Three-membered ring synthetase and its application in the synthesis of chiral cyclopropyl compounds

Abstract

The present invention relates to the technical field of biopharmaceutical preparation, and provides a three-membered ring synthetase and an application thereof in synthesizing chiral cyclopropyl compounds. 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 mutations are made on the basis of the amino acid sequence shown in SEQ ID NO: 1: T67Q or T67Q / Y68S; or the following mutations are made on the basis of the amino acid sequence shown in SEQ ID NO: 2: Q66T, Q66T / S67Y, or the 63rd-72nd amino acid PYGQSRKYNT is mutated to DYTTYKKFNP. The three-membered ring synthase of the present invention can achieve the technical effect of catalyzing 6-nitrodemethylleucine to synthesize (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, and the conversion rate in 3 hours can reach more than 92%, which is of great significance for the synthesis of chiral cyclopropyl compounds.

Description

Technical Field

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

[0002] Cyclopropane is the smallest cycloalkane with high steric strain. Polysubstituted chiral cyclopropyl rings are commonly found in natural products with diverse biological activities, including antitumor, antiviral, antibacterial, and antifungal properties. These structures have inspired medicinal chemists to consider polysubstituted chiral cyclopropyl rings as key pharmacodynamic carriers for clinical drugs. Furthermore, the introduction of a cyclopropane backbone into a molecule can significantly enhance various properties of the compound, notably increasing its bioactivity, metabolic stability, receptor selectivity, bioavailability, half-life, and reducing off-target effects, among other druggable properties. Therefore, the synthesis of cyclopropyl rings is of great significance in the field of medicinal chemistry.

[0003] Although there are various chemical synthesis strategies for the synthesis of 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, making it difficult to control chemical and stereoselectivity. And due to the special geometric structure of cyclopropane, it has strong ring strain and is relatively unstable. It is easy to undergo ring-opening reactions to generate relatively stable linear 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, giving 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 capable of changing the product configuration for the 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] Alternatively, the following mutations may be performed based on the amino acid sequence shown in SEQ ID NO: 2: Q66T, Q66T / S67Y, or the amino acids PYGQSRKYNT at positions 63-72 may be 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 synthase.

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

[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 solution, 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 solution, 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 solution, 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 synthase of the present invention and its application in the synthesis of chiral cyclopropyl compounds have the following advantages over the prior art: the three-membered ring synthase 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 resulting complex enzymes, generated by mutating positions 67 and / or 68 of the ScBelL protein, positions 66 and / or 67, or positions 63-72 of the MiBelL protein, catalyze the conversion of 6-nitrodemethylleucine to chiral cyclopropyl compounds: (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. The product conformation of the MiBelL mutants at positions 63-72 was converted to that of the wild-type ScBelL protein. ScBelL, MiBelL, and their mutants all achieved conversions exceeding 92% after a 3-h reaction with substrates, demonstrating their significant potential 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any 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 This is the HPLC detection chart of substrate 6-nitrodemethylleucine;

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

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

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

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

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

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

[0025] Figure 9 HPLC detection chart of MiBelL-Q66T / S67Y;

[0026] Figure 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 embodiments described 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 making creative efforts 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, with its amino acid sequence shown in SEQ ID NO: 1 and its nucleotide sequence shown in SEQ ID NO: 3. The ScBelL gene comprises 690 bases and encodes 230 amino acids. MiBelL was isolated from Micromonosporainaquosa, with a sequence similarity of 54.4%. 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 comprises 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 diastereomeric three-membered ring products, (1'R, 2'S) and (1'S, 2'R) 3-(2-nitrocyclopropyl)alanine, as shown in the reaction formula. Figure 1 .

[0031] To alter the conformation of the products of ScBelL and MiBelL, the present invention utilized X-ray crystallography to analyze the tertiary structure of these two proteins and elucidate their detailed structural information. Modifications were then made targeting their active regions or sites with key properties. Following structural analysis and comparison with other similar structures, the following mutations were made to ScBelL and MiBelL proteins:

[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 at position 67 (Thr67) and the tyrosine at position 68 (Tyr68) 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 were mutated to PYGQSRKYNT. Since this mutation is a loop region on ScBelL, the present invention named this mutation ScBelL-loop. Its amino acid sequence is shown in SEQ ID NO: 7.

[0035] The glutamine at position 66 (Gln66) 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 were mutated to DYTTYKKFNP. Since this mutation is a loop region on MiBelL, the present invention named 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 that produce products with different configurations.

