WO2022253111A1 - 一种利用还原方式制备(s)-尼古丁的方法 - Google Patents

一种利用还原方式制备(s)-尼古丁的方法 Download PDF

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WO2022253111A1
WO2022253111A1 PCT/CN2022/095423 CN2022095423W WO2022253111A1 WO 2022253111 A1 WO2022253111 A1 WO 2022253111A1 CN 2022095423 W CN2022095423 W CN 2022095423W WO 2022253111 A1 WO2022253111 A1 WO 2022253111A1
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compound
nicotine
formula
seq
reaction
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PCT/CN2022/095423
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French (fr)
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林文清
郑宏杰
刘小波
陈泽聪
李凌宇
周卿君
王松鹤
乐庸堂
胡集铖
张跃
苗珊珊
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重庆博腾制药科技股份有限公司
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Priority to EP22815157.7A priority Critical patent/EP4349995A1/en
Priority to KR1020237045453A priority patent/KR20240019176A/ko
Publication of WO2022253111A1 publication Critical patent/WO2022253111A1/zh
Priority to US18/522,203 priority patent/US20240124908A1/en

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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
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Definitions

  • the application belongs to the technical field of organic synthesis, and in particular relates to a method for synthesizing (S)-nicotine, in particular to a method for preparing (S)-nicotine by means of reduction.
  • (S)-Nicotine is an alkaloid found in plants of the Solanaceae family (Solanum genus) and an important constituent of tobacco. Tobacco leaves contain 1.5%-3.5% (S)-nicotine. Extracting nicotine from tobacco leaves is currently the most important method to obtain nicotine. Although there have been related reports on chemical synthesis methods, the chemical synthesis methods are still immature. The cost is much higher than the extraction method. The chemical synthesis methods that have been reported so far include chemical resolution, asymmetric hydrogenation, and chiral auxiliary reagents.
  • Patent WO2014174505 discloses the use of imine reductase (ketoreductase enzyme) to catalyze pseudooxynicotine to obtain nicotine.
  • the imine reductase used in this patent is derived from Streptomyces sp. GF3546 and Streptomyces sp. GF3587 respectively, and its catalysis can only produce (R)-nicotine , does not disclose how to prepare (S)-nicotine.
  • Patent US10913962B discloses the preparation of (S)-nornicotine by enzymatic catalysis of Mesmin, and then the methylation of (S)-nornicotine to finally obtain (S)-nicotine.
  • the invention uses biocatalysis to prepare (S)-nornicotine to solve the problem of high cost, and can obtain high-yield (S)-nornicotine.
  • formaldehyde is required to provide a methyl source in the methylation process , requiring formic acid as a reducing agent.
  • the present application provides a method for synthesizing (S)-nicotine, in particular to a method for preparing (S)-nicotine by means of reduction.
  • the application provides a method for preparing (S)-nicotine by means of reduction, the method comprising:
  • the reduction specifically includes the following two strategies:
  • the reduction reaction adopts a biological enzyme-catalyzed method, including:
  • the catalytic reduction reaction is carried out with imine reductase as a catalyst, and the enamine compound shown in formula I and/or the iminium cationic compound shown in formula II is catalytically reduced to obtain (S)-nicotine .
  • the coenzyme circulatory system includes coenzyme, glucose and glucose dehydrogenase.
  • the coenzyme includes NADP salt and/or NAD salt, preferably NADP salt.
  • the glucose dehydrogenase comprises the amino acid sequence shown in SEQ ID No.1.
  • the imine reductase comprises SEQ ID No.2-SEQ ID No.6, SEQ ID No.8, SEQ ID No.11, SEQ ID No.12, or has at least 95 % identity amino acid sequence; preferably SEQ ID No.2-SEQ ID No.4, SEQ ID No.12, or an amino acid sequence having at least 95% identity with SEQ ID No.12.
  • the amino acid sequence at least 95% identical to SEQ ID No.12 has any one or a combination of at least two of the following differences in amino acid residues compared with the sequence of SEQ ID No.12: L73, S148, V171 and A172.
  • the 73rd L is mutated to Q or V
  • the 148th S is mutated to R
  • the 171st V is mutated to Y, N, A or S
  • the 172nd A is mutated to V or F.
  • amino acid sequence at least 95% identical to SEQ ID No.12 has any one or a combination of at least two of the following differences in amino acid residues compared with the sequence of SEQ ID No.12: A57, A176, Y230 and S241.
  • the 57th A is mutated to R
  • the 176th A is mutated to G
  • the 230th Y is mutated to G
  • a or T the 241st S is mutated to G or A.
  • amino acid sequence at least 95% identical to SEQ ID No.12 may be:
  • imine reductase mutants according to any combination of the above mutation points combined with conventional technical means in the field, and screen out the substrates involved in this application to obtain (S)-nicotine, and the conversion rate is 99%.
  • the catalytic reduction reaction is carried out at 15-45°C, such as 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, etc. Other specific points within this numerical range can be selection, which will not be repeated here.
  • the catalytic reduction reaction is carried out in a buffer system
  • the buffer includes phosphate buffer, trimethylolmethylamine-hydrochloric acid buffer or triethanolamine-hydrochloric acid buffer.
  • the reduction reaction is catalyzed by a chiral metal catalyst, including:
  • the enamine compound represented by formula I and/or the iminium compound represented by formula II is catalyzed and reduced to (S)-nicotine by a chiral metal catalyst.
  • the chiral metal catalyst includes a chiral iridium catalyst, a chiral ruthenium catalyst or a chiral rhodium catalyst, preferably a chiral iridium catalyst.
  • a ligand is also added to the catalytic reduction system.
  • the ligand is selected from any one or a combination of at least two in Table 1, preferably (R, R)-f-SpiroPhos and (S, S)-Ph-BPE:
  • the catalytic reduction reaction is carried out at 50-100°C, such as 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C, etc. , other specific point values within this value range can be selected, and will not be repeated here.
  • the hydrogen in the catalytic reduction reaction system is maintained at 1.0-6.0MPa, such as 1.0MPa, 2.0MPa, 3.0MPa, 4.0MPa, 5.0MPa or 6.0MPa, etc. Other specific points within this numerical range can be selection, which will not be repeated here.
  • Other specific point values within this range can be selected, and will not be repeated here.
  • the enamine compound represented by formula I or the iminium compound represented by formula II is obtained by desalting and/or cyclization of the compound represented by formula III or its salt:
  • the salt comprises hydrochloride, dihydrochloride, hydrobromide, dihydrobromide, sulfate or hydrogensulfate.
  • the compound represented by the above formula III or its salt can undergo desalination and/or cyclization reaction under the neutralization condition of the inorganic base and/or organic base to obtain the enamine compound represented by the formula I or the sulfide compound represented by the formula II. Amine cation compounds.
