A method for synthesizing high purity (s)-nornicotine

Abstract

The application discloses a synthesis method of high-purity (S)-nornicotine, which comprises the following steps: (1) taking racemic (R,S)-nornicotine as raw material, and utilizing immobilized enzyme to catalyze (R)-nornicotine in the racemic (R,S)-nornicotine to generate myosmine; (2) taking the myosmine generated in the step (1) as raw material, and reducing the myosmine into (R,S)-nornicotine through a reducing agent; (R)-nornicotine in the racemic (R,S)-nornicotine is continuously consumed through the above steps, so that high-purity (S)-nornicotine is obtained. The application converts the racemic (R,S)-nornicotine into high-concentration (S)-nornicotine through the combined action of the immobilized enzyme and the reducing agent, is a process route for obtaining high-concentration and high-purity chiral (S)-nornicotine with low cost, and has high industrial application value and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, in particular to a synthesis method of high-purity (S)-nornicotine. BACKGROUND

[0002] Alkaloids are an important class of chemical components in tobacco plants, and nicotine accounts for more than 95% of total alkaloids in tobacco. Nicotine, also known as nicotine (chemical name: 1-methyl-2-(3-pyridyl) pyrrolidine, CAS number: 54-11-5), has a chiral center and exists in chiral enantiomers, i.e. (S)-nicotine and (R)-nicotine, which are important organic intermediates with potential clinical value. (S)-nicotine and its derivatives are effective drugs for treating Parkinson's syndrome, Alzheimer's disease, schizophrenia, epilepsy and depression. In agriculture, nicotine is used as an insecticide and herbicide, which is a highly effective and low-toxic broad-spectrum pesticide. In addition, (S)-nicotine can be used as a raw material for health cigarettes, smoking cessation paste, external medicine for treating joint pain and muscle spasm. Common administration methods include chewable tablets, creams, transdermal patches, tablets, nasal sprays and electronic cigarettes. In recent years, the electronic cigarette industry has developed rapidly, and the market demand is very large. As an important active ingredient of electronic cigarettes, the market demand for (S)-nicotine has also increased sharply.

[0003] Currently, (S)-nicotine used in the market is mainly extracted and purified from plants such as tobacco, which is affected by factors such as raw materials, climate, and period. In addition, (S)-nicotine extracted and purified from plants such as tobacco usually contains many impurities that are unhealthy and potentially carcinogenic to the human system. In contrast, racemic nicotine, i.e. (R,S)-nicotine, obtained by artificial synthesis from chemical raw materials, does not contain impurities present in (S)-nicotine purified from nature, and has irreplaceable advantages such as high purity, low cost, large yield, and wide application. However, the prepared (R,S)-nicotine still needs to be further separated and the non-target product (R)-nicotine needs to be destroyed to obtain high-purity (S)-nicotine, which requires expensive reagents, complicated steps, and long period. Low-temperature reactions are often used, and each step of separation and purification is complex and difficult to handle. Therefore, the cost is high, large-scale production is difficult, and the product price is high.

[0004] The chemical synthesis methods for preparing (S)-nicotine reported so far include chemical resolution, asymmetric hydrogenation, chiral auxiliary reagent method, etc. For example, Chinese patent CN107406411A discloses a synthesis method of (R,S)-nicotine, which is prepared by using nicotinic acid ester and N-vinyl-2-pyrrolidone as starting materials, and then through base-catalyzed Claisen condensation, acidic decarboxylation and ring closure to obtain intermediate mesamine (CAS No.: 532-12-7), and then mesamine is subjected to palladium-carbon hydrogenation reduction and Eschweiler-Clarke methylation to obtain racemic mixture (R,S)-nicotine. This process is the mainstream process for artificial synthesis of nicotine at present, but it has a major drawback that the prepared product (R,S)-nicotine is a mixture of (S)-nicotine and (R)-nicotine two chiral enantiomers, which is essentially different from natural (S)-nicotine, and has a large difference in physicochemical properties, drug efficacy and experience of electronic cigarette. (S)-nicotine can also be obtained by synthesis method, and the existing technology for synthesizing (S)-nicotine usually prepares racemic mixture of (R)-nicotine and (S)-nicotine and then separates to obtain. For example, patent CN111511726A reports the synthesis of racemic nicotine, and chiral separation is carried out by using L-DBTA to obtain (S)-nicotine; but this method needs to separate the mixture, and the process is complex, the reaction conditions are harsh, and the yield of the obtained product is low. Therefore, it is of high industrial application value and economic benefit to develop a process route for obtaining high-purity chiral (S)-nicotine.

