Ene-reductase mutants and use thereof in preparation of brivaracetam intermediate

Abstract

An ene-reductase mutant and the use thereof in the preparation of a brivaracetam intermediate. By using wild-type ene-reductase NnER set forth in SEQ ID NO. 1 as a template, alanine at position 57, tryptophan at position 67 and phenylalanine at position 271 are mutated to obtain multiple ene-reductase mutants: A57G, W67K, W67H and F271Y. The ene-reductase mutants exhibit an enhanced stereoselectivity and catalytic activity in catalyzing the conversion of 4-propylfuran-2(5H)-one into a brivaracetam intermediate, i.e. (R)-4-propyldihydrofuran-2(3H)-one, thus enabling the synthesis of the brivaracetam intermediate under mild conditions with a higher efficiency.

Description

An olefin reductase mutant and its application in the preparation of buvasidan intermediates Technical Field

[0001] This invention belongs to the field of biocatalysis technology, and more specifically relates to an olefin reductase mutant and its encoding gene, genetically engineered bacteria, and its application in the preparation of (R)-4-propyldihydrofuran-2(3H)-one (R-PDFO). Background Technology

[0002] Alkene reductases (ER, EC1.6.99.1) are mainly from the flavin mononucleotide (FMN)-containing oxoenzymes (OYEs) family, belonging to the redox enzyme class. They catalyze the asymmetric reduction of α,β-unsaturated ketones, aldehydes, nitroalkenes, carboxylic acids, and their derivatives. The products can serve as chiral components of many drugs, such as pregabalin, baclofen, and L-menthol. Due to their broad substrate spectrum, photoexcitation reactivity, and excellent characteristic of generating two chiral centers at once, they have been widely used in the asymmetric synthesis of chiral intermediates and have high application value in the synthesis of chiral compounds.

[0003] Brivacera is a novel antiepileptic drug with a structure similar to the second-generation antiepileptic drug levetiracetam. Its antiepileptic effect is primarily achieved through binding to synaptic vesicle protein 2A (SV2A), and is also related to the inhibition of specific sodium ion channels in neurons. It offers advantages in drug therapy, including rapid absorption, high bioavailability, and fewer drug interactions. The synthesis of brivacera involves the introduction of chiral centers, which is both a key and challenging aspect of the process. Many existing synthetic routes for brivacera suffer from high costs, complex processes, low yields, and difficulties in product separation, significantly hindering large-scale production. Therefore, designing a simple and efficient synthetic route for brivacera is of great importance.

[0004] (R)-4-propyl-dihydrofuran-2-one (R-PDFO) is a key chiral intermediate in the synthesis of the antiepileptic drug brivacertan, with a relative molecular mass of 128.17. Several synthetic routes for obtaining this chiral intermediate have been developed, including: starting with dimethyl propylmalonate as a starting material, proceeding through substitution, decarboxylation, reduction, and cyclization reactions to obtain the compound (R)-4-propyl-dihydrofuran-2(3H)-one; starting with succinic anhydride, first reacting with a chiral chelating agent, then undergoing an Evans Aldol reaction with propionaldehyde to introduce the R-configuration propyl group, followed by a Barton-McCombie deoxygenation reaction to remove the hydroxyl group, and finally obtaining the target intermediate through carboxyl reduction and lactone formation; and using glyoxylic acid and n-pentanal as starting materials, obtaining the intermediate 4-propylfuran-2(5H)-one through a series of reactions. Using hydrogen as a reducing agent and palladium / carbon as a catalyst, the C=C double bond was catalytically reduced to obtain a racemic mixture. The racemic mixture was then chirally resolved by (1R,2S)-2-amino-1,2-diphenylethanol to prepare (R)-4-propyldihydrofuran-2(3H)-one. Using polymethyl-hydrosiloxane (PMHS) as a reducing agent, enantioselective reduction of 4-propylfuran-2(5H)-one was achieved under the chiral catalysis of Cu(I).

