Three-membered ring synthetase and application thereof in synthesis of chiral cyclopropyl compound

Abstract

The invention relates to the technical field of biological medicine preparation, and provides a three-membered ring synthetase and application thereof in synthesis of a chiral cyclopropyl compound, and the amino acid sequence of the three-membered ring synthetase is as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or the following mutation is carried out on the basis of the amino acid sequence as shown in SEQ ID NO: 1: T67Q or T67Q / Y68S; or the following mutations are carried out on the basis of the amino acid sequence as shown in SEQ ID NO: 2: Q66T, Q66T / S67Y or the 63rd to 72nd amino acids PYGQSRKYNT are mutated into DYTTYKKFNP. The three-membered ring synthetase disclosed by the invention can be used for catalyzing 6-nitro demethyleucine; the method has the technical effects of synthesizing (1 'R, 2' R)-3-(2-nitryl cyclopropyl) alanine, (1 'S, 2' S)-3-(2-nitryl cyclopropyl) alanine, (1 'R, 2' S)-3-(2-nitryl cyclopropyl) alanine and (1 'S, 2' R)-3-(2-nitryl cyclopropyl) alanine, the conversion rate within 3 hours can reach 92% or above, and the method has great significance in synthesis of chiral cyclopropyl compounds.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine preparation, in particular to a three-membered ring synthetase and an application thereof in synthesizing chiral cyclopropyl compounds. Background Art

[0002] Cyclopropane is the smallest cycloalkane with high steric strain. Multi-substituted chiral cyclopropyl rings are often found in natural products with a variety of biological activities, including anti-tumor, anti-viral, anti-bacterial and anti-fungal properties. These structures have inspired medicinal chemists to use multi-substituted chiral cyclopropyl rings as key pharmacodynamic carriers for clinical drugs. In addition, the introduction of a cyclopropane skeleton into a molecule can significantly improve the various properties of the compound, among which it is worth noting that the biological activity, metabolic stability, receptor type selectivity, bioavailability, half-life, and reduction of off-target effects of the molecule are increased. Therefore, the synthesis of cyclopropyl is of great significance in the field of medicinal chemistry.

[0003] Although there are various chemical synthesis strategies to synthesize chiral cyclopropyl compounds, they often face major challenges, including the need for rare metals and the difficulty of producing chiral ligands on a commercial scale. In addition, these methods usually require extremely harsh reaction conditions, and it is difficult to control chemical selectivity and stereoselectivity. And due to the particularity of the geometric structure of cyclopropane, it has strong ring tension and is relatively unstable. It is easy to undergo ring-opening reactions to generate relatively stable straight-chain compounds, or undergo ring expansion reactions to obtain four-membered rings, five-membered rings, etc. In contrast, the biosynthetic pathways for producing chiral cyclopropyl-containing natural products or drugs have significant chemical selectivity, stereospecificity, and regioselectivity, which give them significant advantages. Therefore, the biosynthesis of cyclopropyl compounds has attracted great attention. Summary of the invention

[0004] In view of this, the present invention provides a three-membered ring synthase with a changed product configuration for biosynthesis of cyclopropyl compounds and its application.

[0005] The technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a three-membered ring synthetase with a changed product configuration, the amino acid sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0006] or the following mutations are made based on the amino acid sequence shown in SEQ ID NO: 1: T67Q or T67Q / Y68S;

[0007] Or the following mutations are made based on the amino acid sequence shown in SEQ ID NO: 2: Q66T, Q66T / S67Y or the amino acids PYGQSRKYNT at positions 63-72 are mutated to DYTTYKKFNP.

[0008] In a second aspect, the present invention provides a gene encoding a three-membered ring synthase.

[0009] In a third aspect, the present invention provides an expression vector of a gene encoding a three-membered ring synthetase.

[0010] In a fourth aspect, the present invention provides a recombinant cell containing a gene encoding a three-membered ring synthetase.

[0011] In a fifth aspect, the present invention provides the use of a three-membered ring synthetase capable of changing product configuration in the synthesis of chiral cyclopropyl compounds.