  • the synthetic method of the salt of the compound shown in formula III comprises:
  • the synthetic method of the salt of the compound shown in formula III comprises:
  • the synthesis method of compound A includes: mixing nicotinic acid and methanol, and carrying out esterification reaction in a strongly acidic environment to obtain the compound A.
  • the method for preparing (S)-nicotine includes:
  • Fig. 1 is the NMR spectrum of (S)-nicotine prepared by the synthesis method involved in the present application.
  • Fig. 2 is the NMR spectrum of compound III hydrochloride prepared by the synthesis method involved in the present application.
  • Fig. 3 is the NMR spectrum of compound B prepared by the synthesis method involved in the present application.
  • the purity detection method of Compound A in the following preparations and examples is high performance liquid chromatography; the yield calculation method is:
  • the purity detection method of compound B is high performance liquid chromatography; the yield calculation method is:
  • the purity detection method of the compound shown in formula III or its salt is high performance liquid chromatography; the yield calculation method is:
  • the purity detection method of (S)-nicotine is high performance liquid chromatography;
  • the optical purity detection method is high performance liquid chromatography;
  • the yield calculation method is:
  • Compound A is prepared in this preparation example:
  • the organic layer and the aqueous layer were extracted once with 250 g of ethyl acetate, the combined organic layers were dried with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 55.2 g of a colorless transparent liquid with a purity of 99% and a yield of 99%. Solidify after cooling.
  • Compound A is prepared in this preparation example:
  • Compound A is prepared in this preparation example:
  • Compound A is prepared in this preparation example:
  • Compound A is prepared in this preparation example:
  • Compound A is prepared in this preparation example:
  • This preparation example prepares compound B:
  • the product coexists in two forms of ketone form and enol form, and the ketone form is the main form.
  • the ratio of ketone form and enol form is about 10/1.
  • the H NMR spectrum is shown in Fig.3.
  • This preparation example prepares compound B:
  • the product coexists in two forms of ketone form and enol form, and the ketone form is the main form.
  • the ratio of ketone form and enol form is about 10/1.
  • This preparation example prepares compound B:
  • the crude product was purified by column chromatography and detected by NMR.
  • the product coexisted in two forms, ketone form and enol form, with the ketone form being the main form, and the ratio of ketone form and enol form was about 10/1.
  • This preparation example prepares compound B:
  • the crude product was purified by column chromatography and detected by NMR.
  • the product coexisted in two forms, ketone form and enol form, with the ketone form being the main form, and the ratio of ketone form and enol form was about 10/1.
  • This preparation example prepares compound B:
  • the crude product was purified by column chromatography and detected by NMR.
  • the product coexisted in two forms, ketone form and enol form, with the ketone form being the main form, and the ratio of ketone form and enol form was about 10/1.
  • This preparation example prepares compound B:
  • the crude product was purified by column chromatography and detected by NMR.
  • the product coexisted in two forms, ketone form and enol form, with the ketone form being the main form, and the ratio of ketone form and enol form was about 10/1.
  • compound B is used as a raw material to prepare the hydrochloride of the compound shown in formula III:
  • compound B is used as a raw material to prepare the compound shown in formula III:
  • compound B is used as a raw material to prepare the compound shown in formula III:
  • compound B is used as a raw material to prepare the compound shown in formula III:
  • compound B is used as a raw material to prepare the compound shown in formula III:
  • compound A is used as a raw material to prepare compound D first, and then prepare the compound shown in formula III:
  • compound A is used as a raw material to prepare compound D first, and then prepare the compound shown in formula III:
  • This preparation example prepares the glucose dehydrogenase of the amino acid sequence shown in SEQ ID No.1:
  • the amino acid sequence (SEQ ID No.1) of glucose dehydrogenase derived from Priestia megaterium (NCBI accession number AUO12718.1) was sent to Nanjing GenScript Company for codon optimization and whole gene synthesis, and ligated into plasmid pET30a(+) Middle; the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells to obtain the recombinant bacteria containing the glucose dehydrogenase gene.
  • the amino acid sequence of imine reductase (SEQ ID No.2-SEQ ID No.12, see the table below) reported on NCBI was sent to Nanjing GenScript Company for codon optimization and whole gene synthesis, and ligated into plasmid pET30a( In +); the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells to obtain the recombinant bacteria containing the imine reductase gene.
  • Inoculate the above-mentioned recombinant bacteria into 5 mL of LB liquid medium containing 50 ⁇ g/mL kanamycin and culture overnight at 37°C; inoculate 1 mL of the bacterial liquid into 125 mL of LB liquid medium containing 50 ⁇ g/mL kanamycin cultured at 37°C for 3h, then added 125 ⁇ L 1M IPTG, induced at 25°C for 16h; centrifuged (4000rpm, 4°C, 10min) to collect the bacteria, added 4 times the volume of phosphate buffer (pH 7.0) to resuspend , after resuspension, the cells were ultrasonically disrupted, centrifuged (4000 rpm, 4° C., 10 min) to take the supernatant solution and freeze-dried to obtain imine reductase enzyme powder.
  • This preparation example also prepares the following 10 kinds of imine reductases, which are recorded as enzyme 14-enzyme 23, that is, the amino acid sequence SEQ ID NO:12 is subjected to site-directed mutation to obtain the imine reductase amino acids shown in SEQ ID No.13-22
  • the sequence was sent to Nanjing GenScript Company for codon optimization and whole gene synthesis, and was ligated into the plasmid pET30a(+); the recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain the imine reductase gene containing recombinant bacteria. Carry out the above-mentioned operation, finally obtain imine reductase enzyme powder.
  • This preparation example prepares the following enzymes, denoted as enzyme 12:
  • Imine reductase (amino acid sequence SEQ ID No.2) and glucose dehydrogenase (amino acid sequence SEQ ID No.1) were subcloned into plasmid pETDuet-1 in sequence; the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to sense state cells, and obtain recombinant bacteria containing both imine reductase gene and glucose dehydrogenase gene.
  • (S)-nicotine is prepared by catalytic reduction of a chiral metal catalyst:
  • the prepared (S)-nicotine was purified by column chromatography and then characterized by proton nuclear magnetic spectrum, as shown in Figure 1, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d), 8.50( 1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24( 1H,m), 2.16(3H,m), 1.91-2.02(1H,m), 1.79-1.87(1H,m), 1.68-1.76(1H,m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by catalytic reduction of a chiral metal catalyst:
  • the prepared (S)-nicotine was characterized by H NMR spectrum after column chromatography purification, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d),8.50(1H,dd),7.70 (1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by catalytic reduction of a chiral metal catalyst:
  • the prepared (S)-nicotine was characterized by H NMR spectrum after column chromatography purification, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d),8.50(1H,dd),7.70 (1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by catalytic reduction of a chiral metal catalyst:
  • Example 3 The difference between its preparation method and Example 3 is only that the catalyst 1,5-cyclooctadiene iridium chloride dimer 16.7 mg, ligand (R, R)-f-SpiroPhos 38 mg is replaced by Rh [(R, R )-DIPAMP](COD)BF 4 45mg, other conditions remained unchanged.