[0039] Example 1 Construction of mutants

[0040] First, the ScBelL and MiBelL genes were obtained by whole gene synthesis, with sequences shown in SEQ ID NOs: 1-4. The genes were then 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] To obtain mutant proteins with altered product conformations, the present invention utilizes site-directed mutagenesis, using wild-type ScBelL and MiBelL recombinant plasmids as templates for polymerase chain reaction (PCR). The original template DNA is removed using DpnI. The mutant plasmids are then introduced into competent E. coli cells, and the mutant genes are 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] Protein expression and purification in E. coli were then performed: 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 E. coli BL21(DE3) cells and selected on LB plates containing 100 μg / mL kanamycin sulfate. The strains were then inoculated into 5 mL of LB and cultured overnight, then scaled up to 200 mL of LB, and finally to 10 L of LB medium. When the OD reached 0.6-0.8, the culture was cooled to 16°C and 0.5 mmol / L IPTG was added to induce high-level expression of the enzyme protein. After 18 hours of protein expression induction, the cells were harvested by centrifugation at 8000 rpm for 8 minutes. The cells were then 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 purification step.

[0046] To obtain a highly pure enzyme protein, the present invention uses a fast protein liquid chromatography (FPLC) instrument to sequentially elute the target protein using a nickel ion chromatography column. The target protein is then dialyzed against 5L (20mmol / L HEPES, 50mmol / L NaCl, pH 7.5) at 4°C overnight. Simultaneously, 500μL of TEV protease is added for enzymatic cleavage to remove the His tag from the protein. The cleaved target protein is then passed through the nickel column again, and the His-tag-free target protein in the flow-through is collected. Finally, the purified target protein is concentrated in 20mmol / L HEPES, 50mmol / L NaCl, pH 7.5 buffer and stored at -80°C.

[0047] Example 2 Synthesis of Chiral Cyclopropyl Compounds Catalyzed by 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 determined the configurations of the products generated by the reaction of these enzymes with the substrate 6-nitrodemethylleucine.

[0049] Activity assays were performed as follows: Each reaction mixture (100 μL) contained 1 mmol / L substrate, 2 mmol / L α-ketoglutarate, 2 mmol / L ascorbic acid, 0.4 mmol / L FeSO₄·7H₂O, and 5.3 μL enzyme (10 mg / mL) (enzyme concentration: 20 μmol / L) in 20 mmol / L HEPES buffer, pH 8.0. The mixture was incubated in a metal bath at 25°C for 3 h. Each reaction was repeated three times. The reaction mixture was terminated by the addition of 200 μL acetonitrile, followed by derivatization with 40 μL 1 mol / L boric acid (pH 8.0) and 40 μL 10 mmol / L dansyl chloride. After incubation for 1 h, the reaction mixture was filtered through a 0.22 μm filter.

[0050] The products of each reaction group were then determined 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 as follows: 0-4 min, acetonitrile linear gradient of 30%; 4-34 min, acetonitrile linear gradient of 30% to 100%; 34-38 min, acetonitrile linear gradient of 100%; 38-39 min, acetonitrile linear gradient of 100% to 30%; 39-45 min, acetonitrile linear gradient of 30%.

[0051] Figure 2-10 The following are HPLC detection charts of the substrate and the above-mentioned three-membered ring synthase. The substance with a retention time of 16.39 min is the substrate 6-nitrodemethylleucine, 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 newly 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 produced (1'S,2'R)-3-(2-nitrocyclopropyl)alanine (15.87 min), MiBelL-Q66T and MiBelL-Q66T / S67Y mutants both produced 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 mutant produced 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 all successfully converted, and after 3 h of reaction, the conversion rate was as high as over 92%.

[0058] Example 3

[0059] The reaction solution system of this example includes: 1 mmol / L substrate, 1.5 mmol / L α-ketoglutarate, 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. The remaining contents are the same as those of Example 2. The final product is the same as the final product corresponding to the ScBelL-T67Q enzyme in Example 2. After 3 hours of reaction, the conversion rate is 95.7%.

[0060] Example 4

[0061] The reaction solution system of this example includes: 1 mmol / L substrate, 2.5 mmol / L α-ketoglutarate, 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. The remaining 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. After 3 hours of reaction, the conversion rate is 96.3%.

[0062] The present invention successfully designed mutants ScBelL-T67Q, ScBelL-T67Q / Y68S, MiBelL-Q66T, MiBelL-Q66T / S67Y and MiBelL-loop that 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 scope of protection of the present invention.

Claims

1. A three-membered ring synthase, characterized in that: The amino acid sequence is shown in SEQ ID NO: 1, or the following mutations are made based on the amino acid sequence shown in SEQ ID NO: 1: T67Q or T67Q / Y68S.

2. The gene encoding the three-membered ring synthase according to claim 1.

3. An expression vector containing the gene encoding the three-membered ring synthase according to 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 synthase according to claim 1 in synthesizing chiral cyclopropyl compounds, characterized in that: The amino acid sequence is as shown in SEQ ID NO: 1, wherein the three-membered ring synthase is synthesized (1 ’R ,2 ’S )-3-(2-nitrocyclopropyl)alanine, the mutants T67Q and T67Q / Y68 were synthesized (1 ’R ,2 ’R )-3-(2-nitrocyclopropyl)alanine and (1 ’S ,2 ’S )-3-(2-nitrocyclopropyl)alanine.

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

7. The use according to claim 6, 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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