A method for photocatalytic generation of NADPH and photoenzymatic synthesis of (S)-3-hydroxytetrahydrofuran

Abstract

The application provides a method for photocatalytic generation of NADPH and photoenzymatic catalytic synthesis of (S)-3-hydroxytetrahydrofuran, and belongs to the technical field of NADPH regeneration and (S)-3-hydroxytetrahydrofuran preparation. + The method for photocatalytic generation of NADPH comprises the following steps: mixing deaza-riboflavin dRf, EDTA, FDR, NADP The application regenerates coenzyme factor NADPH by using clean and renewable light energy, and realizes the reduction of tetrahydrofuran-3-ketone by alcohol dehydrogenase TbADH under a mild environment by using photocatalytic regenerated NADPH, and then synthesizes (S)-3-hydroxytetrahydrofuran with high optical purity, and 12.93mM (S)-3-hydroxytetrahydrofuran can be synthesized by taking 20mM tetrahydrofuran-3-ketone as a substrate, and the conversion rate reaches 64.65%.

Description

Technical Field

[0001] This invention belongs to the technical field of NADPH regeneration and (S)-3-hydroxytetrahydrofuran preparation, specifically relating to a method for photocatalytic generation of NADPH and photoenzymatic synthesis of (S)-3-hydroxytetrahydrofuran. Background Technology

[0002] (S)-3-hydroxytetrahydrofuran is an important pharmaceutical and chemical intermediate with wide applications in anticancer drugs, hypoglycemic drugs, and AIDS drugs. For example, it can be used to synthesize the antiarrhythmic drug ticardisone, the hypoglycemic drug empagliflozin, the antiAIDS drug arenavir, and the anticancer drug afatinib. (S)-3-hydroxytetrahydrofuran has high market value. Compared with the R configuration, many drugs contain (S)-3-hydroxytetrahydrofuran, so it is often used as an important intermediate for the preparation of chiral drugs. There are many methods for synthesizing (S)-3-hydroxytetrahydrofuran: (1) chemical synthesis using chiral substrates such as L-malic acid and its derivatives, 2,3-dihydrofuran, and 2,5-dihydrofuran as raw materials; (2) asymmetric synthesis using chiral catalysts; and (3) enzyme-catalyzed methods and bio-fermentation methods. These methods each have their own advantages and disadvantages. Enzyme catalysis has received widespread attention due to its environmental friendliness, simple synthesis operation, and high product selectivity.

[0003] Since enzyme-catalyzed redox reactions generally require the participation of coenzyme factors, directly adding coenzyme factors exogenously is an effective method, but it increases production costs. Another approach is to construct a coenzyme factor recycling system: utilizing specific enzymes or enzyme systems to achieve the recycling of coenzyme factors and maintain their concentration. For example, glucose dehydrogenase (GDH) catalyzes the oxidation of glucose to gluconic acid, while simultaneously regenerating nicotinamide adenine dinucleotide phosphate (NADP). + The NADPH is reduced to reduced nicotinamide adenine dinucleotide phosphate (NADPH), which can then participate in other reactions requiring reducing power, creating a cyclical process. However, the byproduct gluconic acid produced in this process accumulates and affects the pH of the system, thus hindering the continuation of the reaction. This limits the range of options available for NADPH regeneration.

[0004] In summary, in order to reduce the cost of using coenzyme factors, it is urgent to develop a technical system that utilizes clean and renewable light energy to regenerate coenzyme factors and synthesize (S)-3-hydroxytetrahydrofuran under mild conditions. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a method for photocatalytic generation of NADPH, which utilizes clean and sufficient light energy to regenerate the coenzyme factor NADPH.

[0006] The second objective of this invention is to provide a method for photoenzymatic synthesis of (S)-3-hydroxytetrahydrofuran, which can synthesize high optical purity (S)-3-hydroxytetrahydrofuran under mild conditions and achieve a conversion rate of 64.65%.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for photocatalytic generation of NADPH, comprising the following steps: reacting denitrified riboflavin dRf, EDTA, FDR, and NADP... + When mixed with phosphate buffer, NADP is reduced by photocatalysis. + Regenerated NADPH; the amino acid sequence of the FDR is shown in SEQ ID NO.1.

[0009] Preferably, the light source for the photocatalysis includes a xenon lamp.

[0010] Preferably, the photocatalytic light power intensity is 90mW to 110mW.