[0005] There is also a method for biosynthesis of (S)-nicotine by using enzymes as biocatalysts to catalytically reduce mesmene in a biological enzyme system to obtain intermediate (S)-nornicotine (CAS No.: 494-97-3) with high optical purity, and then perform aminomethylation reaction to obtain (S)-nicotine. Patent CN112409327A reports a preparation method of high optical purity nicotine. The method first prepares mesmene from nicotinic acid ester and vinyl pyrrolidone, catalytically reduces mesmene in a biological enzyme system to obtain intermediate (S)-nornicotine with high optical purity, and then performs aminomethylation reaction to obtain (S)-nicotine. However, this method has the problems of complex and multiple types of enzymes used, and the enzymes cannot be reused after use. Patent CN113272289A discloses reducing mesmene to (S)-nornicotine using imine reductase, and methylating (S)-nornicotine to form (S)-nicotine. However, this method also has the problems of needing to prepare two types of enzymes and the enzymes cannot be reused. Patent WO2020098978 A1 discloses using imine reductase to reduce 3-(1-pyrrolin-2-yl)pyridine, and using glucose dehydrogenase / glucose as a coenzyme regeneration system to perform catalytic reaction of (S)-nicotine. When the concentration is 400 mM, the conversion rate is 99.6% in 24 h, and the ee value is 99.8%. The optimal imine reductase has a conversion rate of only 52.4% in 24 h and an ee value of 99.6% when the substrate concentration is 1 M. It is not resistant to high-concentration substrate 3-(1-pyrrolin-2-yl)pyridine, which is not conducive to scale-up production, and the production cost is also high.

[0006] To develop a process route for obtaining high-concentration, high-purity chiral (S)-nornicotine at a relatively low cost, which has high industrial application value and economic benefits. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a synthesis method of high-purity (S)-nornicotine, which converts racemic (R,S)-nornicotine into high-concentration (S)-chiral nornicotine through the combined action of immobilized enzyme and reducing agent.

[0008] To solve the above technical problems, the present application provides a synthesis method of high-purity (S)-nornicotine, which comprises the following steps:

[0009] (1) using racemic (R,S)-nornicotine as raw material, and using immobilized enzyme to catalyze (R)-nornicotine to generate mesmene;

[0010] (2) using mesmene generated in step (1) as raw material, and using a reducing agent to reduce mesmene to generate racemic (R,S)-nornicotine;

[0011] Through continuous circulation of the above steps, (R)-nornicotine in the reaction system is consumed to obtain high-purity (S)-nornicotine.

[0012] Specifically, the immobilized enzyme is a high (R) selective monoamine oxidase covalently bound with a hydrogen peroxidase and a resin.

[0013] Specifically, the reducing agent is one or a mixture of two or more of sodium borohydride, sodium cyanoborohydride, sodium acetyloxyborohydride or lithium aluminum hydride.

[0014] Specifically, the resin is an epoxy resin, and the epoxy resin is any one of resin LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 of Xi'an Lanxiao, and resin ES-1, ES-107, ES-108, ES-103B of Tianjin Nankai Hengcheng.

[0015] Specifically, the high (R) selective monoamine oxidase is selected from the following group:

[0016] (1) the amino acid sequence shown in SEQ ID NO. 1; and

[0017] (2) an amino acid sequence obtained by substitution, deletion, modification, insertion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1 within the range of maintaining the enzyme activity.

[0018] Specifically, the high (R) selective monoamine oxidase is selected from the following group:

[0019] (3) the amino acid sequence shown in SEQ ID NO. 2; and

[0020] (4) an amino acid sequence obtained by substitution, deletion, modification, insertion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO. 2 within the range of maintaining the enzyme activity.

[0021] Specifically, the high (R) selective monoamine oxidase is selected from the following group:

[0022] (a) the nucleotide sequence shown in SEQ ID NO. 3;

[0023] (b) a polynucleotide complementary to the sequence defined in (a); or

[0024] (c) any polynucleotide or complementary sequence having at least 70%, or 90% or more sequence identity to the sequence defined in (a).