[0005] Compared to other methods, enzymatic catalysis is the most environmentally friendly and efficient route for the synthesis of (R)-4-propyl-dihydrofuran-2-one (R-PDFO). Using recombinant olefin reductase, a coenzyme, and a coenzyme regeneration system, the chiral reduction of the substrate 4-propylfuran-2(5H)-one to the key intermediate buvascartan is catalyzed. This method offers advantages such as mild reaction conditions, high efficiency, simple operation, no need for chiral resolution, high yield, and low cost. However, currently discovered olefin reductases suffer from low catalytic activity and low enantioselectivity in the asymmetric catalytic synthesis of R-PDFO. Summary of the Invention

[0006] The purpose of this invention is to provide an olefin reductase mutant, its encoding gene, a recombinant plasmid, a recombinant genetically engineered bacterium, and its application in the preparation of buvasidan intermediates, thereby solving the problem that existing olefin reductases have low catalytic activity and corresponding low selectivity, resulting in low yields of buvasidan intermediates that are difficult to use in industrial production.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] According to a first aspect of the present invention, an olefin reductase mutant for catalyzing the production of the buvasidan intermediate (R)-4-propyldihydrofuran-2(3H)-one from 4-propylfuran-2(5H)-one is provided, comprising an olefin reductase mutant formed by mutations at positions 57 (alanine), 67 (tryptophan), and 271 (phenylalanine) using the wild-type olefin reductase shown in SEQ ID NO.1 as a template: a mutant A57G is formed by mutating alanine A at position 57 to glycine G, with the amino acid sequence shown in SEQ ID NO.3; a mutant W67K is formed by mutating tryptophan W at position 67 to lysine K, with the amino acid sequence shown in SEQ ID NO.4; a mutant W67H is formed by mutating tryptophan W at position 67 to histidine H, with the amino acid sequence shown in SEQ ID NO.5; and a mutant F271Y is formed by mutating phenylalanine F at position 271 to tyrosine Y, with the amino acid sequence shown in SEQ ID NO.6.

[0009] According to the present invention, the wild-type olefin reductase is derived from the genus *Novosphingobium nitrogenifigens*.

[0010] According to a second aspect of the present invention, a gene encoding the olefin reductase mutant is provided.

[0011] According to a third aspect of the present invention, a recombinant vector comprising the coding gene of the said olefin reductase mutant and a genetically engineered bacterium are provided. According to a preferred embodiment of the present invention, the host bacterium may be Escherichia coli BL21(DE3).

[0012] According to a fourth aspect of the present invention, a method for preparing an olefin reductase mutant is provided, comprising the following steps: 1) culturing the recombinant genetically engineered bacteria and inducing the expression of the olefin reductase mutant; 2) isolating the olefin reductase mutant from the culture obtained in step 1).

[0013] According to a fifth aspect of the invention, there is provided the use of the olefin reductase mutant described above in the catalytic generation of (R)-4-propyldihydrofuran-2(3H)-one from the substrate 4-propylfuran-2(5H)-one.

[0014] This invention utilizes a laboratory bacterial strain resource bank to obtain multiple olefin reductases (ERs). Through in vitro enzyme activity testing, the olefin reductase NnER derived from *Novosphingobium nitrogenifigens* was screened and found to exhibit the highest enzyme activity, catalytic efficiency, and enantioselectivity towards the substrate 4-propylfuran-2(5H)-one. To date, there have been no reports on the mutation of olefin reductases derived from *Novosphingobium nitrogenifigens*.

[0015] Preferably, the present invention uses a coenzyme cycling system coupled with the olefin reductase mutants A57G, W67K, W67H, and F271Y to catalyze the reaction of PFO to R-PDFO. Glucose dehydrogenase (BsGDH) derived from Bacillus subtilis is used to achieve the cycling of the coenzyme NADPH, thereby saving costs.

[0016] In the application described: the olefin reductase mutant and glucose dehydrogenase were cultured in LB medium until OD200 was reached. 600 =0.6-0.8, IPTG was added to induce expression, wet bacterial cells were obtained, and after freeze treatment, they were lyophilized to obtain dry bacterial powder. PFO was used as a substrate, and flavin mononucleotides FMN and NADP were added. + Using glucose as an auxiliary substrate and 100mM PBS buffer at pH 6-8 as the reaction medium, the reaction system was constructed. The mixture was shaken in a shaker at 25-35℃ and 180-220 rpm for 18-24 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate containing an internal standard (o-xylene), dried with anhydrous sodium sulfate, and detected by gas chromatography.