  • the target compound was obtained; purity 45%, optical purity 27%.
  • the prepared (S)-nicotine was characterized by H NMR spectrum after column chromatography purification, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d),8.50(1H,dd),7.70 (1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by catalytic reduction of a chiral metal catalyst:
  • the prepared (S)-nicotine was characterized by H NMR spectrum after column chromatography purification, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d),8.50(1H,dd),7.70 (1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by catalytic reduction of a chiral metal catalyst:
  • the prepared (S)-nicotine was characterized by H NMR spectrum after column chromatography purification, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d),8.50(1H,dd),7.70 (1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by catalytic reduction of a chiral metal catalyst:
  • the prepared (S)-nicotine was characterized by H NMR spectrum after column chromatography purification, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d),8.50(1H,dd),7.70 (1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by biological enzyme catalytic reduction:
  • (S)-nicotine is prepared by biological enzyme catalytic reduction:
  • the prepared (S)-nicotine was characterized by proton nuclear magnetic spectrum, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d), 8.50(1H,dd), 7.70(1H,dt) ,7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m) ,1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by biological enzyme catalytic reduction:
  • the prepared (S)-nicotine was characterized by proton nuclear magnetic spectrum, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d), 8.50(1H,dd), 7.70(1H,dt) ,7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m) ,1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m). Indicating that (S)-nicotine was successfully synthesized.
  • (S)-nicotine is prepared by biological enzyme catalytic reduction:
  • the prepared (S)-nicotine was characterized by proton nuclear magnetic spectrum, the data is: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.54(1H,d), 8.50(1H,dd), 7.70(1H,dt) ,7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m) ,1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m). Indicating that (S)-nicotine was successfully synthesized.

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Abstract

一种利用还原方式制备(S)-尼古丁的方法，所述方法包括：将式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物进行还原反应，得到(S)-尼古丁。鉴于现有技术中已公开的制备(S)-尼古丁的策略大多需要甲基化方式才能得到(S)-尼古丁，或者不经甲基化使用酶催化得不到(S)-尼古丁，(S)-尼古丁的制备策略还十分有限，创造性地开发了全新的不需使用甲基化过程即可高效制备(S)-尼古丁的方法，即对上述式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物进行还原，即可。该合成方法操作简单、安全可靠，收率高，纯度高。

Description

一种利用还原方式制备(S)-尼古丁的方法 技术领域
本申请属于有机合成技术领域，具体涉及一种(S)-尼古丁的合成方法，尤其涉及一种利用还原方式制备(S)-尼古丁的方法。
背景技术
(S)-尼古丁(烟碱)是一种存在于茄科植物(茄属)中的生物碱，也是烟草的重要成分。烟叶中含有1.5％-3.5％的(S)-尼古丁，从烟叶从提取烟碱是目前获取烟碱的最主要方法，虽然已经有化学合成法的相关报道，但是化学合成方法还不成熟，其成本远高于提取法。目前已经报道的化学合成方法包括化学拆分法、不对称氢化法、手性辅助试剂法等。
专利WO2014174505公开了使用亚胺还原酶(ketoreductase enzyme)催化pseudooxynicotine得到尼古丁，该专利使用的亚胺还原酶分别来源Streptomyces sp.GF3546和Streptomyces sp.GF3587，且其催化只能制备得到(R)-尼古丁，没有公开如何制备(S)-尼古丁。