[0012] Based on the above technical scheme, preferably, the chiral cyclopropyl compound is (1'R, 2'R)-3-(2-nitrocyclopropyl)alanine, (1'S, 2'S)-3-(2-nitrocyclopropyl)alanine, (1'R, 2'S)-3-(2-nitrocyclopropyl)alanine and (1'S, 2'R)-3-(2-nitrocyclopropyl)alanine.

[0013] On the basis of the above technical scheme, preferably, 6-nitrodemethylleucine is used as a substrate, an enzyme purified after fermentation and culture of an engineered bacterium expressing the coding gene of the three-membered ring synthase is used as a catalyst, and a buffer, α-ketoglutaric acid, ascorbic acid, and FeSO4·7H2O are used as reaction media for the reaction.

[0014] Based on the above technical scheme, preferably, the substrate concentration is 1 mmol / L, the enzyme amount is 18-22 μmol / L, the α-ketoglutaric acid concentration is 1.5-2.5 mmol / L, the ascorbic acid concentration is 1.5-2.5 mmol / L, the FeSO4·7H2O concentration is 0.3-0.5 mmol / L, and the pH value of the reaction system is 7-8.

[0015] The three-membered ring synthetase of the present invention and its application in synthesizing chiral cyclopropyl compounds have the following beneficial effects compared with the prior art: the three-membered ring synthetase gene ScBelL in the present invention is isolated from Streptomyces cavourensis, and MiBelL is isolated from Micromonospora inaquosa, and their sequence similarity is 54.4%. The composite enzyme obtained by mutating the 67th and / or 68th positions of ScBelL protein, the 66th and / or 67th positions of MiBelL protein, or the 63rd-72th positions can catalyze the synthesis of chiral cyclopropyl compounds from 6-nitrodemethylleucine: (1'R,2'R)-3-(2-nitrocyclopropyl)alanine, (1'S,2'S)-3-(2-nitrocyclopropyl)alanine, (1'R,2'S)-3-(2-nitrocyclopropyl)alanine, and (1'S,2'R)-3-(2-nitrocyclopropyl)alanine; among them, the product configuration of the 63rd-72th position mutation of MiBelL protein is converted into the product configuration of the ScBelL wild type. The conversion rate of ScBelL, MiBelL protein and its mutants with substrates can reach more than 92% after 3h reaction, which is of great significance for the synthesis of chiral cyclopropyl compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 is the reaction formula for synthesizing a chiral cyclopropyl compound from the ScBelL protein and the MiBelL protein of the present invention;

[0018] Figure 2 It is the HPLC detection chart of substrate 6-nitrodemethylleucine;

[0019] Figure 3 is the HPLC detection chart of ScBelL-WT;

[0020] Figure 4 It is the HPLC detection chart of ScBelL-T67Q;

[0021] Figure 5 It is the HPLC detection chart of ScBelL-T67Q / Y68S;

[0022] Figure 6 This is the HPLC detection diagram of ScBelL-loop;

[0023] Figure 7 is the HPLC detection chart of MiBelL-WT;

[0024] Figure 8 This is the HPLC detection chart of MiBelL-Q66T;

[0025] Fig. 9 This is the HPLC detection chart of MiBelL-Q66T / S67Y;

[0026] Fig.10 This is the HPLC detection chart of MiBelL-loop.

[0027] Figure 3-10 In the figure, Figure (a) is a 2h conversion data diagram, and Figure (b) is a 3h conversion data diagram. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The three-membered ring synthase gene ScBelL in the present invention is isolated from Streptomyces cavourensis, and its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 3; the ScBelL gene contains 690 bases and encodes 230 amino acids. MiBelL is isolated from Micromonosporainaquosa, and their sequence similarity is 54.4%, and its amino acid sequence is shown in SEQ ID NO: 2, and its nucleotide sequence is shown in SEQ ID NO: 4; the MiBelL gene contains 699 bases and encodes 233 amino acids.

[0030] Both ScBelL and MiBelL proteins can catalyze the oxidative cyclization of the same substrate 6-nitrodemethylleucine to generate two diastereoisomers of three-membered ring products, namely (1'R, 2'S) and (1'S, 2'R) 3-(2-nitrocyclopropyl)alanine. The reaction formula is shown in Figure 1 .