[0011] Preferred components include denitrified riboflavin dRf, EDTA, FDR, and NADP. + The concentration ratio of phosphate buffer is (60-90 μM): (20-25 mM): (0.2-0.5 mg / mL): (1-2 mM): 50 mM.

[0012] Preferably, the phosphate buffer comprises 10.67 g / L K2HPO4·3H2O and 0.44 g / L KH2PO4, and the pH of the phosphate buffer is 8.0.

[0013] The present invention also provides the application of the above method in the synthesis of (S)-3-hydroxytetrahydrofuran.

[0014] The present invention also provides a method for photoenzymatic synthesis of (S)-3-hydroxytetrahydrofuran, wherein TbADH and tetrahydrofuran-3-one are added to the above method.

[0015] Preferably, the amino acid sequence of the TbADH is shown in SEQ ID NO.2.

[0016] Preferably, the concentration of TbADH is 1.25–1.8 mg / mL.

[0017] The present invention also provides the application of the above method in the preparation of drugs, including anticancer drugs, hypoglycemic drugs, or anti-AIDS drugs.

[0018] The beneficial effects of this invention are:

[0019] This invention utilizes clean and renewable light energy to regenerate the coenzyme factor NADPH, and under mild conditions, it enables the photocatalytic regeneration of NADPH by the alcohol dehydrogenase TbADH to synthesize high-optical-purity (S)-3-hydroxytetrahydrofuran from tetrahydrofuran-3-one. Using 20 mM tetrahydrofuran-3-one as a substrate, 12.93 mM (S)-3-hydroxytetrahydrofuran can be synthesized with a conversion rate of 64.65%. Attached Figure Description

[0020] Figure 1 This is a route diagram for the photocatalytic NADPH regeneration synthesis of (S)-3-hydroxytetrahydrofuran in this invention;

[0021] Figure 2 This is a schematic diagram of the recombinant plasmid pET-20b(+);

[0022] Figure 3 This is a curve showing the regeneration of photocatalytic coenzyme factor NADPH.

[0023] Figure 4 Schematic diagram of recombinant plasmid pET-20b-TbADH;

[0024] Figure 5 The results of gas chromatography determination of the optical purity of (S)-3-hydroxytetrahydrofuran;

[0025] Figure 6 Graphs showing the catalytic formation of (S)-3-hydroxytetrahydrofuran from substrates with different tetrahydrofuran-3-one concentrations;

[0026] Figure 7 To optimize the amount of photocatalyst and reactive enzyme used, the (S)-3-hydroxytetrahydrofuran generation curve was obtained. Detailed Implementation

[0027] This invention provides a method for photocatalytic generation of NADPH, comprising the following steps: reacting denitrified riboflavin dRf, EDTA, FDR, and NADP... + When mixed with phosphate buffer, NADP is reduced by photocatalysis. + Regenerated NADPH; the amino acid sequence of the FDR is shown in SEQ ID NO.1.

[0028] In this invention, denitrified riboflavin dRf serves as a photocatalyst, and ethylenediaminetetraacetic acid (EDTA) acts as an electron donor, also known as a photo-hole sacrificial agent. These electrons, transferred through the electron transport chain, provide a sufficient electron source for the reduction and regeneration of NADPH. In this invention, electrons from the photocatalytic electron donor are transferred through the photocatalyst dRf, and further transferred to NADP using flavin disulfide reductase (FDR). +It enables the regeneration of the expensive coenzyme factor NADPH and maintains a stable NADPH concentration.

[0029] In this invention, the photocatalytic light source preferably includes a xenon lamp, and the photocatalytic light power intensity is preferably 90mW to 110mW, more preferably 95mW to 105mW. In this invention, denitrified riboflavin dRf, EDTA, FDR, and NADP are used. + The preferred concentration ratio of the phosphate buffer is (60–90 μM):(20–25 mM):(0.2–0.5 mg / mL):(1–2 mM):50 mM, more preferably (70–80 μM):(22–24 mM):(0.3–0.4 mg / mL):(1.2–1.6 mM):50 mM. In this invention, the phosphate buffer preferably comprises 10.67 g / L K₂HPO₄·3H₂O and 0.44 g / L KH₂PO₄, and the pH of the phosphate buffer is preferably 8.0. In this invention, the nucleotide sequence encoding the FDR is preferably as shown in SEQ ID NO.3.

[0030] The present invention also provides the application of the above method in the synthesis of (S)-3-hydroxytetrahydrofuran.

[0031] The present invention also provides a method for photoenzymatic synthesis of (S)-3-hydroxytetrahydrofuran, wherein TbADH and tetrahydrofuran-3-one are added to the above method.

