A method for synthesizing high purity (s)-nornicotine catalyzed by immobilized enzyme

Abstract

The application discloses a kind of synthesis method of high-purity (S) -nicotine which is catalyzed by immobilized enzyme, it includes the following steps: (1) with racemic (R, S) -nicotine as raw material, (R) -myersmine is generated by using immobilized enzyme catalysis in it;(2) with the myersmine generated in step (1) as raw material, (R, S) -nicotine is reduced into by using immobilized enzyme catalysis;(R) -nicotine in racemic (R, S) -nicotine is continuously consumed by the above steps to obtain high-purity (S) -nicotine.The application generates high-concentration (S) -nicotine by immobilized enzyme catalytic conversion racemic (R, S) -nicotine, it is a kind of process route to obtain high-concentration, high-purity chiral (S) -nicotine and relatively low cost, with higher industrial application value and economic benefits.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to a method for synthesizing high-purity (S)-nornicotinic acid using immobilized enzyme catalysis. Background Technology

[0002] Alkaloids are an important class of chemical components found in tobacco plants, with nicotine accounting for over 95% of the total alkaloids. Nicotine (chemical name: 1-methyl-2-(3-pyridyl)pyrrolidine, CAS number: 54-11-5) has a chiral center and exists as chiral enantiomers, namely levorotatory (S)-nicotine and dextrorotatory (R)-nicotine, and is an organic intermediate with significant clinical potential. (S)-nicotine and its derivatives are effective drugs for treating Parkinson's disease, Alzheimer's disease, schizophrenia, epilepsy, and as antidepressants. In agriculture, nicotine is used as an insecticide and herbicide, and is a highly effective, low-toxicity, broad-spectrum pesticide. Furthermore, (S)-nicotine can be used as a raw material in health cigarettes, smoking cessation pastes, and topical medications for treating joint pain and muscle spasms. Common application methods include chewing tablets, creams, transdermal patches, tablets, nasal sprays, and e-cigarettes. In recent years, the e-cigarette industry has developed rapidly, with huge market demand. As an important active ingredient in e-cigarettes, (S)-nicotine has also seen a surge in market demand.

[0003] Currently, the (S)-nicotine used in the market is mainly extracted and purified from plants such as tobacco, and is therefore affected by factors such as raw materials, climate, and production cycle. Furthermore, (S)-nicotine extracted and purified from plants such as tobacco often contains many impurities that are unhealthy for the human body and potentially carcinogenic. In contrast, racemic nicotine, i.e., (R,S)-nicotine, obtained through targeted artificial synthesis from chemical raw materials, does not contain the impurities present in naturally purified (S)-nicotine. It has irreplaceable advantages such as high purity, low cost, large yield, and wide application. However, the reagents used are expensive, the steps are cumbersome, and the cycle is long; it often employs low-temperature reactions, and each step of separation and purification is complex and difficult to process. At the same time, the prepared (R,S)-nicotine needs to be further separated and destroyed to obtain high-purity (S)-nicotine, resulting in high cost, difficulty in large-scale production, and high product price.

[0004] Currently reported chemical synthesis methods for (S)-nicotine include chemical resolution, asymmetric hydrogenation, and chiral auxiliary reagent methods. For example, Chinese patent CN107406411A discloses a method for synthesizing (R,S)-nicotine. The reaction uses nicotinic acid esters and N-vinyl-2-pyrrolidone as starting materials, undergoing alkaline-catalyzed Claysen condensation and acidic decarboxylation and cyclization to obtain the intermediate mesmin (CAS No.: 532-12-7). Mesmin is then subjected to palladium-on-carbon hydrogenation reduction and Eschweiler–Clarke methylation to obtain a racemic mixture of (R,S)-nicotine. This process is currently the mainstream method for the artificial synthesis of nicotine. However, this process has a significant drawback: the prepared product (R,S)-nicotine is a mixture of two chiral enantiomers, (S)-nicotine and (R)-nicotine, which is fundamentally different from natural (S)-nicotine. This results in significant differences in its physicochemical properties, efficacy, and the user experience of e-cigarettes. (S)-nicotine can also be obtained synthetically. Current techniques for synthesizing (S)-nicotine typically involve preparing a racemic mixture of (R)-nicotine and (S)-nicotine, followed by resolution. For example, patent CN111511726A reports the synthesis of racemic nicotine and utilizes L-DBTA for chiral resolution to obtain (S)-nicotine; however, this method requires resolution of the mixture, is complex, has stringent reaction conditions, and results in low product yields. Therefore, developing a process route to obtain high-purity chiral (S)-nicotine has significant industrial application value and economic benefits.

