Application of three-membered ring synthetase gene MiBelL and mutant thereof in synthesis of chiral cyclopropyl compound

Abstract

The invention provides a three-membered ring synthetase gene MiBelL and application of a mutant of the three-membered ring synthetase gene MiBelL in synthesis of a chiral cyclopropyl compound, a nucleotide sequence of the MiBelL is as shown in SEQ ID NO: 1, and the MiBelL mutant is subjected to the following mutations on the basis of the nucleotide sequence as shown in SEQ ID NO: 1: MiBelL-Q66T, MiBelL-Q66T / S67Y and MiBelL-loop (coded 63-72nd amino acid PYGQSRKYNT is mutated into DYTTYKKFNP). The three-membered ring synthetase gene MiBelL and the mutant thereof can catalyze 6-nitryl demethyleucine to synthesize all four stereoisomers of 3-(2-nitryl cyclopropyl) alanine, the conversion rate within 3 hours can reach 92% or above, and the three-membered ring synthetase gene MiBelL and the mutant thereof have great significance in synthesis of chiral cyclopropyl compounds.

Description

Technical Field

[0001] This invention is based on a divisional application of “Application date: 2024-12-11, Application number: 2024118176596, Title: Three-membered ring synthase and its application in the synthesis of chiral cyclopropyl compounds”.

[0002] This invention relates to the field of biopharmaceutical preparation technology, and more particularly to a three-membered ring synthase gene. Mi Application of BelL and its mutants in the synthesis of chiral cyclopropyl compounds. Background Technology

[0003] Cyclopropane is the smallest cycloalkanes 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 use polysubstituted chiral cyclopropyl rings as key pharmacodynamic carriers for clinical drugs. Furthermore, introducing a cyclopropane skeleton into a molecule can significantly improve various properties of the compound, notably increasing its biological activity, metabolic stability, receptor type selectivity, bioavailability, half-life, and reducing off-target effects—a range of drug-like properties. Therefore, the synthesis of cyclopropyl groups is of great significance in the field of medicinal chemistry.

[0004] While various chemical synthesis strategies exist for the synthesis of chiral cyclopropyl compounds, they often face significant challenges, including the need for rare metals and the difficulty in producing chiral ligands on a commercial scale. Furthermore, these methods typically require extremely demanding reaction conditions, making it difficult to control chemoselectivity and stereoselectivity. Additionally, due to the unique geometry of cyclopropane, it exhibits strong ring strain and relative instability, readily undergoing ring-opening reactions to generate relatively stable straight-chain compounds, or ring-expansion reactions to yield four- or five-membered rings. In contrast, biosynthetic pathways for producing chiral cyclopropyl-containing natural products or drugs offer significant advantages in terms of chemoselectivity, stereospecificity, and regioselectivity. Therefore, the biosynthesis of cyclopropyl compounds has attracted considerable attention. Summary of the Invention

[0005] In view of this, the present invention proposes a three-membered ring synthase gene. Mi Application of BelL and its mutants in the synthesis of chiral cyclopropyl compounds.

[0006] The technical solution of this invention is implemented as follows: In a first aspect, this invention provides a three-membered ring synthase gene. Mi BelL and its mutants, the Mi The nucleotide sequence of BelL is shown in SEQ ID NO: 1. MiThe BelL mutant is based on the nucleotide sequence shown in SEQ ID NO: 1, with the following mutations: Mi BelL-Q66T: The 66th amino acid encoded by BelL-Q66T is mutated from Q to T; Mi BelL-Q66T / S67Y: The QS mutation at amino acid positions 66-67 is replaced by TY; Mi BelL-loop: The amino acid PYGQSRKYNT encoded at positions 63-72 is mutated to DYTTYKKFNP.

[0007] Secondly, this invention provides a three-membered ring synthase gene. Mi The encoding genes of BelL and its mutants.

[0008] Thirdly, this invention provides a three-membered ring synthase gene. Mi Expression vectors for the encoding genes of BelL and its mutants.

[0009] Fourthly, this invention provides a three-membered ring synthase gene. Mi Recombinant cells encoding the genes of BelL and its mutants.