[0032] In this invention, the preferred amino acid sequence of alcohol dehydrogenase (TbADH) is shown in SEQ ID NO.2, and the preferred nucleotide sequence encoding TbADH is shown in SEQ ID NO.4. In this invention, the concentration of TbADH is preferably 1.25–1.8 mg / mL, more preferably 1.4–1.6 mg / mL; in a specific embodiment of this invention, the concentration of tetrahydrofuran-3-one is preferably 10–50 mM, more preferably 15–30 mM. This invention uses tetrahydrofuran-3-one as a substrate, and TbADH utilizes photocatalytically regenerated NADPH to catalyze the synthesis of (S)-3-hydroxytetrahydrofuran from tetrahydrofuran-3-one. A schematic diagram of the synthesis of (S)-3-hydroxytetrahydrofuran in this invention is shown below. Figure 1 As shown. After synthesizing (S)-3-hydroxytetrahydrofuran using the method of this invention, ethyl acetate containing 3 mM octanoic acid as an internal standard was used as the extractant, and gas chromatography was used to detect and quantify (S)-3-hydroxytetrahydrofuran.

[0033] The present invention also provides the application of the above method in the preparation of drugs, wherein the drugs preferably include anticancer drugs, hypoglycemic drugs or anti-AIDS drugs.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Unless otherwise specified, the following embodiments are all conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] The KPi buffer (50 mM, pH = 8.0) in the following examples was obtained by mixing 10.67 g of K2HPO4·3H2O and 0.44 g of KH2PO4 and bringing the volume to 1 L.

[0038] Example 1

[0039] The photocatalytic method for generating NADPH consists of the following steps:

[0040] (1) Flavin disulfide reductase (FDR) was induced and purified in Escherichia coli using NdeI and XbaI endonucleases. The FDR nucleotide sequence, as shown in SEQ ID NO.3, was inserted into the pET-20b expression vector (see [link to original text]). Figure 2 (The purple portion represents the inserted nucleic acid sequence), resulting in a recombinant plasmid containing the FDR gene (this process was performed by Genscript Biotech (Nanjing)). The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells, and after resuscitation culture, it was plated and cultured overnight. After transformants appeared, they were picked and placed in 5 mL LB culture tubes containing 100 μg / mL ampicillin, and cultured overnight at 37℃ and 220 rpm / min to obtain a seed culture. The seed culture was inoculated at a volume ratio of 1% into LB medium supplemented with 1% glycerol and 100 μg / mL ampicillin for expansion culture. When OD... 600After reaching a pH of 0.6–0.8, a final concentration of 500 μM IPTG was added, and the cells were induced at 18°C ​​and 220 rpm / min for 16–20 hours. The induced bacterial cells were collected by centrifugation at 7000 rpm for 10 min at 4°C. The cells were resuspended in KPi buffer (50 mM, pH = 8.0) and then sonicated. The disrupted cells were centrifuged at 12000 rpm for 30 min at 4°C to obtain the supernatant. The supernatant was passed through a Ni column equilibrated with KPi buffer at a flow rate of 5 mL / min. The target protein, due to its His tag, was adsorbed onto the Ni column. The Ni column was then washed with KPi buffer containing 30 mM imidazole to elute loosely bound proteins. The Ni column was then washed again with KPi buffer containing 250 mM imidazole to elute the target protein. Finally, the eluent containing the target protein was replaced with KPi buffer to remove the imidazole, yielding purified FDR.

[0041] (2) Photocatalytic regeneration reaction of coenzyme factor NADPH

[0042] The reaction vessel required for photocatalytic NADPH regeneration is a vial. Since the reaction requires anaerobic conditions, the reaction system is configured in an anaerobic glove box, as shown in Table 1.

[0043] Table 1 Photocatalytic NADPH regeneration reaction system

[0044]

[0045] Under anaerobic conditions, the NADPH regeneration reaction system was prepared in a vial according to Table 1. After sealing the vial, it was placed under a xenon lamp, and the light power intensity was adjusted to 100mW to start the photocatalytic reaction.

[0046] (3) NADPH concentration detection

[0047] Every hour, take a sample from reaction (2) for testing. Centrifuge 300 μL of the sample at 12000 rpm for 5 min, and add 200 μL of the supernatant to a 96-well plate. Detect at 340 nm using a multi-plate reader (Thermo Fisher Scientific, Finland) at room temperature. Use the system without FDR as a blank control. NADPH(ε 340 The millimolecular extinction coefficient of ) is 6.22 mM. -1 cm -1 The NADPH concentration was calculated based on the extinction coefficient. All reactions were performed in triplicate.