[0017] The key inventive point of this invention lies in the discovery of an olefin reductase NnER from the genus *Novosphingobium nitrogenifigens* from a laboratory bacterial strain resource bank, and the heterologous expression of the olefin reductase NnER achieved through a series of molecular cloning techniques. Furthermore, through semi-rational design of the wild-type NnER, the expression of the olefin reductase NnER can be achieved at a distance from the substrate. Five residues were selected within the range, including A57, F271, G28, H130, and W67, which were considered "hot spots" for mutation. These five residues were then subjected to saturation mutations. This invention also constructed a dual-enzyme cascade reaction system, cascading an olefin reductase mutant and glucose dehydrogenase to achieve the cyclic regeneration of NADPH, significantly reducing costs. Subsequently, using PFO as a substrate, the catalytic production of R-PDFO by all mutants was determined. Among them, the positive mutants W67K, W67H, A57G, and F271Y showed increased R-PDFO yield and ee value at 8 mM substrate concentration for 24 h compared to the wild type (15.5%, 82.7%), to W67K (17.8%, 95.7%), W67H (31.44%, 96.9%), A57G (15.5%, 93.1%), and F271Y (30.28%, 95.7%), respectively. The ee values ​​of these four positive mutation sites were all improved, indicating that further modification plans can be carried out. Among them, mutant W67H had the highest R-PDFO yield and ee value, making it the optimal mutant obtained in this invention.

[0018] In summary, this invention modifies olefin reductase NnER through semi-rational design, obtaining various olefin reductase mutants, encoding genes, recombinant plasmids, and recombinant genetically engineered bacteria with improved catalytic efficiency and ee value. Furthermore, it constructs a two-enzyme cascade reaction system that can synthesize the buvasidan intermediate R-PDFO more efficiently under mild conditions, overcoming the low yield and low enantioselectivity of wild-type olefin reductase NnER for PFO, and thus possessing broader application value. Attached Figure Description

[0019] Figure 1 shows a schematic diagram of the reaction mechanism of olefin reductase mutant catalyzing the formation of buvacertane intermediate (R)-4-propyldihydrofuran-2(3H)-one from 4-propylfuran-2(5H)-one.

[0020] Figure 2 shows the molecular docking results;

[0021] Figure 3 shows the detection patterns of substrate PFO and product R-PDFO;

[0022] Figure 4 shows the stereoselective detection of the product R-PDFO;

[0023] Figure 5 shows the concentration standard curve of the product R-PDFO. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art, or experimental methods recommended by the reagent kit and instrument manufacturers.

[0025] The LB liquid medium formula is as follows: Take 1L of deionized water, weigh out 5g of yeast extract, 10g of peptone, and 10g of sodium chloride, and stir evenly on a magnetic stirrer until the pH is at its natural value. Measure 50mL of the medium into 250mL Erlenmeyer flasks using a graduated cylinder, and autoclave the prepared medium at 121℃ for 20min.

[0026] LB solid medium: Measure 150 mL of LB liquid medium into 250 mL Erlenmeyer flasks using a graduated cylinder, add 2.25 g of agarose to each flask, and sterilize at 121 °C for 20 min.

[0027] Example 1: Construction of genetically engineered bacteria

[0028] The NnER sequence from *Novosphingobium nitrogenifigens* DSM 19370 was cloned and inserted into the expression plasmid pET28a(+) to obtain pET28a(+)-NnER. The amino acid sequence of the wild-type NnER is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. After confirming that the sequencing was correct, the plasmid was transformed into the expression host *Escherichia coli* BL21(DE3) for subsequent recombinase expression.

[0029] Example 2: Bacterial Culture

[0030] The correctly sequenced bacterial culture was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin resistance and incubated in a shaker at 37°C and 200 rpm for 12 h. Then, 500 μL of the bacterial culture was added to 50 mL of LB liquid medium (containing kanamycin resistance) and incubated in a shaker at 37°C for 2.5 h until OD reached. 600 When the bacterial culture concentration reaches 0.6-0.8, add IPTG (0.1 mM) as an inducer to a final concentration of 0.1 mM, and transfer to a shaker at 20°C and 200 rpm for 16-20 h. After culturing, transfer the culture medium to a 50 mL centrifuge tube, balance the liquid, and centrifuge at 4°C and 8000 rpm for 10 min. Discard the supernatant, add 15 mL of physiological saline to wash the bacterial cells, centrifuge again to collect the cells, and then freeze at -40°C for at least 3 h for freeze-drying. After freeze-drying the bacterial cells in a vacuum freeze dryer for 10 h, remove them, grind them evenly to obtain freeze-dried bacterial powder.