[0010] Fifthly, this invention provides a three-membered ring synthase gene. Mi Application of BelL and its mutants in the synthesis of chiral cyclopropyl compounds.

[0011] Based on the above technical solutions, preferably, the... Mi BelL Synthesis (1) ’S ,2 ’R 3-(2-nitrocyclopropyl)alanine, the mutant Mi BelL-Q66T and Mi BelL-Q66T / S67Y are both newly synthesized (1) ’R ,2 ’R )-3-(2-nitrocyclopropyl)alanine and (1 ’S ,2 ’S 3-(2-nitrocyclopropyl)alanine, the mutant Mi BelL-loop synthesis (1) ’ S ,2 ’S )-3-(2-nitrocyclopropyl)alanine and (1 ’R ,2 ’S 3-(2-nitrocyclopropyl)alanine.

[0012] Based on the above technical solutions, preferably, 6-nitrodemethylleucine is used as the substrate, and an enzyme obtained by fermentation and purification of engineered bacteria that express the encoding gene of the three-membered ring synthase is used as the catalyst, with buffer, α-ketoglutarate, ascorbic acid, and FeSO4·7H2O as the reaction medium.

[0013] Based on the above technical solutions, preferably, the substrate concentration is 1 mmol / L, the enzyme amount is 18-22 µmol / L, the α-ketoglutarate 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.

[0014] 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 present invention... Mi BelL is a small monosporous bacterium ( Micromonospora inaquosa It was separated from ) by means of Mi Point mutants obtained by mutations at positions 66, 66 / 67, or 63-72 of the BelL protein 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. Mi BelL protein and its mutants can achieve a conversion rate of over 92% when reacted with substrates for 3 hours, which is of great significance for the synthesis of chiral cyclopropyl compounds. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 For the present invention Mi The reaction formula for the synthesis of chiral cyclopropyl compounds from BelL protein; Figure 2The HPLC chromatogram shows the detection of the substrate 6-nitrodemethylleucine. Figure 3 for Mi HPLC detection chromatogram of BelL-WT; Figure 4 for Mi HPLC detection chromatogram of BelL-Q66T; Figure 5 for Mi HPLC detection chromatogram of BelL-Q66T / S67Y; Figure 6 for Mi HPLC detection chromatogram of BelL-loop.

[0017] Figures 3 - 6 In the figure, Figure (a) shows the data transformed over 2 hours, and Figure (b) shows the data transformed over 3 hours. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] The three-membered ring synthase gene in this invention Mi BelL is a small monosporous bacterium ( Micromonospora inaquosa The nucleotide sequence of the sample is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. Mi The BelL gene contains 699 bases and encodes 233 amino acids.

[0020] Mi BelL protein can catalyze the oxidative cyclization of the substrate 6-nitrodemethylleucine to generate a three-membered ring product: (1) ’S ,2 ’ R 3-(2-nitrocyclopropyl)alanine, reaction formula see Figure 1 .

[0021] In order to change Mi The conformation of the BelL protein was determined using X-ray crystallography, elucidating its detailed structural information. Modifications were then made targeting its active region or sites with key characteristics. Through structural analysis and comparison with other similar structures, the product conformation was further refined. Mi The BelL protein undergoes the following mutations: WillMi The glutamine (Gln66) at position 66 near the active region of BelL is mutated to threonine (Thr). This invention names this mutation as... Mi BelL-Q66T.

[0022] The glutamine at position 66 (Gln66) and the serine at position 67 (Ser67) are mutated to threonine (Thr) and tyrosine (Tyr), respectively. This mutation is named... Mi BelL-Q66T / S67Y.

[0023] Will Mi The amino acid PYGQSRKYNT at positions 63-72 of BelL is mutated to DYTTYKKFNP, because this mutation is... Mi A loop region on BelL, therefore this invention names this mutation as Mi BelL-loop.

[0024] This invention successfully obtained mutant proteins that produce products with different conformations. Mi BelL protein.

[0025] Example 1 Construction of mutants First, it is obtained through whole-genome synthesis. Mi The BelL gene, sequenced as shown in SEQ ID NO: 1-2, was utilized... Nde I and Xho I restriction enzymes were used to construct the gene into the pET28a(+) vector, and then the recombinant plasmid was transformed into competent cells to obtain... Mi BelL recombinant engineered bacteria.