[0005] Currently, there are also biosynthetic methods that utilize enzymes as biocatalysts to catalytically reduce mesmin in a bio-enzymatic system to obtain the intermediate -(S)-nornicotinine (CAS No.: 494-97-3) with high optical purity, and finally undergo aminomethylation to obtain (S)-nicotinine. For example, patent CN112409327A reports a method for preparing high optical purity nicotinine. This method first prepares mesmin using nicotinic acid ester and vinylpyrrolidone, then catalytically reduces mesmin in a bio-enzymatic system to obtain the intermediate -(S)-nornicotinine with high optical purity, and finally undergoes aminomethylation to obtain (S)-nicotinine. However, this method has the problems of using complex and multiple types of enzymes, and the enzymes cannot be reused after use. Patent CN113272289A discloses the reduction of mesmin to (S)-nornicotinine using imine reductase, followed by methylation of (S)-nornicotinine to form (S)-nicotinine. However, this method also has the problem of requiring the preparation of two enzymes and the enzymes cannot be reused. Patent WO2020098978 A1 discloses the use of imine reductase to reduce 3-(1-pyrrololin-2-yl)pyridine, using glucose dehydrogenase / glucose as a coenzyme regeneration system for the catalytic reaction of (S)-nicotine. At a concentration of 400 mM, the conversion rate reaches 99.6% in 24 hours, with an ee value of 99.8%. The optimal imine reductase, at a substrate concentration of 1 M, achieves only a 52.4% conversion rate in 24 hours, with an ee value of 99.6%. However, it is intolerant to high concentrations of the substrate 3-(1-pyrrololin-2-yl)pyridine, which is unfavorable for scale-up production and results in higher production costs.

[0006] Therefore, developing a process route to obtain high concentration and high purity chiral (S)-nornicotine at a low cost has high industrial application value and economic benefits. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for synthesizing high-purity (S) nicotine by immobilized enzyme catalysis, which converts racemic (R,S)-nicotine into high-concentration (S)-chiral nicotine by immobilized enzyme catalysis.

[0008] To address the aforementioned technical problems, this invention provides a method for synthesizing high-purity (S)-nornicotine using immobilized enzyme catalysis, comprising the following steps: (1) Using racemic (R,S)-nornicotinine as raw material, immobilized enzymes are used to catalyze the (R)-nornicotinine to generate mesmin; (2) Using the mesmin generated in step (1) as raw material, the immobilized enzyme catalyzes and reduces it to generate racemic (R,S)-nornicotinic acid; By continuously repeating the above steps, (R)-nornicotinic acid in the reaction system is consumed to obtain high-purity (S)-nornicotinic acid.

[0009] Specifically, the immobilized enzyme is obtained by covalently binding a highly (R) selective monoamine oxidase, an imine reductase, a glucose dehydrogenase, a catalase, and a resin.

[0010] Specifically, the resin is an epoxy resin, and the epoxy resin model is any one of the following: Xi'an Lanxiao's resins LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 and Tianjin Nankai Hecheng's resins ES-1, ES-107, ES-108, ES-103B.

[0011] Specifically, the highly (R)-selective monoamine oxidase is selected from the group consisting of: (1) The amino acid sequence as shown in SEQ ID NO.1; and (2) The amino acid sequence obtained by replacing, deleting, altering, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 within the range of maintaining enzyme activity.