专利US10913962B公开了使用酶催化麦思明制备(S)-去甲烟碱，再对(S)-去甲烟碱甲基化，最后得到(S)-尼古丁。该发明使用生物催化制备(S)-去甲烟碱解决了成本高的问题，而且能够得到高收率的(S)-去甲烟碱，同时在甲基化过程中需要甲醛提供甲基来源，需要甲酸作为还原剂。
Figure PCTCN2022095423-appb-000001
为了解决现有技术需要甲基化方式才能制备(S)-尼古丁，或者不经甲基化使用酶催化得不到(S)-尼古丁的问题，需要开发一种新的无需甲基化制备(S)-尼古丁的方法。
发明内容
本申请提供了一种(S)-尼古丁的合成方法，尤其涉及一种利用还原方式制备(S)-尼古丁的方法。
本申请提供一种利用还原方式制备(S)-尼古丁的方法，所述方法包括：
将式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物进行还原反应，得到(S)-尼古丁：
Figure PCTCN2022095423-appb-000002
鉴于现有技术中已公开的制备(S)-尼古丁的策略大多需要甲基化方式才能得到(S)-尼古丁，或者不经甲基化使用酶催化得不到(S)-尼古丁，(S)-尼古丁的制备策略还十分有限，本申请创造性地开发了全新的不需使用甲基化过程即可高效制备(S)-尼古丁的方法，即对上述式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物进行还原，即可。该合成方法操作简单、安全可靠，收率高，纯度高。
在本申请中，所述还原具体包括如下两种策略：
所述还原反应采用生物酶催化方法，包括：
在辅酶循环系统的条件下，以亚胺还原酶为催化剂进行催化还原反应，催化还原式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物，得到(S)-尼古丁。
优选地，所述辅酶循环系统包括辅酶、葡萄糖和葡萄糖脱氢酶。
优选地，所述辅酶包括NADP盐和/或NAD盐，优选NADP盐。
优选地，所述葡萄糖脱氢酶包括SEQ ID No.1所示的氨基酸序列。
Figure PCTCN2022095423-appb-000003
优选地，所述亚胺还原酶包括SEQ ID No.2-SEQ ID No.6、SEQ ID No.8、SEQ ID No.11、SEQ ID No.12、或具有与SEQ ID No.12至少95％同一性的氨基酸序列；优选SEQ ID No.2-SEQ ID No.4、SEQ ID No.12、或具有与SEQ ID No.12至少95％同一性的氨基酸序列。
Figure PCTCN2022095423-appb-000004
Figure PCTCN2022095423-appb-000005
Figure PCTCN2022095423-appb-000006
本领域技术人员在上述SEQ ID No.2-SEQ ID No.12氨基酸序列的基础上结合本领域的常规技术手段可以对序列中的一个或多个氨基酸进行突变，进一步获得亚胺还原酶突变体，并筛选出能够催化本申请涉及的底物得到(S)-尼古丁、且转化率、对映体过量(ee值)均满足实际需要的亚胺还原酶。
优选地，所述与SEQ ID No.12至少95％同一性的氨基酸序列与SEQ ID No.12序列相比在氨基酸残基上存在下述差异的任意一种或至少两种的组合：L73、S148、V171和A172。
进一步优选地，第73位L突变为Q或V，第148位S突变为R，第171位V突变为Y、N、A或S，以及第172位A突变为V或F。
进一步优选地，所述与SEQ ID No.12至少95％同一性的氨基酸序列与SEQ ID No.12序列相比在氨基酸残基上还存在下述差异的任意一种或至少两种的组合：A57、A176、Y230和S241。
进一步优选地，第57位A突变为R，第176位A突变为G，第230位Y突变为G、A或T，以及第241位S突变为G或A。
示例性地，所述与SEQ ID No.12至少95％同一性的氨基酸序列可以为：
Figure PCTCN2022095423-appb-000007
Figure PCTCN2022095423-appb-000008
Figure PCTCN2022095423-appb-000009
本领域技术人员可以根据上述突变点的任意组合结合本领域的常规技术手段获得亚胺还原酶突变体，并筛选出能够催化本申请涉及的底物得到(S)-尼古丁、且转化率99％以上、ee值99％以上的亚胺还原酶。优选地，所述催化还原反应在15-45℃下进行，例如15℃、20℃、25℃、30℃、35℃、40℃或45℃等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
优选地，所述催化还原反应在缓冲液体系中进行，所述缓冲液包括磷酸盐缓冲液、三羟甲基甲胺-盐酸缓冲液或三乙醇胺-盐酸缓冲液。
优选地，所述催化还原反应在pH＝6.0-8.0下进行，例如pH＝6.0、pH＝6.2、pH＝6.5、pH＝6.8、pH＝7.0、pH＝7.2、pH＝7.5、pH＝7.8或pH＝8.0等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
在本申请中，所述还原反应采用手性金属催化剂催化方法，包括：
在氢气气氛中，式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物被手性金属催化剂催化还原为(S)-尼古丁。
优选地，所述手性金属催化剂包括手性铱催化剂、手性钌催化剂或手性铑催化剂，优选手性铱催化剂。
优选地，在所述催化还原体系中还添加配体。
所述配体选自表1中任意一种或至少两种的组合，优选(R,R)-f-SpiroPhos和(S,S)-Ph-BPE：
表1
Figure PCTCN2022095423-appb-000010
Figure PCTCN2022095423-appb-000011
Figure PCTCN2022095423-appb-000012
优选地，所述催化还原反应在50-100℃下进行，例如50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃、90℃、95℃或100℃等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
优选地，所述催化还原反应体系中的氢气维持在1.0-6.0MPa，例如1.0MPa、2.0MPa、3.0MPa、4.0MPa、5.0MPa或6.0MPa等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
优选地，所述催化还原反应在pH＝4.0-13.0下进行，例如pH＝4.0、5.0、6.0、pH＝6.2、pH＝6.5、pH＝6.8、pH＝7.0、pH＝7.2、pH＝7.5、pH＝7.8、pH＝8.0、pH＝10.0、pH＝12.0或pH＝13.0等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