[0026] To obtain mutant proteins with altered product conformation, this invention utilizes site-directed mutagenesis, using wild-type genes... Mi The BelL recombinant plasmid was used as a template for polymerase chain reaction (PCR). The original template DNA was then used... Dpn The mutated plasmid was then removed. Next, it was introduced into competent E. coli cells, and the mutated gene was confirmed by DNA sequencing.

[0027] Table 1 Mutant Primers

[0028] Here, the present invention constructs three mutant strains. Mi BelL-Q66T, Mi BelL-Q66T / S67Y and MiBelL-loop.

[0029] Next, the protein was expressed and purified in E. coli: the constructed recombinant plasmid was then... Mi BelL、 Mi BelL-Q66T, Mi BelL-Q66T / S67Y and Mi BelL-loop cells were transformed into *E. coli* BL21(DE3) competent cells, and strains were screened in LB broth containing 50 µg / mL kanamycin sulfate. The strains were then inoculated into 200 mL LB medium for scale-up culture, eventually reaching 10 L LB medium. During scale-up, the LB medium always contained 50 µg / mL kanamycin sulfate. 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 high expression of the enzyme protein. After 18 hours of protein induction, the bacterial culture was centrifuged at 8000 rpm for 8 min to collect the cells. The cells were then resuspended in buffer (20 mmol / L HEPES, 50 mmol / L NaCl, pH 7.5), lysed using a cell disruptor, and centrifuged at 17000 rpm for 50 min. The supernatant was collected for further purification.

[0030] To obtain high-purity enzyme protein, this invention employs a rapid protein liquid chromatography (FPLC) system, sequentially eluting the target protein using a nickel ion chromatography column. The target protein is then collected via a dialysis membrane and dialyzed overnight at 4°C in 10 L of dialysis buffer (20 mmol / L HEPES, 50 mmol / L NaCl, pH 7.5). Simultaneously, 3 mL of TEV protease is added for enzymatic digestion to remove the His tag from the protein. The digested target protein is then purified again using a nickel column, and the His-tag-free protein is collected during flow-through. Finally, the high-purity target protein is concentrated in 20 mmol / L HEPES, 50 mmol / L NaCl, pH 7.5 buffer and stored at -80°C.

[0031] Example 2 utilizes Mi BelL and its mutants catalyze the synthesis of chiral cyclopropyl compounds. For comparison Mi BelL、 Mi BelL-Q66T, Mi BelL-Q66T / S67Y and Mi The present invention further determined the configuration of the products generated by the reaction of these enzymes with the substrate 6-nitrodemethylleucine to differentiate the BelL-loop products.

[0032] The activity assay was performed as follows: Each reaction mixture (100 µL) was placed in a 20 mmol / L HEPES buffer at pH 8.0, comprising 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). After mixing, the mixture was incubated in a metal bath at 25°C for 3 h. Each reaction was performed in triplicate. After the reaction, 200 µL of acetonitrile was added to terminate the enzyme reaction, followed by the addition of 40 µL of 1 mol / L boric acid (pH 8.0) and 40 μL of 10 mmol / L dansyl chloride for derivatization. After mixing and incubation for 1 h, the reaction solution was filtered through a 0.22 µm filter membrane.

[0033] Next, the products of each reaction group were 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 wavelength 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%–100%; 34-38 min, acetonitrile linear gradient of 100%; 38-39 min, acetonitrile linear gradient of 100%–30%; 39-45 min, acetonitrile linear gradient of 30%.

[0034] Figures 2 - 6 The HPLC chromatograms show the substrate and the catalytic product of the above-mentioned three-membered ring synthase. The substance with a retention time of 16.39 min is the product of dansyl chloride derivatization of the substrate 6-nitrodemethylleucine, and the substance with a retention time of 15.96 min is (1 ’R ,2 ’S The product of 3-(2-nitrocyclopropyl)alanine derivatized with dansyl chloride, with a retention time of 15.40 min, is (1 ’S ,2 ’S The product of 3-(2-nitrocyclopropyl)alanine derivatized with dansyl chloride, with a retention time of 15.87 min, is (1 ’S ,2 ’R The product of 3-(2-nitrocyclopropyl)alanine derivatized with dansyl chloride, with a retention time of 15.75 min, is (1 ’R ,2 ’R The product of 3-(2-nitrocyclopropyl)alanine derivatized with dansyl chloride.