[0012] Specifically, the imine reductase is selected from the following group: (3) The amino acid sequence as shown in SEQ ID NO.2; and (4) The amino acid sequence obtained by replacing, deleting, altering, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 within the range of maintaining enzyme activity.

[0013] Specifically, the highly (R)-selective monoamine oxidase is selected from the group consisting of: (a) A nucleotide sequence as shown in SEQ ID NO.3; (b) A polynucleotide complementary to the sequence defined in (a); or (c) Any polynucleotide or complementary sequence that has at least 70% or more 90% sequence identity with the sequence defined in (a).

[0014] Specifically, the imine reductase is selected from the following group: (d) The nucleotide sequence shown in SEQ ID NO.4; (e) A polynucleotide complementary to the sequence defined by (d); or (f) and (d) define any polynucleotide or complementary sequence having at least 70% or more 90% sequence identity.

[0015] Specifically, the immobilized enzyme is prepared by adding 140-600g of K2HPO4•3H2O and 14-60g of KH2PO4 to 500-2000ml of the enzyme solution of the highly (R)-selective monoamine oxidase, 200-500ml of the enzyme solution of the imine reductase, 100-300ml of the enzyme solution of the glucose dehydrogenase, and 100-300ml of the enzyme solution of the catalase. The mixture is stirred to dissolve, and then 50-300g of epoxy resin is added. The mixture is stirred at 25°C and 180rpm for 24 hours to adsorb the enzyme. After filtration, the mixture is washed three times with deionized water, and then washed twice with 500ml of PBS buffer (200mM, pH 8.0, containing 0.5M NaCl). The immobilized enzyme is then obtained by filtration.

[0016] Specifically, the enzyme activity range of the immobilized enzyme is 200–400 U / g.

[0017] Specifically, the molar ratio (g / mol) of the immobilized enzyme to the racemic (R,S)-nornicotinic acid is (80-200):1.

[0018] Specifically, the reaction temperature of the reaction system is 15–40°C.

[0019] Specifically, the reaction time of the reaction system is 16 to 48 hours.

[0020] Specifically, the reaction system also includes using PBS buffer (200mM, pH 7.0) to maintain the reaction pH at 6.8–7.2.

[0021] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a method that utilizes an immobilized enzyme with high selectivity and activity to catalyze the conversion of racemic (R,S)-nornicotinine to a high concentration of (S)-nornicotinine. This immobilized enzyme does not catalyze (S)-nornicotinine but only (R)-nornicotinine. Through this invention, high-purity (S)-nornicotinine is obtained by continuously consuming (R)-nornicotinine from the racemic (R,S)-nornicotinine. Attached Figure Description

[0022] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a route diagram for producing high-purity (S) nicotine from racemic (R,S)-nornicotine. Figure 2This is an SDS-PAGE image of highly (R) selective monoamine oxidase E1, imine reductase E2, catalase E3, and glucose dehydrogenase E4. Detailed Implementation

[0024] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] Example 1.

[0026] In this invention, the highly (R)-selective monoamine oxidase E1 is derived from *Arthrobacter nicotinovorans*, with the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.3; imine reductase E2 is derived from *Amycolatopsis orientalis*, with the amino acid sequence shown in SEQ ID NO.2 and the nucleotide sequence shown in SEQ ID NO.4; catalase E3 is derived from *Bacillus pumilus* (Gene ID: WP_144458738.1), and glucose dehydrogenase E4 is derived from *Exiguobacterium sibiricum* (Gene ID: ACB59697.1). The genes for all four enzymes were synthesized, and each gene was ligated into the vector pET28a. The strains were cultured on plates, and finally, single clones were selected for stepwise liquid culture. First, the cells were transferred to 3 ml of LB medium containing 50 μM kanamycin (37°C, 220 rpm) and cultured overnight. Then, 1% of the cells were inoculated into 100 ml of LB medium containing 50 μM kanamycin and cultured for 4–8 hours. Finally, the cells were transferred to a 10 L fermenter for further culture. When the cell OD reached approximately 25, 0.5 mM isopropyl-β-D-thiopyranoside (IPTG) was added, and protein expression was induced at 30°C for 18 hours. The fermentation broth was then centrifuged at high speed (6000 rpm, 30 min) to collect 600–1000 g of wet cells, which were stored at -20°C for later use. 10 g of the wet cells were resuspended in 100 ml of PBS buffer (50 mM, pH 8.0) and mixed thoroughly. The cells were then homogenized using high-pressure homogenization, centrifuged at 12000 rpm for 20 min, and the supernatant was collected to obtain four enzyme solutions.