在本申请中，所述式Ⅰ所示的烯胺化合物或式Ⅱ所示的亚胺正离子化合物是由式Ⅲ所示的化合物或其盐经过脱盐和/或环化得到的：
Figure PCTCN2022095423-appb-000013
优选地，所述盐包括盐酸盐、二盐酸盐、氢溴酸盐、二氢溴酸盐、硫酸盐或硫酸氢盐。
上述式Ⅲ所示的化合物或其盐在无机碱和/或有机碱中和的条件下即可发生脱盐和/或环化反应，得到式Ⅰ所示的烯胺化合物或式Ⅱ所示的亚胺正离子化合物。
在本申请中，式Ⅲ所示化合物的盐的合成方法包括：
将化合物A与N-甲基吡咯烷酮、有机碱混合反应，得到化合物D；化合物D再与酸混合反应，得到式Ⅲ所示化合物的盐；其反应式如下所示：
Figure PCTCN2022095423-appb-000014
或者，式Ⅲ所示化合物的盐的合成方法包括：
将化合物A与N-甲基吡咯烷酮、有机碱混合反应后，加入酸中和至pH＝7-8，得到化合物B；化合物B再与酸混合反应，得到式Ⅲ所示化合物的盐；其反应式如下所示：
Figure PCTCN2022095423-appb-000015
在本申请中，所述化合物A的合成方法包括：将烟酸与甲醇混合，在强酸性环境中进行酯化反应，即得。
作为本申请的优选技术方案，所述制备(S)-尼古丁的方法包括：
将烟酸与甲醇混合，在强酸性环境中进行酯化反应，得到化合物A；化合物A与N-甲基吡咯烷酮、有机碱混合反应后，可选地加入酸进行中和，得到化合物B或D；然后再与酸混合反应，得到式Ⅲ所示化合物的盐；脱盐及发生环化，最后采用生物酶催化方法或手性金属催化剂催化方法进行还原反应，得到(S)-尼古丁；其反应式如下所示：
Figure PCTCN2022095423-appb-000016
相对于现有技术，本申请具有以下有益效果：
鉴于现有技术中已公开的制备(S)-尼古丁的策略大多需要甲基化方式才能得到(S)-尼古丁，或者不经甲基化使用酶催化得不到(S)-尼古丁，(S)-尼古丁的制备策略还十分有限，本申请创造性地开发了全新的不需使用甲基化过程即可高效制备(S)-尼古丁的方法，即对上述式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物进行还原，即可。该合成方法操作简单、安全可靠，收率高，纯度高，并且丰富了(S)-尼古丁的合成策略。
附图说明
图1是采用本申请所涉及的合成方法制得的(S)-尼古丁的核磁图谱。
图2是采用本申请所涉及的合成方法制得的化合物Ⅲ盐酸盐的核磁图谱。
图3是采用本申请所涉及的合成方法制得的化合物B的核磁图谱。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
下述制备例和实施例中化合物A的纯度检测方法为高效液相色谱法；收率计算方法为：
Figure PCTCN2022095423-appb-000017
化合物B的纯度检测方法为高效液相色谱法；收率计算方法为：
Figure PCTCN2022095423-appb-000018
式Ⅲ所示化合物或其盐的纯度检测方法为高效液相色谱法；收率计算方法为：
Figure PCTCN2022095423-appb-000019
(S)-尼古丁的纯度检测方法为高效液相色谱法；光学纯度检测方法为高效液相色谱法；收率计算方法为：
Figure PCTCN2022095423-appb-000020
制备例1-1
本制备例制备化合物A：
Figure PCTCN2022095423-appb-000021
向500mL反应瓶中加入烟酸50g，甲醇250g，启动搅拌，向反应瓶中滴加浓硫酸60g，滴加完毕后升温至回流反应18小时后降温到45℃减压浓缩，蒸出甲醇，待无馏分蒸出后，浓缩残留物降温至20℃，向浓缩残留物中加入乙酸乙酯250g，水250g，搅拌溶清后用20％氢氧化钠水溶液调节pH至7.5，静置分层，收集有机层，水层再用乙酸乙酯250g萃取一次，合并有机层，用无水硫酸钠干燥后，减压蒸除溶剂得无色透明液体55.2g，纯度99％，收率99％，该液体冷却后固化即得。
制备例1-2
本制备例制备化合物A：
向500mL反应瓶中加入烟酸50g，甲醇250g，开启搅拌，升温至回流后向反应瓶中滴加氯化亚砜120.5g(2.5eq)，滴加完毕后保持回流反应9小时后，40℃减压浓缩，蒸出甲醇，待无馏分蒸出后，浓缩残留物降温至10℃，向浓缩残留物中加入二氯甲烷200g，水100g，搅拌溶清后用15％氢氧化钠水溶液调节pH至8，静置分层，收集有机层，水层用200g二氯甲烷再萃取一次，合并有机层，用5％氢氧化钠水溶液100g洗涤一次，有机层用无水硫酸钠干燥后，减压蒸除溶剂得浅黄色透明液体44.8g，纯度99％，收率81％，该液体冷却后固化即得。
制备例1-3
本制备例制备化合物A：
向100mL反应瓶中加入烟酸5g，原甲酸三甲酯5.2g(1.2eq)，甲醇25g，四氯化锆0.48g(0.05eq)，启动搅拌，升温至回流反应17小时后降温到25℃，加入乙醇钠0.55g(0.2eq)中和后，过滤，滤液减压浓缩，蒸出甲醇，得无色透明液体5.5g，纯度98.1％，收率99％，该液体冷却后固化即得。
制备例1-4
本制备例制备化合物A：
向100mL反应瓶中加入烟酸5g，原甲酸三甲酯5.2g(1.2eq)，甲醇25g，四氯化锆0.1g(0.01eq)，启动搅拌，升温至回流反应36小时后降温到30℃，加入乙醇钠0.11g(0.04eq)中和后，过滤，滤液减压浓缩，蒸出甲醇，得无色透明液体5.3g，纯度91.6％，收率95％，该液体冷却后固化即得。
制备例1-5
本制备例制备化合物A：
向100mL反应瓶中加入烟酸5g，原甲酸三甲酯5.2g(1.2eq)，甲醇25g，四氯化锆0.19g(0.02eq)，启动搅拌，升温至回流反应30小时后降温到20℃，加入乙醇钠0.22g(0.08eq)中和后，过滤，滤液减压浓缩，蒸出甲醇，得无色透明液体5.4g，纯度93.9％，收率97％，该液体冷却后固化即得。
制备例1-6
本制备例制备化合物A：
向2L反应瓶中加入烟酸200g，原甲酸三甲酯207g(1.2eq)，甲醇1000g，四氯化锆9.5g(0.025eq)，启动搅拌，升温至回流反应20小时后降温到25℃，加入乙醇钠11.06g(0.1eq)中和后，过滤，滤液减压浓缩，蒸出甲醇，得无色透明液体220.6g，纯度98.5％，收率99％，该液体冷却后固化即得。
制备例2-1
本制备例制备化合物B：
Figure PCTCN2022095423-appb-000022
向反应瓶中加入制备例1-1制得的化合物A 102.0g，N-甲基吡咯烷酮88.5g(1.2eq)，甲苯1.2kg，叔丁醇钾133.6g(1.6eq)，加完物料后，启动搅拌，升温至回流；反应合格后，降温至25℃，用5％盐酸中和至pH＝7.5，静置分层，水相用二氯甲烷萃取萃取后，合并有机相，减压浓缩除去溶剂得到黄棕色液体121.0g，即为化合物B，纯度94％，收率80％；不用纯化处理，直接进入下一步反应。
粗产品经柱层析纯化后，用核磁检测，产品以酮式和烯醇式两种形态共存，以酮式为主，酮式和烯醇式的比例约为10/1。酮式形态化合物的核磁氢谱数据如下：δppm(400MHz,CDCl 3)9.30(s,1H),8.77-8.80(m,1H),8.44(1H,dt,J＝8.0,2.0Hz),7.43-7.47(m,1H)，4.46(1H,dd,J＝8.8,3.2Hz),3.36-3.46(m,2H),2.87(s,3H),2.67-2.75(m,1H),2.22-2.32(m,1H)。核磁氢谱谱图如图3 所示。