[0048] The results are as follows Figure 3As shown, photocatalysis successfully achieved the reduction and regeneration of coenzyme factor NADPH. At a light power intensity of 100 mW, the reaction lasted 3 hours, resulting in the regeneration of 2 mM NADPH. + Completely reduce to generate NADPH.

[0049] Example 2

[0050] Photocatalytic regeneration of coenzyme factor NADPH to synthesize (S)-3-hydroxytetrahydrofuran

[0051] (1) Induction and purification of alcohol dehydrogenase (TbADH) in Escherichia coli

[0052] The nucleic acid and amino acid sequences of TbADH were obtained from the NCBI database. TbADH was modified by replacing isoleucine I at position 86 with valine V, tryptophan W at position 110 with leucine L, and leucine L at position 294 with glutamate Q. Modifications were performed at the corresponding sites in the nucleic acid sequence according to the codon table to obtain the modified nucleic acid sequence. After codon optimization, the modified nucleic acid sequence SEQ ID NO.4 was inserted into the pET-20b expression vector using NdeI and XbaI restriction enzymes (see [link to original text]). Figure 4 (The blue part is the inserted nucleic acid sequence, which was handled by GenScript (Nanjing)) to obtain a recombinant plasmid containing the modified TbADH gene.

[0053] The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and TbADH was induced and purified. The induction and purification methods were the same as in Example 1 (1) to obtain purified TbADH.

[0054] (2) Photocatalytic regeneration of coenzyme factor NADPH to synthesize (S)-3-hydroxytetrahydrofuran

[0055] The reaction vessel required for the photocatalytic NADPH regeneration synthesis of (S)-3-hydroxytetrahydrofuran is a vial. The reaction requires anaerobic conditions, so the reaction system is prepared in an anaerobic glove box. The reaction system is based on (2) of Example 1, with the addition of tetrahydrofuran-3-one and TbADH. The reaction system is shown in Table 2.

[0056] Table 2. Photocatalytic NADPH regeneration and synthesis of (S)-3-hydroxytetrahydrofuran reaction system

[0057]

[0058]

[0059] The reaction system was prepared in an anaerobic glove box and placed in a vial. After sealing the vial, it was placed under a xenon lamp, and the light power intensity was adjusted to 100mW to start the photocatalytic reaction.

[0060] Example 3

[0061] The difference from Example 2 is that the final concentration of tetrahydrofuran-3-one was adjusted to 10 mM or 50 mM, respectively, while the rest is the same as in Example 2.

[0062] Detection and quantification of (S)-3-hydroxytetrahydrofuran obtained in Examples 2 and 3

[0063] (S)-3-hydroxytetrahydrofuran was extracted with an extractant (ethyl acetate containing 3 mM octanoic acid) and detected by gas chromatography. The gas chromatography conditions were as follows: chromatograph: Fuli GC9790 plus gas chromatograph; detector: flame ionization detector (FID); injection: Fuli FL1092T autosampler; injection volume: 1 μL; column: HYDRODEX BETA-TBDAC (25 m × 0.25 mM × 0.25 μm, Macherey-Nagel, Germany); temperature program: injection port temperature 240 °C, column flow rate 1.0 mL / min, carrier gas nitrogen, column oven temperature increased from 125 °C to 135 °C at a rate of 3 °C / min, then increased to 220 °C at a rate of 50 °C / min, and held at 220 °C for 3 min.

[0064] Furthermore, the (S)-3-hydroxytetrahydrofuran obtained in Example 2 was detected by gas chromatography and compared with (S)-3-hydroxytetrahydrofuran standards and (R)-3-hydroxytetrahydrofuran standards. The results are as follows: Figure 5 As shown, the reaction product obtained in Example 2 is a high-optical-purity (S)-3-hydroxytetrahydrofuran.