[0027] Take 40 μL of the supernatant enzyme solution from E1, E2, E3, and E4 respectively, add 10 μL of 5* staining solution, treat at 100°C for 10 min, centrifuge at 12000 rpm for 30 s, and take 10 μL for SDS-PAGE to verify the protein expression and quantity. The results are as follows. Figure 2 As shown, the molecular weights of proteins E1, E2, E3, and E4 are 49 kDa, 30 kDa, 57 kDa, and 29 kDa, respectively, which is in line with expectations.

[0028] Example 2.

[0029] The highly (R)-selective monoamine oxidase enzyme solution (1000 ml, enzyme activity 69.4 U / mL), imine reductase enzyme solution (300 ml, enzyme activity 73.4 U / mL), glucose dehydrogenase (200 ml, enzyme activity 81.4 U / mL), and catalase enzyme solution (150 ml, enzyme activity 82.7 U / mL) obtained in Example 1 were mixed. Then, 250 g of K₂HPO₄•3H₂O and 25 g of KH₂PO₄ were added, and the mixture was stirred to dissolve. 100 g of epoxy resin carrier was added, and the mixture was stirred at 25°C and 180 rpm for 24 h for adsorption. The mixture was then filtered, washed three times with deionized water, and then washed twice with 500 ml of PBS buffer (200 mM, pH 8.0, containing 0.5 M NaCl). The immobilized complex enzyme was obtained by filtering different types of resins to select the resin with the highest immobilized enzyme activity.

[0030] The method for detecting immobilized enzyme activity is as follows: Using racemic (R,S)-nornicotinic acid as a substrate, the activity of the immobilized enzyme was detected. The enzyme activity detection method was as follows: 5 mL of 0.5 mol / L racemic (R,S)-nornicotinic acid and 3 mmol (0.54 g) of glucose were dissolved in PBS buffer (200 mM, pH 7.0) and the volume was adjusted to 50 mL. The mixture was incubated at 30 °C and 200 rpm for 20 min on a shaker. 1.0 g of the immobilized enzyme was weighed and added to the reaction system. The reaction was carried out at 30 °C and 200 rpm for 20 min on a shaker. After standing for 30 s, 2 mL of the supernatant was taken and centrifuged at 12000 rpm for 3 min. The enzyme activity was detected by HPLC. The concentration of the product (S)-nornicotinic acid in the enzyme activity reaction system was calculated according to the standard curve of (S)-nornicotinic acid concentration, and the immobilized enzyme activity was calculated.

[0031] The activities of immobilized enzymes prepared with different types of resins were detected, and the results are shown in Table 1 below: Table 1. Immobilized enzyme activities prepared with different types of resins

[0032] Note: Resins LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, and LXTE-609 are sourced from Xi'an Lanxiao New Material Technology Co., Ltd.; Resins ES-1, ES-107, ES-108, and ES-103B are sourced from Tianjin Nankai Hecheng Technology Co., Ltd.

[0033] Example 3. In this embodiment, the immobilized enzymes with high enzyme activity (amine oxidase, imine reductase, glucose dehydrogenase and catalase, and immobilized enzymes prepared with ES-108 resin) prepared in Example 2 were used to catalyze the conversion of racemic (R,S)-nornicotine to (S)-nornicotine.