制备例2-2
本制备例制备化合物B：
向反应瓶中加入制备例1-2制得的化合物A 102.0g，N-甲基吡咯烷酮77.4g(1.05eq)，甲苯1.2kg，叔丁醇钾91.8g(1.1eq)，加完物料后，启动搅拌，升温至回流；反应合格后，降温至20℃，用5％盐酸中和至pH＝7，静置分层，水相用甲苯萃取后，合并有机相，减压浓缩除去溶剂得到黄棕色液体118.0g，即为化合物B，纯度93.5％，收率78％；不用纯化处理，直接进入下一步反应。
粗产品经柱层析纯化后，用核磁检测，产品以酮式和烯醇式两种形态共存，以酮式为主，酮式和烯醇式的比例约为10/1。酮式形态化合物的核磁氢谱数据如下：δppm(400MHz,CDCl 3)9.30(s,1H),8.77-8.80(m,1H),8.44(1H,dt,J＝8.0,2.0Hz),7.43-7.47(m,1H)，4.46(1H,dd,J＝8.8,3.2Hz),3.36-3.46(m,2H),2.87(s,3H),2.67-2.75(m,1H),2.22-2.32(m,1H)。
制备例2-3
本制备例制备化合物B：
向反应瓶中加入制备例1-3制得的化合物A 102.0g，N-甲基吡咯烷酮88.5g(1.2eq)，甲苯1.2kg，叔丁醇钠114.4g(1.6eq)，加完物料后，启动搅拌，升温至回流；反应合格后，降温至30℃，用5％盐酸中和至pH＝8，静置分层，水相用乙酸乙酯萃取后，合并有机相，减压浓缩除去溶剂得到黄棕色液体117.8g，即为化合物B，纯度95％，收率78％；不用纯化处理，直接进入下一步反应。
粗产品经柱层析纯化后，用核磁检测，产品以酮式和烯醇式两种形态共存，以酮式为主，酮式和烯醇式的比例约为10/1。酮式形态化合物的核磁氢谱数据如下：δppm(400MHz,CDCl 3)9.30(s,1H),8.77-8.80(m,1H),8.44(1H,dt,J＝8.0,2.0Hz),7.43-7.47(m,1H)，4.46(1H,dd,J＝8.8,3.2Hz),3.36-3.46(m,2H),2.87(s,3H),2.67-2.75(m,1H),2.22-2.32(m,1H)。
制备例2-4
本制备例制备化合物B：
向反应瓶中加入制备例1-4制得的化合物A 102.0g，N-甲基吡咯烷酮77.4g(1.05eq)，甲苯1.2kg，叔丁醇钠78.6g(1.1eq)，加完物料后，启动搅拌，升温至回流；反应合格后，降温至25℃，用5％盐酸中和至pH＝7.5，静置分层，水相用甲苯萃取后，合并有机相，减压浓缩除去溶剂得到黄棕色液体108.7g，即为化合物B，纯度93％，收率72％；不用纯化处理，直接进入下一步反应。
粗产品经柱层析纯化后，用核磁检测，产品以酮式和烯醇式两种形态共存，以酮式为主，酮式和烯醇式的比例约为10/1。酮式形态化合物的核磁氢谱数据如下：δppm(400MHz,CDCl 3)9.30(s,1H),8.77-8.80(m,1H),8.44(1H,dt,J＝8.0,2.0Hz),7.43-7.47(m,1H)，4.46(1H,dd,J＝8.8,3.2Hz),3.36-3.46(m,2H),2.87(s,3H),2.67-2.75(m,1H),2.22-2.32(m,1H)。
制备例2-5
本制备例制备化合物B：
向反应瓶中加入制备例1-5制得的化合物A 102.0g，N-甲基吡咯烷酮88.5g(1.2eq)，甲苯1.2kg，乙醇钠81.0g(1.6eq)，加完物料后，启动搅拌，升温至回流；反应16小时后，降温至25℃，用5％盐酸中和至pH＝7.5，静置分层，水相用甲苯萃取后，合并有机相，减压浓缩除去溶剂得到黄棕色液体127.0g，即为化合物B，纯度92％，收率84％；不用纯化处理，直接进入下一步反应。
粗产品经柱层析纯化后，用核磁检测，产品以酮式和烯醇式两种形态共存，以酮式为主，酮式和烯醇式的比例约为10/1。酮式形态化合物的核磁氢谱数据如下：δppm(400MHz,CDCl 3)9.30(s,1H),8.77-8.80(m,1H),8.44(1H,dt,J＝8.0,2.0Hz),7.43-7.47(m,1H)，4.46(1H,dd,J＝8.8,3.2Hz),3.36-3.46(m,2H),2.87(s,3H),2.67-2.75(m,1H),2.22-2.32(m,1H)。
制备例2-6
本制备例制备化合物B：
向反应瓶中加入制备例1-6制得的化合物A 102.0g，N-甲基吡咯烷酮88.5g(1.2eq)，甲苯1.2kg，氢化钠47.6g(1.6eq，60％Sodium hydride in oil)，加完物料后，启动搅拌，升温至回流；反应合格后，降温至25℃，用5％盐酸中和至pH＝7，静置分层，水相用甲基叔丁基醚萃取后，合并有机相，减压浓缩除去溶剂得到黄棕色液体111.0g，即为化合物B，纯度90％，收率70％；不用纯化处理，直接进入下一步反应。
粗产品经柱层析纯化后，用核磁检测，产品以酮式和烯醇式两种形态共存，以酮式为主，酮式和烯醇式的比例约为10/1。酮式形态化合物的核磁氢谱数据如下：δppm(400MHz,CDCl 3)9.30(s,1H),8.77-8.80(m,1H),8.44(1H,dt,J＝8.0,2.0Hz),7.43-7.47(m,1H)，4.46(1H,dd,J＝8.8,3.2Hz),3.36-3.46(m,2H),2.87(s,3H),2.67-2.75(m,1H),2.22-2.32(m,1H)。
制备例3-1
本制备例以化合物B为原料制备式Ⅲ所示化合物的盐酸盐：
Figure PCTCN2022095423-appb-000023
向反应瓶中加入制备例2-1制得的化合物B 100.0g，浓盐酸500.0g，加完物料后，启动搅拌，升温至回流；反应合格后，降温至55℃，减压浓缩，残余物中加入甲醇300.0g，降温到4℃，过滤得到类白色固体92.2g，即为式Ⅲ所示化合物，纯度99％，收率75％。
经检测，所得化合物核磁氢谱与所示结构一致，检测数据如下： 1H-NMR(400MHz,D 2O):δppm 9.25(m,1H),9.0(dt,J＝8.4Hz,1.6Hz,1H),8.90-8.91(m,1H),8.13-8.17(m,1H),3.28(t,J＝6.8Hz,2H),3.06(t,J＝8.0Hz,2H),2.66(s,3H),2.02-2.09(m,2H)。谱图如图2所示。
制备例3-2
本制备例以化合物B为原料制备式Ⅲ所示化合物：
向反应瓶中加入制备例2-2制得的化合物B 100.0g，浓盐酸100.0g，加完物料后，启动搅拌，升温至回流；反应合格后，降温至50℃，减压浓缩，残余物中加入甲醇100.0g，降温 到8℃，过滤得到类白色固体95.9g，即为式Ⅲ所示化合物，纯度98.5％，收率78％。
经检测，所得化合物核磁氢谱与所示结构一致，检测数据如下： 1H-NMR(400MHz,D 2O):δppm 9.25(m,1H),9.0(dt,J＝8.4Hz,1.6Hz,1H),8.90-8.91(m,1H),8.13-8.17(m,1H),3.28(t,J＝6.8Hz,2H),3.06(t,J＝8.0Hz,2H),2.66(s,3H),2.02-2.09(m,2H)。