[0025] Specifically, the high (R) selective monoamine oxidase is selected from the following group:

[0026] (d) a nucleotide sequence as shown in SEQ ID NO. 4;

[0027] (e) a polynucleotide complementary to the sequence defined in (d); or

[0028] (f) any polynucleotide or complementary sequence having at least 70%, or 90% sequence identity to the sequence defined in (d).

[0029] Specifically, the preparation method of the immobilized enzyme is that 500-2000 ml of the enzyme solution of the high (R) selective monoamine oxidase, 100-500 ml of the enzyme solution of the catalase, 120-500 g of K2HPO4·3H2O and 12-50 g of KH2PO4 are added, stirred and dissolved, 50-200 g of epoxy resin is added, stirred at 25°C and 180 rpm for 24 h, filtered, washed with deionized water for three times, then 500 ml of PBS buffer (200 mM, pH 8.0, containing 0.5 M NaCl) is added, stirred at 150 rpm for 1 h, filtered, then washed with 500 ml of PBS buffer (200 mM, pH 8.0) for three times, and the immobilized enzyme is obtained by filtration.

[0030] Specifically, the enzyme activity of the immobilized enzyme is 200-500 U / g.

[0031] Specifically, the mass molar ratio (M / mol) of the immobilized enzyme to the racemic (R,S)-nornicotine is (100-150):1.

[0032] Specifically, the molar ratio of the reducing agent to the racemic (R,S)-nornicotine is (0.5-2):1.

[0033] Specifically, the reaction temperature of the reaction system is 15-40°C.

[0034] Specifically, the reaction time of the reaction system is 16-48 hours.

[0035] Specifically, the reaction system further comprises using PBS buffer (200 mM, pH 7.0) to maintain the reaction pH at pH 6.8-7.2.

[0036] Specifically, the resin is any one of LXTE-602, LXTE-604, LXTE-606 and ES-108.

[0037] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a method for converting racemic (R,S)-nornicotine into high-concentration (S)-nornicotine by using the combined action of immobilized enzyme with high (R) selectivity, high activity and high stability and reducing agent. The immobilized enzyme does not catalyze (S)-nornicotine but only (R)-nornicotine, and by continuously consuming (R)-nornicotine in racemic (R,S)-nornicotine, high-purity (S)-nornicotine is obtained. Under the condition of high substrate concentration (2 mol / L), the conversion rate is greater than 90.2%, and the ee value is greater than 99.1%. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the present application, the drawings used in the present application will be briefly introduced as follows. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0039] Figure 1 is a route map for producing high-purity (S)-nornicotine from racemic (R,S)-nornicotine;

[0040] Figure 2 is a SDS-PAGE map of high (R) selectivity monoamine oxidase E1, high (R) selectivity monoamine oxidase E2 and catalase E3. DETAILED DESCRIPTION

[0041] The technical solutions in the present application will be described clearly and completely below in combination with the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0042] Example 1.

[0043] In the present application, two high (R) selective monoamine oxidase (E1 is derived from Arthrobacter nicotinovorans, the amino acid sequence is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 3; E2 is derived from Erythrobacteraceae bacterium, the amino acid sequence is shown as SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO. 4), catalase E3 (Gene ID: WP_144458738.1), the genes of the three enzymes are obtained by gene synthesis, and each gene is connected to the vector pET28a. The strain is cultured on a plate, and single colonies are selected for step-by-step culture. First, transfer 3ml of LB culture solution containing 50μM kanamycin (37℃, 220rpm) overnight culture, inoculate 1% into 100ml of LB culture solution containing 50μM kanamycin, and culture for 4-8h, and finally transfer into a 10L fermenter for culture; when the cell OD reaches about 25, cool to 30℃, add 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG), continue to culture for 18h to induce the expression of the target protein, take the fermentation liquor and centrifuge at high speed (6000rpm, 30min) to collect the cells to obtain wet bacteria 600-1000g, and store at-20℃ for use. Take 10g of wet bacteria and suspend in 100ml of PBS buffer (200mM, pH 8.0) and mix evenly, perform cell disruption by high-pressure homogenization, centrifuge at 12000rpm for 20min, collect the supernatant to obtain three enzyme liquids.

