAKAMGID

[0030] ADLRDVTSQELYDADELIAVTTAGGVTPITSLDGAPVADGTPGSVTVALRDRF

[0031] WALMDEPSPLVEAID

[0032] SEQ ID NO.7:

[0033] MTLADSGTDFSTSNLVAVEPGAIREDTPPGSVIQYSDYELDTSSPYAGGAAWIE

[0034] GEYVPASEARISIFDTGFGHSDLTYTVAHVWHGNIFRLADHIERLLDGARKLRL

[0035] ASPYDETEIAEIAKRCVGLSQLREAYVNITLTRGYGKRKGEKDLSKLTSQIYVY

[0036] AIPYLWAFPPYEQIFGTSAVVPRHVQRAGRNTIDPTIKNYQWGDLTAASFEAK

[0037] DRGARTGILLDADGCVAEGPGFNVVVVKDGALASPSRNALPGITRKTVFEIAH

[0038] ARGISAELRDVTSRELYDADELMAVTTAGGVTPITSLDGAAVGDGEPPGPITVAI

[0039] RDRFWALMDEPSDLIDTIRYDVDR

[0040] The present invention also provides a gene encoding the aforementioned R-aminotransferase.

[0041] In the above technical solution, further, the nucleotide sequence of the gene is shown as SEQ ID NO.2, 4, 6, 8.

[0042] In the above technical solution, further, the R-aminotransferase is used as a biocatalyst in catalyzing the transamination reaction of N-heterocyclic ketone compounds to prepare chiral N-heterocyclic amines.

[0043] In the above technical solution, further, the structure of the N-heterocyclic ketone compound is as shown in Formula 1:

[0044] Formula 1:

[0045] In the above technical solution, further, the R-aminotransferase as a biocatalyst is in the form of free cells, free enzymes, immobilized cells and immobilized enzymes.

[0046] In the above technical scheme, further, the method for synthesizing chiral N-heterocyclic amine compounds is: in a buffer solution, in the presence of an amine donor and a cofactor, using the aforementioned R-aminotransferase as a catalyst, asymmetric amination of N-Boc-3-pyrrolidone (1a) or N-Boc-3-piperidone (2a) to generate chiral N-heterocyclic amines, the reaction formula:

[0047]

[0048] In the above technical scheme, further, the substrate concentration in the reaction system is 0.1-300mM; the final concentration of the amine donor in the reaction system is 1-2000mM, and the amine donor is alanine, phenethylamine or isopropylamine, preferably isopropylamine; the cofactor in the reaction system is pyridoxal phosphate (PLP), and the final concentration of the cofactor is 0.1-5mM.

[0049] In the above technical solution, further, the substrate concentration is 0.1-100 mM; the final concentration of the amine donor is 1-500 mM.

[0050] In the above technical solution, further, the pH value of the reaction system is 7.0-10.0; the temperature is 20-45°C; the concentration of the co-solvent is 0.1-30% (v / v) of the reaction system; and the concentration of the buffer is 0.05-0.2M.

[0051] In the above technical solution, further, the co-solvent is selected from one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile and acetone.

[0052] Beneficial technical achievements of the present invention:

[0053] The present invention obtains four R-transaminases by gene mining and screening, and uses genetic engineering technology to construct and express recombinant Escherichia coli. The R-transaminases obtained by the present invention can synthesize chiral drug intermediates (R)-3-amino-N-Boc-pyrrole (1b) and (R)-3-amino-N-Boc-piperidine (2b) with high stereoselectivity, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The soluble protein expression was analyzed by SDS-PAGE.

[0055] Figure 2 Gas phase detection diagram of substrate 1a and its product 1b.

[0056] Figure 3 Gas phase detection diagram of substrate 2a and its product 2b.

[0057] Figure 4 Chiral detection liquid phase diagram of racemic-1b and (R)-1b.

[0058] Figure 5 Chiral detection liquid phase diagram of racemic-2b and (R)-2b. DETAILED DESCRIPTION

[0059] The R-aminotransferase of the present invention and its application in the asymmetric synthesis of chiral N-heterocyclic amine compounds are further explained below in conjunction with the accompanying drawings and specific examples.

[0060] The present invention obtains four R-transaminase genes from Williamsia marianensis, Aeromicrobium terrae, Pseudonocardia ammonioxydans and Prauserella flavalba through gene mining and screening, and uses genetic engineering technology to construct and express recombinant Escherichia coli. The R-transaminase obtained by the present invention can synthesize chiral drug intermediates (R)-3-amino-N-Boc-pyrrole (1b) and (R)-3-amino-N-Boc-piperidine (2b) with high stereoselectivity, and has good application prospects.

[0061] Example 1 Obtaining transaminase gene

[0062] Potential R-transaminase amino acid sequences (Table 1) were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / genome / ), and a gene synthesis company was commissioned to synthesize the codon-optimized target DNA sequence.