[0034] Add 16g K₂HPO₄•3H₂O, 5g KH₂PO₄, 0.3mol (54g) glucose, and 0.01mol (6.8g) sodium nicotinamide adenine dinucleotide to 500mL of 0.5mol / L racemic (R,S)-nornicotinic acid solution. Stir well, then add 30g immobilized enzyme. The reaction temperature is 30℃, the stirring speed is 200rpm, and the pH is adjusted to approximately 7.0 using 2M NaOH. After reacting for 30h, the conversion rate and product ee value are detected by HPLC. The reaction solution is collected by filtration, and 50mL PBS buffer (200mM, pH 7.0) is added. The immobilized enzyme is washed with stirring, filtered, and the washing solution is collected. The washing solution is repeated twice. The two washing solutions are mixed with the previously collected reaction solution for the next post-processing step. The recovered immobilized enzyme is used for the next batch of reaction. The same operation is repeated for 20 batches of immobilized enzyme.

[0035] The results are shown in Table 2 below. Under the condition of 0.5 mol / L substrate concentration, the conversion rate of the immobilized enzyme in 20 repeated experiments was greater than 99%, and the optical purity (ee) of (S)-nornicotinic acid was greater than 99.8%. The immobilized enzyme can be reused after the reaction is completed.

[0036] Table 2. Conversion rates of immobilized enzymes in 20 repeated experiments at a substrate concentration of 0.5 mol / L.

[0037] Example 4. In this embodiment, the immobilized enzymes with high enzyme activity (amine oxidase, imine reductase, glucose dehydrogenase and catalase, and immobilized enzymes prepared with ES-108 resin) prepared in Example 2 were used to catalyze the conversion of racemic (R,S)-nornicotine to (S)-nornicotine.

[0038] Add 16g K₂HPO₄•3H₂O, 5g KH₂PO₄, 0.6mol (10⁸g) glucose, and 0.02mol (13g) sodium nicotinamide adenine dinucleotide to 500mL of 0.5mol / L racemic (R,S)-nornicotinic acid solution. Stir well, then add 60g immobilized enzyme. Adjust the pH to approximately 7.0 using 2M NaOH. Maintain the reaction temperature at 30℃ and the stirring speed at 200rpm for 30h. After reaction, detect the conversion rate and product ee value using HPLC. Filter and collect the reaction solution. Add 70mL PBS buffer (200mM, pH 7.0), start stirring, and wash the immobilized enzyme. Filter and collect the washing solution. Repeat the washing process twice. Mix the collected washing solutions with the previous reaction solution for the next post-processing step. The recovered immobilized enzyme is used for the next batch reaction. Repeat the same procedure for 20 batches of immobilized enzyme.

[0039] The results are shown in Table 3 below. Under the condition of 1 mol / L substrate concentration, the conversion rate of the immobilized enzyme in 20 repeated experiments was greater than 95.1%, and the optical purity (ee value) of (S)-nornicotinic acid was greater than 99.7%. The immobilized enzyme can be reused after the reaction is completed.

[0040] Table 3. Conversion rates of immobilized enzymes in 20 repeated experiments at a substrate concentration of 1 mol / L.

[0041] Example 5. In this embodiment, the immobilized enzymes with high enzyme activity (amine oxidase, imine reductase, glucose dehydrogenase and catalase, and immobilized enzymes prepared with ES-108 resin) prepared in Example 2 were used to catalyze the conversion of racemic (R,S)-nornicotine to (S)-nornicotine.