[0044] Take 40ul of E1, E2 and E3 enzyme liquids respectively, add 10ul of 5* loading dye, treat at 100℃ for 10min, centrifuge at 12000rpm for 30s, take 10ul for SDS-PAGE to verify the expression of the protein and the size of the amount, the results are shown as Figure 2 The molecular weight of E1 and E2 proteins is about 49kDa, and the molecular weight of E3 protein is about 57kDa, which meets the expectation.

[0045] Example 2.

[0046] Two kinds of high (R) selective monoamine oxidase enzyme solution (1000 ml) obtained from Example 1 were mixed with catalase enzyme solution (300 ml) respectively to obtain two kinds of mixed enzyme solution. 250 g of K2HPO4·3H2O and 25 g of KH2PO4 were added to each mixed enzyme solution, stirred and dissolved, then 100 g of epoxy resin carrier (resin ES-108 from Tianjin Nankai Hengcheng) was added, stirred at 25°C and 180 rpm for 24 h, suction filtered, washed with deionized water three times, then 500 ml of PBS buffer (200 mM, pH 8.0 containing 0.5 M NaCl) was added, stirred at 150 rpm for 1 h, filtered, then washed with 500 ml of PBS buffer (200 mM, pH 8.0) three times, suction filtered, and two kinds of immobilized enzymes were obtained. First, select the one with higher activity from the two kinds of immobilized enzymes, then select different types of resin for immobilization, follow the same steps as above to select the resin with higher immobilized enzyme activity.

[0047] The method for detecting the enzyme activity of the immobilized enzyme is as follows:

[0048] The enzyme activity of the immobilized enzyme was detected using racemic (R, S)-norsicane as the substrate. The enzyme activity detection method was as follows: 5 mL of 0.5 mol / L racemic (R, S)-norsicane was dissolved in PBS buffer (200 mM, pH 7.0) and diluted to 50 mL, incubated at 30°C on a 200 rpm shaker for 20 min, 1.0 g of immobilized enzyme was added to the reaction system, reacted at 30°C on a 200 rpm shaker for 20 min, stood for 30 s, 2 ml of supernatant was taken, centrifuged at 12000 rpm for 3 min, HPLC detection, the concentration of the enzyme activity reaction product mesicine was calculated according to the standard curve of the standard mesicine concentration, and the immobilized enzyme activity was calculated.

[0049] The enzyme activities of the immobilized enzymes prepared from two kinds of high (R) selective monoamine oxidase and different types of resin were detected, and the detection results are shown in Table 1 and Table 2:

[0050] Table 1. Immobilized enzyme activity prepared from two kinds of high (R) selective monoamine oxidase

[0051]

[0052] Table 2. Immobilized enzyme activity prepared from different types of resin

[0053]

[0054]

[0055] Note: Resins LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 are from Xi'an Lanxiao Science and Technology New Material Co., Ltd.; Resins ES-1, ES-107, ES-108, ES-103B are from Tianjin Nankai Hecheng Technology Co., Ltd.

[0056] Example 3.

[0057] In this example, the immobilized enzyme with higher activity prepared in Example 2 is coupled with reducing agent sodium borohydride to catalyze the production of (S)-nornicotine from racemic (R,S)-nornicotine.

[0058] To 500 mL of 0.5 mol / L racemic (R,S)-nornicotine solution, 16 g of K2HPO4·3H2O and 5 g of KH2PO4 were added and stirred uniformly, 30 g of immobilized enzyme (LXTE-604 resin and ES-108 resin were used to prepare immobilized enzyme) was added, and then sodium borohydride (total 5.5 g, 0.145 mol) was added in batches. The reaction temperature was 30°C, the stirring speed was 200 rpm, and the reaction was carried out for 30 h. After that, the conversion rate and the ee value of the product were detected by HPLC. The reaction solution was collected by suction filtration, 50 ml of PBS buffer (200 mM, pH 7.0) was added, and the immobilized enzyme was washed by starting stirring. The washing solution was collected by suction filtration, and the same operation was repeated twice. The two washing solutions collected and the reaction solution collected before were mixed for next post-treatment, and the immobilized enzyme recovered was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0059] The results are shown in Table 3 below. Under the condition of 0.5 mol / L substrate concentration, the conversion rate of 20 batches of repeated experiments of two kinds of immobilized enzyme was greater than 97%, and the optical purity (ee value) of (S)-nornicotine was greater than 99.1%. After the reaction was completed, the immobilized enzyme could be repeatedly used.

