Method for splitting bicyclic compound by using ketoreductase

Abstract

The invention discloses a method for splitting a bicyclic compound by using ketoreductase. According to the method, racemic dicyclic ketone is used as a substrate, specific ketoreductase is used as a catalyst, (1S, 5R)-dicyclic ketone is selectively reduced into alcohol, and a target chiral compound (1R, 5S)-dicyclic ketone is reserved. According to the method disclosed by the invention, the pH is stable in the reaction process, the catalytic activity of the enzyme is not influenced, the concentration of a conversion substrate can reach 200g / L, and the ee value can reach 99.8%. The method is simple to operate and good in selectivity, and can be used for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to a method for splitting a bicyclic compound using ketone reductase. Background Art

[0002] The chirality of drug molecules is closely related to the drug's pharmacological activity, toxicity, and pharmacokinetic properties. The research and development of chiral drugs has become an important direction and field in the development of new drugs worldwide.

[0003] Neuropathic pain is pain caused by damage or disease of the somatic sensory system. The prevalence of neuropathic pain in the general population is as high as 8.0%, and there are approximately 90 million patients with neuropathic pain in my country. In January 2019, according to the results of a Phase 3 clinical trial in patients with diabetic peripheral neuropathic pain (DPNP) and postherpetic neuralgia (PHN), Tablets (Milobalin Besylate Tablets) have been approved in Japan for the treatment of peripheral neuropathic pain. Their chemical structure is shown below. Existing studies have shown that, compared with pregabalin and gabapentin, milobalin has a higher analgesic effect in relieving pain associated with DPNP and PHN, with a wider safety margin and a relatively low incidence of adverse reactions.

[0004]

[0005] Milobalin has multiple chiral centers in its structure, and controlling its chirality is both a key and challenging aspect of its synthesis. Daiichi Sankyo Co., Ltd. disclosed a method for preparing milobalin in patent CN200880118892.8, as shown in Scheme 1. This method uses ethyl 3-oxohexanoate as a starting material, which is first reacted with allyl bromide to produce compound 2. Reduction, hydrolysis, and cyclization reactions yield the racemic bicyclic compound 5. This racemate is then reacted with tert-butyl dimethoxyphosphorylacetate and nitromethane to introduce an ester side chain and a nitro group. The bicyclic compound 7 is then resolved using Chiralpak IC to yield a single chiral compound. Finally, milobalin is obtained through nitro group reduction, Boc protection, and hydrochloric acid hydrolysis. This route involves chromatographic separation, which is costly and complex, making it difficult to commercialize.

[0006]

[0007] Subsequently, Daiichi Sankyo Co., Ltd. attempted to prioritize the construction of optically active compounds in patent CN201380019311.6, using a route shown in Scheme 2. This route uses racemic bicyclic compound 5 as a substrate, which is converted to compound 10 and an undesired alcohol in the presence of reductase, formate dehydrogenase, sodium formate, glucose dehydrogenase, and glucose. The conversion yield was approximately 60 g / L, with a yield of 41% and an ee of 97.7%. The rate of sodium formate in this reaction system continuously affects the reaction pH, which in turn affects the catalytic activity of the enzyme. Therefore, additional reagents were required to adjust the pH to maintain stability, making the process complex and resulting in low yields and ee values.

[0008]

[0009] Patent CN202310331729.6 shows that when the ketoreductase from Leifsonia sp. is used as a catalyst to resolve the racemic bicyclic compound 5, the chiral purity does not reach 99% when the conversion substrate concentration is 50-200 g / L.

[0010] Therefore, we need to develop a method for splitting bicyclic compounds with low production cost, simple operation and good selectivity. Summary of the Invention

[0011] The purpose of the present invention is to address the deficiencies of the prior art and provide a method for separating bicyclic compounds with low production cost, simple operation and good selectivity.

[0012] The technical solution adopted by the present invention is shown in Scheme 3:

[0013]

[0014] Wherein, R is selected from hydrogen, methyl, and ethyl.