[0042] Add 16g K₂HPO₄•3H₂O, 5g KH₂PO₄, 1.2mol (216g) glucose, and 0.04mol (27g) sodium nicotinamide adenine dinucleotide to 500mL of 0.5mol / L racemic (R,S)-nornicotinic acid solution. Stir well, then add 100g immobilized enzyme. Adjust the pH to approximately 7.0 using 2M NaOH. Maintain the reaction temperature at 30℃ and the stirring speed at 200rpm for 30h. After reaction, detect the conversion rate and product ee value using HPLC. Filter and collect the reaction solution. Add 100mL PBS buffer (200mM, pH 7.0), start stirring, and wash the immobilized enzyme. Filter and collect the washing solution. Repeat the washing process twice. Mix the two washing solutions with the previously collected reaction solution for the next post-processing step. The recovered immobilized enzyme is used for the next batch reaction. Repeat the same procedure for 20 batches of immobilized enzyme.

[0043] The results are shown in Table 4 below. Under the condition of 2 mol / L substrate concentration, the conversion rate of the immobilized enzyme in 20 repeated experiments was greater than 86.5%, and the optical purity (ee value) of (S)-nornicotinic acid was greater than 99.1%. The immobilized enzyme can be reused after the reaction is completed.

[0044] Table 4. Conversion rates of immobilized enzymes in 20 repeated experiments at a substrate concentration of 2 mol / L.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing high-purity (S)-nornicotine catalyzed by immobilized enzyme, characterized in that: It includes the following steps: (1) Using racemic (R,S)-nornicotinine as raw material, immobilized enzymes are used to catalyze the (R)-nornicotinine to generate mesmin; (2) Using the mesmin generated in step (1) as raw material, the immobilized enzyme catalyzes and reduces it to generate racemic (R,S)-nornicotinic acid; By continuously repeating the above steps, (R)-nornicotinic acid in the reaction system is consumed to obtain high-purity (S)-nornicotinic acid; The immobilized enzyme is obtained by covalently binding a highly (R) selective monoamine oxidase, imine reductase, glucose dehydrogenase, catalase, and resin. in: The amino acid sequence of the highly (R)-selective monoamine oxidase is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3; The amino acid sequence of the imine reductase is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.4; The gene ID of the catalase is WP_144458738.1; The gene ID of the glucose dehydrogenase is ACB59697.

1.

2. The synthesis method according to claim 1, characterized in that, The resin is an epoxy resin, and the epoxy resin model is any one of the following: Xi'an Lanxiao's resins LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 and Tianjin Nankai Hecheng's resins ES-1, ES-107, ES-108, ES-103B.

3. The synthesis method according to claim 1, characterized in that, The immobilized enzyme is prepared by adding 140-600g of K2HPO4•3H2O and 14-60g of KH2PO4 to 500-2000ml of the enzyme solution of the highly (R)-selective monoamine oxidase, 200-500ml of the enzyme solution of the imine reductase, 100-300ml of the enzyme solution of the glucose dehydrogenase and 100-300ml of the enzyme solution of the catalase, stirring to dissolve, then adding 50-300g of epoxy resin, stirring at 25℃ and 180rpm for 24h for adsorption, filtering, washing three times with deionized water, then washing twice with 500ml of PBS buffer, and filtering to obtain the immobilized enzyme. The PBS buffer has a concentration of 200mM, a pH of 8.0, and includes 0.5M NaCl.

4. The synthesis method according to claim 3, characterized in that, The enzyme activity range of the enzyme solutions of the highly (R) selective monoamine oxidase, imine reductase, glucose dehydrogenase, and catalase is 50–100 U / mL.

5. The synthesis method according to claim 3, characterized in that, The enzyme activity range of the immobilized enzyme is 200–400 U / g.

6. The synthesis method according to claim 1, characterized in that, The molar ratio (g / mol) of the immobilized enzyme to the racemic (R,S)-nornicotinic acid is (80-200):

1.

7. The synthesis method according to claim 1, characterized in that, The reaction temperature of the reaction system is 15–40°C.

8. The synthesis method according to claim 1, characterized in that, The reaction time of the reaction system is 16 to 48 hours.

9. The synthesis method according to claim 1, characterized in that, The reaction system also includes using PBS buffer with a concentration of 200 mM and a pH of 7.0 to maintain the reaction pH at 6.8–7.2.

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