[0060] Table 3. Conversion rate of 20 batches of repeated experiments of two kinds of immobilized enzyme under the condition of 0.5 mol / L substrate concentration

[0061]

[0062]

[0063] Example 4.

[0064] In this example, the immobilized enzyme with higher activity prepared in Example 2 (LXTE-604 resin was used to prepare immobilized enzyme) is coupled with reducing agent sodium borohydride to catalyze the production of (S)-nornicotine from racemic (R,S)-nornicotine.

[0065] To 500 mL of 1 mol / L solution of racemic (R,S)-nicotine, 16 g of K2HPO4·3H2O and 5 g of KH2PO4 were added, stirred uniformly, 75 g of immobilized enzyme (LXTE-604 resin immobilized enzyme) was added, and then sodium borohydride (a total of 14 g, 0.37 mol) was added in batches. The reaction temperature was 30°C, the stirring speed was 200 rpm, and after 30 h of reaction, the conversion rate and the ee value of the product were detected by HPLC. The reaction solution was collected by suction filtration, 80 ml of PBS buffer (200 mM, pH 7.0) was added, the stirring was started to wash the immobilized enzyme, and then the washing solution was collected by suction filtration. The same operation was repeated twice, and the two washing solutions collected and the reaction solution collected previously were mixed for the next post-treatment. The recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0066] The results are shown in Table 4 below. After 20 batches of repeated experiments of the immobilized enzyme prepared by LXTE-604 resin under the condition of 1 mol / L substrate concentration, the conversion rate was greater than 96.8%, and the optical purity (ee value) of (S)-nicotine was greater than 99.1%. The immobilized enzyme could be reused after the reaction was completed.

[0067] Table 4. Conversion rate after 20 batches of repeated experiments of the immobilized enzyme prepared by LXTE-604 resin under the condition of 1 mol / L substrate concentration

[0068] Batch Reaction time / h Conversion / % ee value / % 1 30 99.4 99.5 20 30 96.8 99.1

[0069] Example 5.

[0070] In this example, the immobilized enzyme with higher activity prepared in Example 2 (LXTE-604 resin immobilized enzyme) was coupled with the reducing agent sodium borohydride to catalyze the preparation of (S)-nicotine from racemic (R,S)-nicotine.

[0071] To 500 mL of 2 mol / L solution of racemic (R,S)-nicotine, 16 g of K2HPO4·3H2O and 5 g of KH2PO4 were added, stirred uniformly, 100 g of immobilized enzyme (LXTE-604 resin immobilized enzyme) was added, and then sodium borohydride (a total of 71.7 g, 1.895 mol) was added in batches. The reaction temperature was 30°C, the stirring speed was 200 rpm, and after 30 h of reaction, the conversion rate and the ee value of the product were detected by HPLC. The reaction solution was collected by suction filtration, 100 ml of PBS buffer (200 mM, pH 7.0) was added, the stirring was started to wash the immobilized enzyme, and then the washing solution was collected by suction filtration. The same operation was repeated twice, and the two washing solutions collected and the reaction solution collected previously were mixed for the next post-treatment. The recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0072] The results are shown in Table 5 below. After 20 batches of repeated experiments, the conversion rate of the immobilized enzyme prepared by the LXTE-604 resin was greater than 90.2% and the optical purity (ee value) of (S)-nornicotine was greater than 99.1% under the condition of a 2 mol / L substrate concentration. The immobilized enzyme could be reused after the reaction.

[0073] Table 5. Conversion rate after 20 batches of repeated experiments of the immobilized enzyme prepared by the LXTE-604 resin under the condition of a 2 mol / L substrate concentration

[0074] Batch Reaction time / h Conversion / % ee value / % 1 30 98.6 99.4 20 30 90.2 99.1

[0075] Example 6.

[0076] In this example, the immobilized enzyme (prepared by the ES-108 resin) with higher activity prepared in Example 2 was coupled with the reducing agent sodium borohydride to catalyze the production of (S)-nornicotine from racemic (R,S)-nornicotine.