[0031] In order to change the configuration of the products of ScBelL protein and MiBelL protein, the present invention uses X-ray crystallography to analyze the tertiary structure of these two proteins and clarify their detailed structural information, and then transforms their active regions or sites with key characteristics. After structural analysis and comparison with other similar structures, the following mutations are performed on ScBelL protein and MiBelL protein:

[0032] The threonine (Thr67) at position 67 near the active region of ScBelL is mutated to glutamine (Glu). The present invention names this mutation ScBelL-T67Q, and its amino acid sequence is shown in SEQ ID NO:5.

[0033] The threonine (Thr67) at position 67 and the tyrosine (Tyr68) at position 68 are mutated to glutamine (Glu) and serine (Ser), respectively. The present invention names this mutation ScBelL-T67Q / Y68S, and its amino acid sequence is shown in SEQ ID NO: 6.

[0034] The amino acids DYTTYKKFNP at positions 64-73 of ScBelL are mutated to PYGQSRKYNT. Since this mutation is a loop region on ScBelL, the present invention names this mutation ScBelL-loop. Its amino acid sequence is shown in SEQ ID NO:7.

[0035] The glutamine (Gln66) at position 66 near the active region of MiBelL is mutated to threonine (Thr). The present invention names this mutation MiBelL-Q66T, and its amino acid sequence is shown in SEQ ID NO:8.

[0036] The glutamine at position 66 (Gln66) and the serine at position 67 (Ser67) are mutated to threonine (Thr) and tyrosine (Tyr), respectively. The present invention names this mutation MiBelL-Q66T / S67Y, and its amino acid sequence is shown in SEQ ID NO:9.

[0037] The amino acids PYGQSRKYNT at positions 63-72 of MiBelL are mutated to DYTTYKKFNP. Since this mutation is a loop region on MiBelL, the present invention names this mutation MiBelL-loop. Its amino acid sequence is shown in SEQ ID NO: 10.

[0038] The present invention successfully obtains mutant proteins ScBelL protein and MiBelL protein which produce products with different configurations.

[0039] Example 1 Construction of mutants

[0040] First, the ScBelL and MiBelL genes were obtained by total gene synthesis, the sequences of which are shown in SEQ ID NOs: 1-4, and the genes were constructed into the pET28a(+) vector using NdeI and XhoI restriction enzymes, respectively. The recombinant plasmids were then transformed into competent cells to obtain ScBelL and MiBelL recombinant plasmids.

[0041] In order to obtain mutant proteins with changed product configuration, the present invention uses site-directed mutagenesis technology to perform polymerase chain reaction (PCR) using wild-type gene ScBelL and MiBelL recombinant plasmids as templates. The original template DNA is removed using DpnI. The mutant plasmid is then introduced into E. coli competent cells, and the mutant gene is confirmed by DNA sequencing.

[0042] Table 1 Mutation primers

[0043]

[0044] Here, the present invention constructed six mutants: ScBelL-T67Q, ScBelL-T67Q / Y68S, ScBelL-loop, MiBelL-Q66T, MiBelL-Q66T / S67Y and MiBelL-loop.

[0045] Then express and purify the protein in E. coli: The constructed recombinant plasmids ScBelL, ScBelL-T67Q, ScBelL-T67Q / Y68S, ScBelL-loop, MiBelL, MiBelL-Q66T, MiBelL-Q66T / S67Y and MiBelL-loop were transformed into competent cells of E. coli BL21 (DE3), and the strains were selected in LB culture plates containing 100 μg / mL kanamycin sulfate. Then the strains were inoculated into 5 mL LB and cultured overnight, then expanded to 200 mL LB culture, and finally expanded to 10 L LB culture medium. When the OD value reached 0.6-0.8, the culture temperature was cooled to 16°C and 0.5 mmol / L IPTG was added to induce the massive expression of the enzyme protein. After 18 hours of protein induction expression, the bacterial solution was centrifuged at 8000 rpm for 8 minutes to collect the cells. Then, the cells were resuspended in a buffer solution (20 mmol / L HEPES, 50 mmol / L NaCl, pH 7.5), disrupted using a cell disruptor, and centrifuged at 17,000 rpm for 50 min. The supernatant was collected for the next step of purification.