[0031] Example 3: Selection of mutation sites

[0032] The structure of wild-type olefin reductase was predicted using AlphaFold3. Auto-Dock was used to perform molecular docking of the substrate PFO and cofactor FMN to the binding site of the olefin reductase. The molecular diagrams were visualized and analyzed using PyMOL software, and the results are shown in Figure 2. Based on the docking results, key residues within the NnER catalytic activity pocket of the olefin reductase were screened: A57, F271, G28, H130, and W67. Subsequently, saturation mutagenesis was performed on these five potential sites to construct a mutant library.

[0033] Example 4: Construction of a single-point mutant of olefin reductase

[0034] E. coli containing the pET28a(+)-NnER recombinant plasmid were cultured overnight in LB liquid medium (containing kanamycin resistance). The plasmid was extracted and used as a template for subsequent mutant construction. The primers used for mutation are shown in Table 1.

[0035] Table 1. Primer information for mutants (SEQ ID NO.7-16)

[0036] Table 2 PCR reaction system (20 μL)

[0037] Table 3 PCR reaction conditions

[0038] (2)-(4) steps are set to 35 loops.

[0039] After PCR amplification, the product was verified as positive by agarose gel electrophoresis. Then, 0.5 μL of restriction endonuclease Dpn I was added and the mixture was reacted in a metal bath at 37°C for 2.5 h to achieve specific excision of the methylated template DNA strand.

[0040] The digested PCR product was transferred to competent E. coli BL21(DE3) cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then incubated on ice again for 2 min. 700 μL of LB medium (without antibiotics) was added, and the cells were incubated at 37°C for 45 min on a shaker. The bacterial culture was then plated onto LB agar plates containing kanamycin resistance (Kan, 50 μg / mL) and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium. After 5 h, sequencing was performed. A portion of the correctly sequenced bacterial culture was inoculated into preservation tubes and stored at -40°C, while the other portion was cultured to prepare lyophilized bacterial powders of various mutants. Specific procedures are detailed in Example 2.

[0041] Example 5: Determination of mutant enzyme activity

[0042] Using the lyophilized olefin reductase bacterial powder prepared in Example 4 as a catalyst, the conversion reaction of substrate PFO was carried out to screen for mutants with improved yield.

[0043] The 1 mL reaction system consists of: 100 μL substrate PFO (final concentration 8 mM), 100 μL glucose (final concentration 20 mM), and 100 μL NADP. + 100 μL FMN (final concentration 0.1 mM), 20 mg lyophilized bacterial powder, 20 mg GDH bacterial powder, 600 μL LPB buffer (100 mM pH 7.0). Mix all components thoroughly after addition. Perform three replicates for each reaction.

[0044] The reaction was carried out in a shaker at 30℃ and 250 rpm for 24 h. After the reaction was completed, 750 μL of ethyl acetate was added to terminate the reaction and extract the solution. The mixture was then vortexed at high speed for 10 min and centrifuged at high speed (14000 rpm, 10 min). The upper organic phase was collected, and the above steps were repeated once. The organic phases were combined and evaporated to dryness in a fume hood. 1 mL of ethyl acetate (containing o-xylene) was added to reconstitute the solution, and the mixture was shaken vigorously for 5 min. Anhydrous sodium sulfate was added to remove water, and the mixture was centrifuged again at high speed (14000 rpm, 10 min). 200 μL of the supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by gas chromatography.