[0015] The present invention provides a method for resolving bicyclic compounds using ketoreductase, which specifically comprises the following steps: using a racemic bicyclic ketone compound III as a substrate, selectively reducing a (1S, 5R)-bicyclic ketone to a compound II under the catalytic action of ketoreductase and isopropanol, and retaining compound I, i.e., the target chiral compound (1R, 5S)-bicyclic ketone compound I.

[0016] Furthermore, the ketoreductase is KRED001 and KRED002.

[0017] Furthermore, the ketoreductase KRED001 is derived from Synechocystis, and the NCBI accession number of the amino acid sequence is WP_010872244.1.

[0018] Furthermore, the ketoreductase KRED001 can selectively reduce (1S, 5R)-bicyclic ketone to compound II.

[0019] Furthermore, the ketoreductase KRED002 is derived from Clostridium beijerinckii, and the NCBI accession number of the amino acid sequence is WP_077844196.1.

[0020] Furthermore, the ketoreductase KRED002 can dehydrogenate isopropyl alcohol to ketone.

[0021] Furthermore, the ketoreductase participates in the catalytic reaction in the form of ketoreductase enzyme powder, ketoreductase enzyme solution, ketoreductase homogenate solution, ketoreductase lyophilized powder, ketoreductase-containing cells, etc., preferably ketoreductase cells.

[0022] Furthermore, the ketoreductase expression receptor strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis, preferably Escherichia coli.

[0023] Furthermore, coenzymes can be added to the reaction system to promote the reaction. When cells containing ketoreductase are used, a small amount of coenzyme is contained in the cells, and in this case, no coenzyme is added. In some cases, the prepared ketoreductase powder also contains a small amount of coenzyme, and in this case, no coenzyme is added. However, coenzymes can also be added to the reaction system to further promote the reaction. When coenzymes are added to the reaction system to promote the reaction, the coenzyme is selected from NAD + , NADH, NADP + , NADPH or a combination thereof, preferably NADP + .

[0024] Furthermore, the concentration of the added coenzyme is 0.02 to 0.4 g / L, preferably 0.1 to 0.2 g / L.

[0025] Furthermore, the coenzymes used in this technical solution are all selected from the coenzyme products sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0026] Furthermore, the concentration of the compound III is 1 to 200 g / L.

[0027] Furthermore, the reaction pH is 6.0 to 8.0, preferably 7.0.

[0028] Furthermore, the pH in the reaction is stable and will not affect the catalytic activity of the enzyme.

[0029] Furthermore, the reaction temperature is 15°C to 35°C, preferably 25°C.

[0030] The present invention provides a method for resolving bicyclic compounds using ketoreductase. A racemic bicyclic compound is resolved into the target chiral bicyclic compound I under the action of ketoreductase and isopropanol. The reaction maintains a stable pH, requiring no additional adjustment and without affecting the catalytic activity of the enzyme. The conversion substrate concentration can reach 200 g / L, and the ee value can reach 99.8%. The method is simple to operate, exhibits good selectivity, and does not require the use of glucose dehydrogenase or glucose, thereby reducing wastewater generation and making it more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Electrophoresis diagram of protein expression in Example 1.

[0032] Figure 2 Example 2 Chiral HPLC spectrum. DETAILED DESCRIPTION

[0033] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0034] Example 1 In vitro construction of KRED enzyme

[0035] The gene sequences of wild-type ketoreductases KRED001 and KRED002 from Synechocystis and Clostridium beijerinckii were mined from the NCBI database. The NCBI numbers of the enzymes are WP_010872244.1 and WP_077844196.1, respectively. Both ketoreductase proteins can be expressed soluble in E. coli. The protein electrophoresis is shown in Figure 1 .