[0063] Table 1 R-transaminase sources and NCBI sequence numbers

[0064]

[0065]

[0066] Example 2: Construction of recombinant engineering plasmid

[0067] The above synthesized target DNA sequence and vector pET-28a(+) or pET-28b_NhisMBP-V were double-digested with NdeⅠ and XhoⅠ, respectively (Table 2). After incubation at 37℃ for 4-6h, the digested products were recovered by DNA product gel recovery kit to obtain DNA fragments and vector fragments with the same sticky ends. The digested target gene was connected to the vector plasmid using T4DNA ligase to construct recombinant engineering plasmids of different R-transaminases, and the recombinant engineering plasmids were transformed into competent Escherichia coli Rosetta2(DE3), coated on LB agar plates containing Kana resistance, and cultured at 37℃ overnight.

[0068] Table 2 Enzyme digestion system

[0069]

[0070] Example 3: Transaminase protein expression

[0071] Different positive transformants verified on the LB agar plate were inoculated into 4 mL of LB liquid medium containing Kana resistance, and cultured at 37°C and 200 rpm for 6-8 h to obtain seed liquid of different positive transformants. 1 mL of seed liquid of different positive transformants was inoculated into 100 mL of LB liquid medium containing Kana resistance, and cultured at 37°C and 200 rpm in a shaking incubator until the culture liquid 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 r / min for 10 minutes to collect the cells, wash with physiological saline, and centrifuge again to collect the cells to obtain the whole-cell biocatalyst.

[0072] Example 4: Gas chromatography method for measuring conversion rate

[0073] SCION 456-GC system, Chromatographic column, detection conditions: injection temperature 240℃, split ratio 20:1, FID detector temperature 240℃. Initial temperature 60℃, rising to 240℃ at a rate of 20℃ / min. The retention times of 1a and 1b were 7.86min and 8.08min, respectively, and the retention times of 2a and 2b were 8.49min and 8.18min, respectively.

[0074] Example 5: Liquid chromatography method for determining product ee value

[0075] JASCO LC-1500 system, Daicel AD-H column, sample Boc derivatization, detection conditions: 210nm, n-hexane:isopropanol = 90:10; flow rate 0.7mL / min. The retention times of R-1b and S-1b were 7.62min and 8.61min, respectively, and the retention times of R-2b and S-2b were 7.63min and 8.62min, respectively.

[0076] Example 6: Activity and selectivity test of transaminase 1a and 2a

[0077] Reaction:

[0078]

[0079] Reaction system: 80 mg / mL of the whole-cell biocatalyst as described in Example 4, substrate 1a (20 mM) or 2a (50 mM), isopropylamine hydrochloride (250 mM), PLP (1 mM), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M), and the reaction temperature was 25° C. After 5 hours of reaction, the reaction conversion rate was measured by gas chromatography, and the results are shown in Table 3:

[0080] Table 3 Determination of transaminase-catalyzed activity of substrates 1a and 2a

[0081]

Claims

1. Application of R-aminotransferase in asymmetric synthesis of chiral N-heterocyclic amine compounds, characterized in that The amino acid sequence of the R-transaminase is shown in SEQ ID NO.1, 3, 5, 7.

2. A gene encoding the R-aminotransferase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO. 2, 4, 6, 8.

4. The use according to claim 1, characterized in that: The R-aminotransferase is used as a biocatalyst in catalyzing the transamination reaction of N-heterocyclic ketone compounds to prepare chiral N-heterocyclic amines.

5. The use according to claim 4, characterized in that: The structure of the N-heterocyclic ketone compound is shown in Formula 1: Formula 1:

6. The use according to claim 4, characterized in that: The R-aminotransferase as a biocatalyst is in the form of free cells, free enzymes, immobilized cells and immobilized enzymes.

7. The use according to claim 4, characterized in that: The method for synthesizing chiral N-heterocyclic amine compounds is: in a buffer solution, in the presence of an amine donor and a cofactor, using the R-aminotransferase of claim 1 as a catalyst, asymmetric amination of N-Boc-3-pyrrolidone (1a) or N-Boc-3-piperidone (2a) to generate chiral N-heterocyclic amines, the reaction formula is:

8. The use according to claim 7, characterized in that: The substrate concentration in the reaction system is 0.1-300 mM; the final concentration of the amine donor in the reaction system is 1-2000 mM, and the amine donor is alanine, phenethylamine or isopropylamine, preferably isopropylamine; the cofactor in the reaction system is pyridoxal phosphate (PLP), and the final concentration of the cofactor is 0.1-5 mM.

9. The use according to claim 8, characterized in that: The substrate concentration is 0.1-100 mM; the final concentration of the amine donor is 1-500 mM.

10. The use according to claim 7, characterized in that: The pH value of the reaction system is 7.0-10.0; the temperature is 20-45° C.; the co-solvent concentration is 0.1-30% (v / v) of the reaction system; the buffer concentration is 0.05-0.2 M; preferably, the co-solvent is selected from one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, and acetone.

Images