[0046] In order to obtain a high-purity enzyme protein, the present invention uses a fast protein liquid chromatography (FPLC) to sequentially elute the target protein using a nickel ion chromatography column, and then dialyzes the target protein in 5L (20mmol / L HEPES, 50mmol / L NaCl, pH7.5) at 4°C overnight, and simultaneously adds 500μL of TEV protease to perform enzyme cleavage to remove the His tag on the protein. The target protein after enzyme cleavage is passed through the nickel column again, and the target protein without the His tag in the flow-through is collected. Finally, the purified target protein is concentrated in a 20mmol / L HEPES, 50mmol / L NaCl, pH 7.5 buffer and stored at -80°C.

[0047] Example 2 Synthesis of chiral cyclopropyl compounds using ScBelL, MiBelL and their mutants

[0048] In order to compare the differences in the product configurations of ScBelL, ScBelL-T67Q, ScBelL-T67Q / Y68S, ScBelL-loop, MiBelL, MiBelL-Q66T, MiBelL-Q66T / S67Y and MiBelL-loop, the present invention further determines the configurations of the products generated by the reaction of these enzymes with the substrate 6-nitrodemethylleucine.

[0049] The activity test method is as follows: the mixture (100 μL) of each reaction is in 20 mmol / L HEPES, pH 8.0 buffer, including 1 mmol / L substrate, 2 mmol / L α-ketoglutaric acid, 2 mmol / L ascorbic acid, 0.4 mmol / L FeSO4·7H2O and 5.3 μL enzyme (10 mg / mL) (enzyme concentration: 20 μmol / L). After mixing, place in a metal bath at 25°C for 3 h, and each reaction is repeated 3 times. After the reaction, 200 μL acetonitrile is added to the mixture to terminate the enzyme reaction, and then 40 μL 1 mol / L boric acid (pH 8.0) and 40 μL 10 mmol / L dansyl chloride are added for derivatization. After mixing for 1 h, the reaction solution is filtered with a 0.22 μm filter membrane.

[0050] Then, each group of reactions was measured and analyzed by high performance liquid chromatography (HPLC). The analytical column was an InertSustain C18 column (4.6×250 mm, 5 μm), the mobile phase was acetonitrile / 0.1% formic acid water, the flow rate was 1 mL / min, the detection wave was 330 nm, and the elution conditions were 0-4 min, acetonitrile linear gradient was 30%; 4-34 min, acetonitrile linear gradient was 30% to 100%; 34-38 min, acetonitrile linear gradient was 100%; 38-39 min, acetonitrile linear gradient was 100% to 30%; 39-45 min, acetonitrile linear gradient was 30%.

[0051] Figure 2-10 The HPLC detection chart of the substrate and the above-mentioned three-membered ring synthetase shows that the substance with a retention time of 16.39 min is the substrate 6-nitrodesmethylleucine, the substance with a retention time of 15.96 min is (1'R, 2'S)-3-(2-nitrocyclopropyl)alanine, the substance with a retention time of 15.40 min is (1'S, 2'S)-3-(2-nitrocyclopropyl)alanine, the substance with a retention time of 15.87 min is (1'S, 2'R)-3-(2-nitrocyclopropyl)alanine, and the substance with a retention time of 15.75 min is (1'R, 2'R)-3-(2-nitrocyclopropyl)alanine.

[0052] Figure 3-6 As shown, ScBelL-WT generated (1'R,2'S)-3-(2-nitrocyclopropyl)alanine (15.96 min); ScBelL-T67Q and ScBelL-T67Q / Y68S mutants both generated (1'R,2'R)-3-(2-nitrocyclopropyl)alanine (15.75 min) and (1'S,2'S)-3-(2-nitrocyclopropyl)alanine (15.40 min), while the ScBelL-loop mutant lost its activity.