[0036] Example 2 Enzymatic Preparation of (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one

[0037] 2.0g of substrate racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one and 4.2mL of isopropanol were added to the reactor in sequence. After stirring evenly, 0.001g of coenzyme NADP solid, 0.05g of KRED001 cells and 0.001g of KRED002 cells were added. The reaction volume was made up to 10mL with tap water, and then the pH was adjusted to 7.0 with 10% Na2CO3 solution. The pH did not need to be controlled during the reaction. After the reaction system was stirred at 25°C for 24h, 0.4mL of the reaction solution was taken out, and 0.8mL of n-hexane was added to the reaction solution. After thorough shaking and centrifugation, the sample was sent for testing. The reaction results showed that the ee value of (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one was 99.3%, the conversion rate was 47.3%, and the chiral HPLC spectrum was as shown below. Figure 2 shown.

[0038] Example 3 Enzymatic Preparation of (1R,5S)-3-Methylbicyclo[3.2.0]hept-3-en-6-one

[0039] To a reactor, 2.0 g of racemic 3-methylbicyclo[3.2.0]hept-3-en-6-one and 4.2 mL of isopropanol were added, stirred, and then 0.001 g of solid coenzyme NADP, 0.05 g of KRED001 cells, and 0.001 g of KRED002 cells were added. The reaction volume was brought to 10 mL with tap water, and the pH was adjusted to 7.0 with 10% Na₂CO₃ solution. pH control was not required during the reaction. The reaction system was stirred at 25°C for 24 hours. 0.4 mL of the reaction solution was removed and 0.8 mL of n-hexane was added to the solution. The solution was thoroughly shaken and centrifuged. The sample was then sent for analysis. The reaction results showed an ee value of 97.7% for (1R,5S)-3-methylbicyclo[3.2.0]hept-3-en-6-one, and a conversion rate of 44.7%.

[0040] Example 4 Enzymatic Preparation of (1R,5S)-Bicyclo[3.2.0]hept-3-en-6-one

[0041] To a reactor, 2.0 g of racemic bicyclo[3.2.0]hept-3-en-6-one and 4.2 mL of isopropanol were added, stirred, and then 0.001 g of solid coenzyme NADP, 0.05 g of KRED001 cells, and 0.001 g of KRED002 cells were added. The reaction volume was brought to 10 mL with tap water, and the pH was adjusted to 7.0 with 10% Na₂CO₃ solution. pH control was not required during the reaction. The reaction system was stirred at 25°C for 24 hours. 0.4 mL of the reaction solution was removed and 0.8 mL of n-hexane was added to the solution. The solution was thoroughly shaken and centrifuged. The sample was sent for analysis. The reaction results showed an ee value of 96.2% for (1R,5S)-bicyclo[3.2.0]hept-3-en-6-one, and a conversion rate of 43.2%.

Claims

1. A method for resolving a bicyclic compound using ketoreductase, characterized in that: Using racemic bicyclic ketone compound III as a substrate, under the catalysis of ketoreductase and isopropanol, (1S, 5R)-bicyclic ketone is selectively reduced to compound II, retaining compound I, i.e., the target chiral compound (1R, 5S)-bicyclic ketone compound I. The ketoreductases are KRED001 and KRED002. The route of the preparation method is as follows: Wherein, R is selected from hydrogen, methyl, and ethyl.

2. The preparation method according to claim 1, wherein The ketoreductase participates in the catalytic reaction in the form of ketoreductase enzyme powder, ketoreductase enzyme solution, ketoreductase homogenized solution, ketoreductase freeze-dried powder, cells containing ketoreductase, etc.

3. The preparation method according to claim 1, wherein The ketoreductase expression receptor strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis.

4. The preparation method according to claim 1, wherein The concentration of the racemic bicyclic ketone compound III is 1-200 g / L.

5. The preparation method according to claim 1, wherein The reaction requires the addition of a coenzyme, which is selected from NAD + , NADH, NADP + , NADPH or a combination thereof.

6. The preparation method according to claim 1, wherein The reaction pH is 6.0-8.

0.

7. The preparation method according to claim 1, wherein The reaction temperature is 15°C to 35°C.

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