[0035] Figures 3 - 6 As shownMi BelL-WT generated (1) ’S ,2 ’R )-3-(2-nitrocyclopropyl)alanine (15.87 min), Mi BelL-Q66T and Mi All BelL-Q66T / S67Y mutants were newly generated (1 ’S ,2 ’S )-3-(2-nitrocyclopropyl)alanine (15.40 min) and (1 ’R ,2 ’R The product of 3-(2-nitrocyclopropyl)alanine (15.75 min) with two configurations, Mi The BelL-loop mutation completely generates (1) ’S ,2 ’S )-3-(2-nitrocyclopropyl)alanine (15.40 min) and (1 ’R ,2 ’ S )-3-(2-nitrocyclopropyl)alanine (15.96 min) products of both configurations.

[0036] Table 2 Conversion rates of chiral cyclopropyl compounds

[0037] Table 2 shows that, Mi BelL and its mutants were successfully transformed, and the transformation rate was over 92% after 3 hours of reaction.

[0038] This invention, through structural analysis and modification, successfully designed mutants that alter the product configuration. Mi BelL-Q66T, Mi BelL-Q66T / S67Y and [[ID=7 BelL-loop.

[0039] 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 within the protection scope of the present invention.

Claims

1. Three-membered ring synthase gene Mi BelL and its mutants are characterized by: The Mi The nucleotide sequence of BelL is shown in SEQ ID NO:

1. Mi The BelL mutant is based on the nucleotide sequence shown in SEQ ID NO: 1, with the following mutations: Mi BelL-Q66T: The 66th amino acid encoded by BelL-Q66T is mutated from Q to T; Mi BelL-Q66T / S67Y: The QS mutation at amino acid positions 66-67 is replaced by TY; Mi BelL-loop: The amino acid PYGQSRKYNT encoded at positions 63-72 is mutated to DYTTYKKFNP.

2. The three-membered ring synthase gene as described in claim 1 Mi The encoding genes of BelL and its mutants.

3. Contains the three-membered ring synthase gene as described in claim 2. Mi Expression vectors for the encoding genes of BelL and its mutants.

4. Contains the three-membered ring synthase gene as described in claim 2. Mi Recombinant cells encoding the genes of BelL and its mutants.

5. The three-membered ring synthase gene as described in claim 1 Mi The application of BelL and its mutants in the synthesis of chiral cyclopropyl compounds is characterized by: The Mi BelL Synthesis (1) ’S ,2 ’R 3-(2-nitrocyclopropyl)alanine, the mutant Mi BelL-Q66T and Mi BelL-Q66T / S67Y are both newly synthesized (1) ’R ,2 ’R )-3-(2-nitrocyclopropyl)alanine and (1 ’S ,2 ’S 3-(2-nitrocyclopropyl)alanine, the mutant Mi BelL-loop synthesis (1) ’S ,2 ’S )-3-(2-nitrocyclopropyl)alanine and (1 ’R ,2 ’S 3-(2-nitrocyclopropyl)alanine.

6. The application as described in claim 5, characterized in that: Using 6-nitrodemethylleucine as a substrate, and an enzyme purified by fermentation culture of engineered bacteria expressing the gene encoding the three-membered ring synthase as a catalyst, the reaction was carried out in a buffer solution, α-ketoglutarate, ascorbic acid, and FeSO4·7H2O as the reaction medium.

7. The application as described in claim 5, characterized in that: The substrate concentration was 1 mmol / L, the enzyme concentration was 18-22 µmol / L, the α-ketoglutarate concentration was 1.5-2.5 mmol / L, the ascorbic acid concentration was 1.5-2.5 mmol / L, the FeSO4·7H2O concentration was 0.3-0.5 mmol / L, and the pH of the reaction system was 7-8.

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