[0053] Figure 7-10 As shown, MiBelL-WT generated (1'S,2'R)-3-(2-nitrocyclopropyl)alanine (15.87 min), MiBelL-Q66T and MiBelL-Q66T / S67Y mutants both generated products with two configurations, (1'S,2'S)-3-(2-nitrocyclopropyl)alanine (15.40 min) and (1'R,2'R)-3-(2-nitrocyclopropyl)alanine (15.75 min), and MiBelL-loop mutants generated products with two configurations, (1'S,2'S)-3-(2-nitrocyclopropyl)alanine (15.40 min) and (1'R,2'S)-3-(2-nitrocyclopropyl)alanine 15.96 min.

[0054] Table 2 Conversion rate of chiral cyclopropyl compounds

[0055]

[0056]

[0057] As shown in Table 2, except for ScBelL-loop which lost its activity and failed to be converted, the others were successfully converted, and after 3 hours of reaction, the conversion rate was as high as over 92%.

[0058] Example 3

[0059] The reaction liquid system of this example includes: 1 mmol / L substrate, 1.5 mmol / L α-ketoglutaric acid, 1.5 mmol / L ascorbic acid, 0.3 mmol / L FeSO4·7H2O and 18 μmol / L enzyme (ScBelL-T67Q), the pH value of the reaction system is 7, and the rest of the contents are the same as those in Example 2. The final product is the same as the final product corresponding to the ScBelL-T67Q enzyme in Example 2, and the conversion rate is 95.7% after 3 hours of reaction.

[0060] Example 4

[0061] The reaction liquid system of this example includes: 1 mmol / L substrate, 2.5 mmol / L α-ketoglutaric acid, 2.5 mmol / L ascorbic acid, 0.5 mmol / L FeSO4·7H2O and 22 μmol / L enzyme (ScBelL-T67Q), the pH value of the reaction system is 7.5, and the rest of the contents are the same as those in Example 2. The final product is the same as the final product corresponding to the ScBelL-T67Q enzyme in Example 2, and the conversion rate is 96.3% after 3 hours of reaction.

[0062] The present invention successfully designs mutants ScBelL-T67Q, ScBelL-T67Q / Y68S, MiBelL-Q66T, MiBelL-Q66T / S67Y and MiBelL-loop which change the product configuration through structural analysis and modification design.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A three-membered ring synthetase, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2; or the following mutations are made based on the amino acid sequence shown in SEQ ID NO: 1: T67Q or T67Q / Y68S; Or the following mutations are made based on the amino acid sequence shown in SEQ ID NO: 2: Q66T, Q66T / S67Y or the amino acids PYGQSRKYNT at positions 63-72 are mutated to DYTTYKKFNP.

2. A gene encoding the three-membered ring synthase as claimed in claim 1.

3. An expression vector containing the gene encoding the three-membered ring synthase as claimed in claim 2.

4. A recombinant cell containing the gene encoding the three-membered ring synthase according to claim 2.

5. Use of the three-membered ring synthetase as claimed in claim 1 in synthesizing chiral cyclopropyl compounds.

6. The use according to claim 5, characterized in that: The chiral cyclopropyl compounds are (1'R, 2'R)-3-(2-nitrocyclopropyl)alanine, (1'S, 2'S)-3-(2-nitrocyclopropyl)alanine, (1'R, 2'S)-3-(2-nitrocyclopropyl)alanine and (1'S, 2'R)-3-(2-nitrocyclopropyl)alanine.

7. The use according to claim 5, characterized in that: The method uses 6-nitrodemethylleucine as a substrate, an enzyme purified from an engineered bacterium expressing a gene encoding the three-membered ring synthase through fermentation culture as a catalyst, and a buffer, α-ketoglutaric acid, ascorbic acid and FeSO4·7H2O as reaction media for the reaction.

8. The use according to claim 7, characterized in that: The substrate concentration is 1 mmol / L, the enzyme amount is 18-22 μmol / L, the α-ketoglutaric acid concentration is 1.5-2.5 mmol / L, the ascorbic acid concentration is 1.5-2.5 mmol / L, the FeSO4·7H2O concentration is 0.3-0.5 mmol / L, and the pH value of the reaction system is 7-8.

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