[0077] To a 500 mL solution of 0.5 mol / L racemic (R,S)-nornicotine, 16 g of K2HPO4·3H2O and 5 g of KH2PO4 were added and stirred uniformly. Then, 30 g of the immobilized enzyme (prepared by the ES-108 resin) was added, followed by the addition of sodium borohydride (a total of 5.5 g, 0.145 mol) in batches. The reaction was carried out at a temperature of 30°C and a stirring speed of 200 rpm for 30 h. After the reaction, the conversion rate and the ee value of the product were detected by HPLC. The reaction solution was collected by suction filtration. Then, 50 mL of PBS buffer (200 mM, pH 7.0) was added, and the immobilized enzyme was washed by stirring. The washing solution was collected by suction filtration. The same operation was repeated twice, and the two washing solutions and the previously collected reaction solution were mixed for subsequent treatment. The recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0078] The results are shown in Table 6 below. After 20 batches of repeated experiments, the conversion rate of the immobilized enzyme prepared by the ES-108 resin was greater than 95.7% and the optical purity (ee) of (S)-nornicotine was greater than 97.9% under the condition of a 0.5 mol / L substrate concentration. The immobilized enzyme could be reused after the reaction.

[0079] Table 6. Conversion rate after 20 batches of repeated experiments of the immobilized enzyme prepared by the ES-108 resin under the condition of a 0.5 mol / L substrate concentration

[0080] Batch Reaction time / h Conversion / % ee value / % 1 30 99.6 99.1 20 30 95.7 97.9

[0081] Example 7.

[0082] The immobilized enzyme (ES-108 resin prepared immobilized enzyme) with higher activity prepared in Example 2 was coupled with reducing agent sodium borohydride to catalyze the production of (S)-nornicotine from racemic (R,S)-nornicotine.

[0083] To 500 mL of 1 mol / L solution of racemic (R,S)-nornicotine, 16 g of K2HPO4·3H2O and 5 g of KH2PO4 were added and stirred uniformly, 75 g of immobilized enzyme (ES-108 resin prepared immobilized enzyme) was added, and sodium borohydride (total 14 g, 0.37 mol) was added in batches. The reaction temperature was 30°C, the stirring speed was 200 rpm, and the reaction was carried out for 30 h. After the reaction, the conversion rate and the ee value of the product were detected by HPLC. The reaction solution was collected by suction filtration, 80 mL of PBS buffer (200 mM, pH 7.0) was added, and the immobilized enzyme was washed by stirring. The washing solution was collected by suction filtration, and the same operation was repeated twice. The two washing solutions collected and the reaction solution collected before were mixed for the next post-treatment, and the recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0084] The results are shown in Table 7 below. Under the condition of 1 mol / L substrate concentration, the conversion rate of the immobilized enzyme prepared by ES-108 resin after 20 batches of repeated experiments was greater than 93.5%, and the optical purity (ee) of (S)-nornicotine was greater than 96.3%. The immobilized enzyme can be reused after the reaction is completed.

[0085] Table 7. Conversion rate of immobilized enzyme prepared by ES-108 resin after 20 batches of repeated experiments under the condition of 1 mol / L substrate concentration

[0086] Batch Reaction time / h Conversion / % ee value / % Batch Reaction time / h Conversion / % ee value / % 1 30 95.6 98.2 20 30 93.5 96.3

[0087] Example 8.

[0088] The immobilized enzyme (ES-108 resin prepared immobilized enzyme) with higher activity prepared in Example 2 was coupled with reducing agent sodium borohydride to catalyze the production of (S)-nornicotine from racemic (R,S)-nornicotine.

[0089] To 500 mL of 2 mol / L of racemic (R,S)-Nornicotine solution, 16 g of K2HPO4·3H2O and 5 g of KH2PO4 were added, stirred uniformly, 100 g of immobilized enzyme (ES-108 resin prepared immobilized enzyme) was added, and then sodium borohydride (a total of 71.7 g, 1.895 mol) was added in batches. The reaction temperature was 30°C, the stirring speed was 200 rpm, and after 30 h of reaction, the conversion rate and product ee value were detected by HPLC. The reaction solution was collected by suction filtration, 100 ml of PBS buffer (200 mM, pH 7.0) was added, and the immobilized enzyme was washed by starting stirring. The washing solution was collected by suction filtration, and the same operation was repeated twice. The two washing solutions collected and the reaction solution collected before were mixed for the next post-treatment, and the recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0090] The results are as shown in Table 8. After 20 batches of repeated experiments of ES-108 resin prepared immobilized enzyme under the condition of 2 mol / L substrate concentration, the conversion rate was greater than 88.9%, and the optical purity (ee) of (S)-Nornicotine was greater than 96.1%. The immobilized enzyme can be reused after the reaction is completed.