[0065] A standard curve was constructed for the quantification of (S)-3-hydroxytetrahydrofuran. (S)-3-hydroxytetrahydrofuran standards were prepared into samples with final concentrations of 1, 2, 4, 6, 8, and 10 mM using Kpi buffer. Ethyl acetate containing 3 mM n-octanoic acid as an internal standard was used as the extraction solvent. 100 μL of each concentration standard sample was extracted with 1 mL of the extraction solvent, thoroughly shaken, and centrifuged at 12000 rpm for 5 min. The supernatant was collected to obtain the standard extract containing (S)-3-hydroxytetrahydrofuran, and 1 μL was injected into the sample using an automated gas chromatograph for detection. A standard curve was constructed by plotting the ratio of (S)-3-hydroxytetrahydrofuran standard concentration to the internal standard n-octanoic acid concentration as the x-axis and the ratio of the peak area of ​​different concentrations of (S)-3-hydroxytetrahydrofuran standard to the peak area of ​​the internal standard n-octanoic acid concentration as the y-axis. The resulting standard curve was y = 0.1966x + 0.03439 (R²). 2 =0.999).

[0066] Concentration detection of the reaction product (S)-3-hydroxytetrahydrofuran: Reaction samples from Examples 2 and 3 were taken every hour for detection. 100 μL of each reaction was extracted with 1 mL of extractant, thoroughly shaken and mixed, then centrifuged at 12000 rpm for 5 min. The supernatant containing the reaction product (S)-3-hydroxytetrahydrofuran was collected, and 1 μL was injected using an automated gas chromatograph for detection. The concentration of (S)-3-hydroxytetrahydrofuran was calculated using a standard curve. All reactions were performed in triplicate.

[0067] The results are as follows Figure 6 As shown, photoenzymatic NADPH regeneration successfully synthesized (S)-3-hydroxytetrahydrofuran. The concentration of (S)-3-hydroxytetrahydrofuran reached its peak after 7 hours of reaction. 6.48 mM (S)-3-hydroxytetrahydrofuran could be synthesized using 10 mM tetrahydrofuran-3-one as a substrate, 9.71 mM (S)-3-hydroxytetrahydrofuran could be synthesized using 20 mM tetrahydrofuran-3-one as a substrate, and 10.23 mM (S)-3-hydroxytetrahydrofuran could be synthesized using 50 mM tetrahydrofuran-3-one as a substrate.

[0068] Example 4

[0069] Optimize the amount of photocatalyst and reactive enzyme to increase the concentration of (S)-3-hydroxytetrahydrofuran.

[0070] The difference from Example 2 is that the final concentration of dRf was adjusted to 90 μM, the final concentration of FDR was adjusted to 0.5 mg / mL, the final concentration of TbADH was adjusted to 1.8 mg / mL, and the final concentration of tetrahydrofuran-3-one was adjusted to 20 mM. All other aspects were the same as in Example 2.

[0071] The concentration of the reaction product (S)-3-hydroxytetrahydrofuran was determined using the method described in Example 3, and the results are as follows: Figure 7 As shown, increasing the amount of photocatalyst and reactive enzyme can increase the concentration of the product (S)-3-hydroxytetrahydrofuran. Using 20 mM tetrahydrofuran-3-one as a substrate, 12.93 mM (S)-3-hydroxytetrahydrofuran can be synthesized with a conversion rate of 64.65%, which is 16.10% higher.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for photocatalytic generation of NADPH, characterized in that, The steps include: adding denitrified riboflavin dRf, EDTA, FDR, and NADP. + When mixed with phosphate buffer, NADP is reduced by photocatalysis. + Regenerated NADPH; the amino acid sequence of the FDR is shown in SEQ ID NO. 1; the reaction conditions are anaerobic.

2. The method according to claim 1, characterized in that, The light source for the photocatalysis includes a xenon lamp.

3. The method according to claim 1, characterized in that, The photocatalytic light power intensity is 90mW~110mW.

4. The method according to claim 1, characterized in that, Denitrified riboflavin dRf, EDTA, FDR, NADP + The concentration ratio of phosphate buffer is (60~90μM): (20~25mM): (0.2~0.5mg / mL): (1~2mM): 50mM.

5. The method according to claim 1, characterized in that, The phosphate buffer comprises 10.67 g / L K2HPO4·3H2O and 0.44 g / L KH2PO4, and the pH of the phosphate buffer is 8.

0.

6. The use of the method according to any one of claims 1 to 5 in the synthesis of (S)-3-hydroxytetrahydrofuran.

7. A method for photoenzymatic synthesis of (S)-3-hydroxytetrahydrofuran, characterized in that, TbADH and tetrahydrofuran-3-one are added to the method described in any one of claims 1 to 5.

8. The method according to claim 7, characterized in that, The amino acid sequence of the TbADH is shown in SEQ ID NO.

2.

9. The method according to claim 7, characterized in that, The concentration of TbADH is 1.25~1.8 mg / mL.

Images