制备例3-3
本制备例以化合物B为原料制备式Ⅲ所示化合物：
向反应瓶中加入制备例2-3制得的化合物B 100.0g，浓盐酸300.0g，加完物料后，启动搅拌，升温至回流；反应合格后，降温至50℃，减压浓缩，残余物中加入乙醇300.0g，降温到0℃，过滤得到类白色固体100.8g，即为式Ⅲ所示化合物，纯度99％，收率82％。
经检测，所得化合物核磁氢谱与所示结构一致，检测数据如下： 1H-NMR(400MHz,D 2O):δppm 9.25(m,1H),9.0(dt,J＝8.4Hz,1.6Hz,1H),8.90-8.91(m,1H),8.13-8.17(m,1H),3.28(t,J＝6.8Hz,2H),3.06(t,J＝8.0Hz,2H),2.66(s,3H),2.02-2.09(m,2H)。
制备例3-4
本制备例以化合物B为原料制备式Ⅲ所示化合物：
向反应瓶中加入制备例2-4制得的化合物B 100.0g，浓盐酸300.0g，加完物料后，启动搅拌，升温至回流；反应合格后，降温至55℃，减压浓缩，残余物中加入异丙醇500.0g，降温到4℃，过滤得到类白色固体93.4g，即为式Ⅲ所示化合物，纯度97.5％，收率76％。
经检测，所得化合物核磁氢谱与所示结构一致，检测数据如下： 1H-NMR(400MHz,D 2O):δppm 9.25(m,1H),9.0(dt,J＝8.4Hz,1.6Hz,1H),8.90-8.91(m,1H),8.13-8.17(m,1H),3.28(t,J＝6.8Hz,2H),3.06(t,J＝8.0Hz,2H),2.66(s,3H),2.02-2.09(m,2H)。
制备例3-5
本制备例以化合物B为原料制备式Ⅲ所示化合物：
向反应瓶中加入制备例2-5制得的化合物B 100.0g，浓盐酸500.0g，加完物料后，启动搅拌，升温至回流；反应合格后，降温至55℃，减压浓缩，残余物中加入异丙醇100.0g，降温到4℃，过滤得到类白色固体103.3g，即为式Ⅲ所示化合物，纯度96％，收率84％。
经检测，所得化合物核磁氢谱与所示结构一致，检测数据如下： 1H-NMR(400MHz,D 2O):δppm 9.25(m,1H),9.0(dt,J＝8.4Hz,1.6Hz,1H),8.90-8.91(m,1H),8.13-8.17(m,1H),3.28(t,J＝6.8Hz,2H),3.06(t,J＝8.0Hz,2H),2.66(s,3H),2.02-2.09(m,2H)。
制备例4-1
本制备例以化合物A为原料先制备化合物D，再制备式Ⅲ所示化合物：
向反应瓶中加入制备例1-1制得的化合物A 120.0g，N-甲基吡咯烷酮96.2g，甲苯1.6kg，叔丁醇钾147g，加完物料后，启动搅拌，升温至回流；反应合格后，降温至25℃，过滤，得化合物D(钾盐)，直接用于下一步反应。
将过滤得到的D(钾盐)，加入到650g浓盐酸中，升温至100℃反应，直至反应完全。减压蒸馏除去大部分水，向残余物中加入95％乙醇650mL，充分搅拌后降温到10℃，过滤，烘干，得到类白色固体86g，即为式Ⅲ所示化合物，收率55.2％，纯度97.2％。
经检测，所得化合物核磁氢谱与所示结构一致，检测数据如下： 1H-NMR(400MHz,D 2O): δppm 9.25(m,1H),9.0(dt,J＝8.4Hz,1.6Hz,1H),8.90-8.91(m,1H),8.13-8.17(m,1H),3.28(t,J＝6.8Hz,2H),3.06(t,J＝8.0Hz,2H),2.66(s,3H),2.02-2.09(m,2H)。
制备例4-2
本制备例以化合物A为原料先制备化合物D，再制备式Ⅲ所示化合物：
向反应瓶中加入制备例1-2制得的化合物A 153.0g，N-甲基吡咯烷酮165g，甲苯2kg，叔丁醇钠214.5g，加完物料后，启动搅拌，升温至回流；反应合格后，降温至15℃，过滤，得化合物D(钠盐)，直接用于下一步反应。
将过滤得到的D(钠盐)，加入到960g浓盐酸中，升温至100℃左右反应，直至反应完全。减压蒸馏除去大部分水，向残余物中加入异丙醇800mL，充分搅拌后除温到20℃，过滤，烘干，得到类白色固体127g，即为式Ⅲ所示化合物，收率63.5％，纯度96.7％。
经检测，所得化合物核磁氢谱与所示结构一致，检测数据如下： 1H-NMR(400MHz,D 2O):δppm 9.25(m,1H),9.0(dt,J＝8.4Hz,1.6Hz,1H),8.90-8.91(m,1H),8.13-8.17(m,1H),3.28(t,J＝6.8Hz,2H),3.06(t,J＝8.0Hz,2H),2.66(s,3H),2.02-2.09(m,2H)。
制备例5-1
本制备例制备如SEQ ID No.1所示氨基酸序列的葡萄糖脱氢酶：
将来源于Priestia megaterium(NCBI登录号AUO12718.1)的葡萄糖脱氢酶氨基酸序列(SEQ ID No.1)送至南京金斯瑞公司进行密码子优化和全基因合成，连接入质粒pET30a(+)中；将重组质粒转入大肠杆菌BL21(DE3)感受态细胞，获得含有葡萄糖脱氢酶基因的重组菌。
将上述重组菌接种于5mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养过夜；取1mL菌液接种于125mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养3h，然后加入125μL 1M IPTG，25℃下诱导过夜；离心(4000rpm，4℃，10min)收集菌体，加入4倍体积的磷酸缓冲液(pH＝7.0)重悬，重悬后对细胞进行超声破碎，离心(4000rpm，4℃，10min)取上清溶液进行冷冻干燥，获得葡萄糖脱氢酶酶粉。
制备例5-2
本制备例制备以下11种亚胺还原酶，依次记为酶1、酶3-11、酶13：
将NCBI上报道的亚胺还原酶氨基酸序列(SEQ ID No.2-SEQ ID No.12，信息见下表)送至南京金斯瑞公司进行密码子优化和全基因合成，连接入质粒pET30a(+)中；将重组质粒转入大肠杆菌BL21(DE3)感受态细胞，获得含有亚胺还原酶基因的重组菌。
氨基酸序列 NCBI登录号 来源
SEQ ID No.2 WP_201992507.1 Aeromonas veronii
SEQ ID No.3 WP_139729886.1 Aeromonas sobria
SEQ ID No.4 WP_010862236.1 Plesiomonas shigelloides
SEQ ID No.5 WP_064339362.1 Aeromonas veronii
SEQ ID No.6 QNF15299.1 Aeromonas jandaei
SEQ ID No.7 WP_073480922.1 Streptoalloteichus hindustanus
SEQ ID No.8 WP_015347361.1 Myxococcus stipitatus
SEQ ID No.9 WP_198539790.1 Rhizobium sp.