[0091] Table 8. Conversion rate of ES-108 resin prepared immobilized enzyme after 20 batches of repeated experiments under the condition of 2 mol / L substrate concentration

[0092]

[0093]

[0094] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of protection of the present application.

Claims

1. A method of synthesizing high purity (S)-nornicotine, characterized by: It comprises the following steps: (1) using racemic (R, S)-norsalol as raw material, (R)-norsalol in it is catalyzed to form mesamide by immobilized enzyme; (2) using mesamide generated in step (1) as raw material, it is reduced by reducing agent to form racemic (R, S)-norsalol; Through the continuous circulation of the above steps, (R)-norsalol in the reaction system is consumed to obtain high-purity (S)-norsalol; The immobilized enzyme is high (R) selectivity monoamine oxidase, hydrogen peroxidase and resin covalent combination; The high (R) selectivity monoamine oxidase is selected from the amino acid sequence shown in SEQ ID NO. 1 or the amino acid sequence shown in SEQ ID NO.

2.

2. The method of synthesis of claim 1, wherein, The reducing agent is one or a mixture of two or more of sodium borohydride, sodium cyanoborohydride, sodium acetoxyborohydride or lithium aluminum hydride.

3. The method of synthesis of claim 2, wherein, The resin is an epoxy resin, and the model of the epoxy resin is any one of resin LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 of Xi'an Lanxiao and resin ES-1, ES-107, ES-108, ES-103B of Tianjin Nankai Hengcheng.

4. The method of synthesis of claim 1, wherein, The nucleotide sequence of the high (R) selectivity monoamine oxidase is shown in SEQ ID NO.

3.

5. The method of synthesis of claim 1, wherein, The nucleotide sequence of the high (R) selectivity monoamine oxidase is shown in SEQ ID NO.

4.

6. The method of synthesis according to claim 1 or 4 or 5, wherein, The preparation method of the immobilized enzyme is that 500-2000 ml of enzyme solution of the high (R) selectivity monoamine oxidase, 100-500 ml of enzyme solution of the hydrogen peroxidase, 120-500 g of K2HPO4·3H2O and 12-50 g of KH2PO4 are added, stirred and dissolved, 50-200 g of epoxy resin is added at 25°C, 180 rpm stirring adsorption is carried out for 24 h, suction filtration is carried out, deionized water is washed for three times, 500 ml of PBS buffer solution with a concentration of 200 mM and pH 8.0 is used for flushing three times, 150 rpm stirring is carried out for 1 h, filtration is carried out, then 500 ml of PBS buffer solution with a concentration of 200 mM and pH 8.0 is used for flushing three times, and suction filtration is carried out to obtain the immobilized enzyme.

7. The method of synthesis of claim 6, wherein, The enzyme activity of the immobilized enzyme is 200-500 U / g.

8. The method of synthesis of claim 1, wherein, The mass molar ratio (M / mol) of the immobilized enzyme to the racemic (R, S)-norsalol is (100-150):

1.

9. The method of synthesis of claim 1, wherein, The molar ratio of the reducing agent to the racemic (R, S)-norsalol is (0.5-2):

1.

10. The method of synthesis of claim 1, wherein, The reaction temperature of the reaction system is 15-40°C.

11. The method of synthesis of claim 1, wherein, The reaction time of the reaction system is 16-48 hours.

12. The method of synthesis of claim 1, wherein, The reaction system further comprises using PBS buffer solution with a concentration of 200 mM and pH 7.0 to maintain the reaction pH at pH 6.8-7.

2.

13. The method of synthesis of claim 3, wherein, The resin is any one of LXTE-602, LXTE-604, LXTE-606, and ES-108. The resin is any one of LXTE-602, LXTE-604, LXTE-606, and ES-108.

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