[0045] The yield was determined by gas chromatography (GC) using an internal standard method, with o-xylene selected as the internal standard. An Agilent DB-1701 capillary column (30 m × 0.25 mm × 0.25 μm) was used. The analytical method was as follows: the injector and detector temperatures were set to 250 °C, the column oven temperature to 80 °C, the carrier gas to be N2, the flow rate to be 1 mL / min, the split ratio to be 20:1, and the injection volume to be 1 μL. The temperature program was 80 °C for 3 min, then increased to 230 °C at a rate of 15 °C / min and held for 2 min. Under these analytical conditions, the retention time of the substrate PFO was 9.473 min, the retention time of the product R-PDFO was 8.350 min, and the retention time of the internal standard o-xylene was 2.943 min, as shown in Figure 3.

[0046] The stereoselectivity of the product was determined by gas chromatography (GC) using an internal standard method with o-xylene as the internal standard. The GC column used was an Agilent Beta DEXTM 225 capillary column (30 m × 0.25 mm × 0.25 μm). The analytical method was as follows: the injection port temperature was set to 220 °C, the detector temperature to 250 °C, the initial column oven temperature to 40 °C, the carrier gas to be N2, the flow rate to be 0.5 mL / min, the split ratio to be 50:1, and the injection volume to be 1 μL. The temperature program was: 40 °C for 1 min, then increased to 170 °C at a rate of 15 °C / min and held for 1 min, then increased to 180 °C at a rate of 1 °C / min and held for 1 min. As shown in Figure 4, under these analytical conditions, the retention time of product R-PDFO was 16.703 min, and the retention time of product S-PDFO was 16.917 min. The ee value is calculated using the following formula: ee value (%) = [(RS) / (R+S)] × 100%, where R and S represent the peak areas of the R-type and S-type of the product in the gas chromatogram, respectively.

[0047] The catalytic activity of the mutant expression system constructed above was evaluated by the conversion of the product R-PDFO. The content of R-PDFO was calculated using a standard curve (as shown in Figure 5).

[0048] Using PFO as the substrate for the catalytic reaction, the constructed mutant library was screened and analyzed using the above method. The mutants with higher R-PDFO generating capacity than the wild type are shown in Table 4. As can be seen from the table, mutants with significantly improved yield or ee value include W67K, W67H, A57G, and F271Y. Among them, mutant W67H has the highest R-PDFO yield and ee value, making it the optimal mutant obtained in this invention.

[0049] Table 4. Yields and ee values ​​of ene reductase NnER and mutants (3 replicates per experiment)

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. An olefin reductase mutant for catalyzing the production of buvasidan intermediate (R)-4-propyldihydrofuran-2(3H)-one (R-PDFO) from 4-propylfuran-2(5H)-one (PFO), characterized in that, The olefin reductase mutant includes an olefin reductase mutant formed by using the wild-type olefin reductase shown in SEQ ID NO.1 as a template and making the following mutations to alanine at position 57, tryptophan at position 67, and phenylalanine at position 271: The mutant A57G is formed by mutating alanine A at position 57 to glycine G, and the amino acid sequence is shown in SEQ ID NO.3; The mutant W67K is formed by mutating tryptophan W at position 67 to lysine K, and its amino acid sequence is shown in SEQ ID NO.4; The mutant W67H is formed by mutating tryptophan W at position 67 to histidine H, and the amino acid sequence is shown in SEQ ID NO.

5. The mutant F271Y is formed by mutating phenylalanine F at position 271 to tyrosine Y, and its amino acid sequence is shown in SEQ ID NO.

6.

2. The olefin reductase mutant according to claim 1, characterized in that, The wild-type olefin reductase is derived from the genus *Novosphingobium nitrogenifigens*.

3. A coding gene encoding an olefin reductase mutant as described in any one of claims 1-2.

4. A recombinant plasmid comprising the encoding gene of the olefin reductase mutant as described in claim 3.

5. A recombinant genetically engineered bacterium containing the recombinant plasmid as described in claim 4.

6. The recombinant genetically engineered bacteria according to claim 5, characterized in that, The host cell was Escherichia coli BL21(DE3).

7. A method for preparing an olefin reductase mutant, characterized in that, Includes the following steps: 1) The recombinant genetically engineered bacteria of claim 5 are cultured and alkene reductase mutant expression is induced; 2) The olefin reductase mutant was isolated from the culture obtained in step 1).

8. The use of an olefin reductase mutant according to claim 1 in the catalytic production of (R)-4-propyldihydrofuran-2(3H)-one from the substrate 4-propylfuran-2(5H)-one.

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