SEQ ID No.10 WP_073459042.1 Pseudonocardia thermophila
SEQ ID No.11 WP_020496004.1 Sciscionella marina
SEQ ID No.12 WP_005335883.1 Aeromonas veronii
将上述重组菌接种于5mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养过夜；取1mL菌液接种于125mL含有50μg/mL卡那霉素的LB液体培养基中，置于37℃下培养3h，然后加入125μL 1M IPTG，25℃下诱导16h；离心(4000rpm，4℃，10min)收集菌体，加入4倍体积的磷酸缓冲液(pH＝7.0)重悬，重悬后对细胞进行超声破碎，离心(4000rpm，4℃，10min)取上清溶液进行冷冻干燥，获得亚胺还原酶酶粉。
本制备例还制备以下10种亚胺还原酶，记为酶14-酶23，即对氨基酸序列SEQ ID NO:12进行定点突变，获得SEQ ID No.13-22所示的亚胺还原酶氨基酸序列，送至南京金斯瑞公司进行密码子优化和全基因合成，连接入质粒pET30a(+)中；将重组质粒转入大肠杆菌BL21(DE3)感受态细胞，获得含有亚胺还原酶基因的重组菌。进行如上述操作，最终获得亚胺还原酶酶粉。
制备例5-3
本制备例制备以下酶，记为酶12：
将亚胺还原酶(氨基酸序列SEQ ID No.2)和葡萄糖脱氢酶(氨基酸序列SEQ ID No.1)依次亚克隆至质粒pETDuet-1中；将重组质粒转入大肠杆菌BL21(DE3)感受态细胞，获得同时含有亚胺还原酶基因和葡萄糖脱氢酶基因的重组菌。
将上述重组菌接种于5mL含有100μg/mL氨苄青霉素的LB液体培养基中，置于37℃下培养过夜；取1mL菌液接种于125mL含有100μg/mL氨苄青霉素的LB液体培养基中，置于37℃下培养3h，然后加入125μL 1M IPTG，25℃下诱导过夜；离心(4000rpm，4℃，10min)收集菌体，加入4倍体积的磷酸缓冲液(pH＝7.0)重悬，重悬后对细胞进行超声破碎，离心(4000rpm，4℃，10min)取上清溶液进行冷冻干燥，获得酶12酶粉。
实施例1
本实施例采用手性金属催化剂催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000024
向100mL氢化釜中加入制备例3-1制得的化合物C 1.25g，甲醇50mL，三乙胺1.0g，再加入催化剂1,5-环辛二烯氯化铱二聚体16.7mg，配体(S)-(-)-1-[(R)-2-二苯基膦二茂铁乙基-二叔丁基膦30mg，加完物料后，通入氢气维持在2.5MPa，温度控制在60℃。反应完毕后，浓缩除去溶剂，残余物再用正庚烷打浆，过滤，浓缩，即得到目标化合物；纯度96％，光学纯度72％。
对制备得到的(S)-尼古丁柱层析纯化后进行核磁氢谱表征，如图1所示，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例2
本实施例采用手性金属催化剂催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000025
向100mL氢化釜中加入制备例3-2制得的化合物C 1.25g，甲醇50mL，三乙胺1.0g，再加入催化剂1,5-环辛二烯氯化铱二聚体16.7mg，配体(+)-1,2-双((2S,5S)-2,5-二苯基膦)乙烷27mg，加完物料后，通入氢气维持在3.0MPa，温度控制在60℃。反应完毕后，浓缩除去溶剂，残余物再用正庚烷打浆，过滤，浓缩，即得到目标化合物；纯度94％，光学纯度80％。
对制备得到的(S)-尼古丁柱层析纯化后进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例3
本实施例采用手性金属催化剂催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000026
向100mL氢化釜中加入制备例3-3制得的化合物C 1.25g，甲醇50mL，三乙胺1.0g，再加入催化剂1,5-环辛二烯氯化铱二聚体16.7mg，配体(R,R)-f-SpiroPhos 38mg，加完物料后，通入氢气维持在5.5MPa，温度控制在80℃。反应完毕后，反应液直接浓缩，除去溶剂，残余物再用正庚烷打浆，过滤，浓缩，即得到目标化合物；纯度90％，光学纯度88％。
对制备得到的(S)-尼古丁柱层析纯化后进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例4
本实施例采用手性金属催化剂催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000027
其制备方法与实施例3的区别仅在于将催化剂1,5-环辛二烯氯化铱二聚体16.7mg，配体(R,R)-f-SpiroPhos 38mg替换为Rh[(R,R)-DIPAMP](COD)BF 4 45mg，其他条件均保持不变。得到目标化合物；纯度45％，光学纯度27％。
对制备得到的(S)-尼古丁柱层析纯化后进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例5
本实施例采用手性金属催化剂催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000028
向100mL氢化釜中加入制备例3-4制得的化合物C 1.25g，乙醇60mL，三乙胺1.0g，再加入催化剂Rh[(R,R)-DIPAMP](COD)BF 4 45mg，加完物料后，通入氢气维持在4.0MPa，温度维持在90℃。反应合格后，浓缩除去溶剂，残余物再用正庚烷打浆，过滤，浓缩，即得到目标化合物；纯度85％，光学纯度58％。
对制备得到的(S)-尼古丁柱层析纯化后进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例6
本实施例采用手性金属催化剂催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000029
向100mL氢化釜中加入制备例3-5制得的化合物C 1.25g，甲醇50mL，二异丙基乙胺1.2g，再加入催化剂(-)-1,2-双((2S,5S)-2,5-二甲基磷)乙烷(环辛二烯)四氟硼酸铑39mg，加完物料后，通入氢气维持在1.5MPa，温度控制在100℃。反应完成后，浓缩除去溶剂，残余物再用甲基叔丁基醚打浆，过滤，浓缩，即得到目标化合物；纯度64％，光学纯度82％。
对制备得到的(S)-尼古丁柱层析纯化后进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例7
本实施例采用手性金属催化剂催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000030
向100mL氢化釜中加入制备例4-1制得的化合物C 1.25g，甲醇50mL，三乙胺1.0g，再加入催化剂[(R)-(+)-2,2'-双(二苯基磷)-1,1'-联萘]二氯化钌35mg，加完物料后，通入氢气维持在3.0MPa，温度控制在90℃，中控反应合格后，反应液直接浓缩，除去溶剂，残余物再用正庚烷打浆，过滤，浓缩，即得到目标化合物；纯度59％，光学纯度66％。
对制备得到的(S)-尼古丁柱层析纯化后进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例8
本实施例采用生物酶催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000031
向10个5mL离心管中加入30mg制备例4-2制得的化合物C，加入2mL 0.1M的磷酸盐缓冲液，调节pH至6.0。再向反应瓶中加入32mg葡萄糖，搅拌至完全溶解，再向其中分别加入30mg制备例5-2制得的氨基酸序列依次为SEQ ID No.2-SEQ ID No.22所示的亚胺还原酶。在另一个5mL离心管中加入3mL 0.1M的磷酸盐缓冲液，40mg制备例5-1制得的氨基酸序列为SEQ ID No.1所示的酶2和40mg NADP盐，搅拌至完全溶解。然后将第二个离心管中的溶液分别取0.1mL缓慢加入第一个离心管中，升温至25℃，以300r/min搅拌反应16h。分别取反应液0.1mL加入0.9mL的甲醇中振荡1min后过滤至1mL液相瓶中供高效液相分析，以(S)-尼古丁的面积比为转化率。
转化率数据如表2所示：
表2
酶 转化率(％) ee值(％) 构型
1 99.5 99.4 S
3 99.6 100 S
4 94.6 99.1 S
5 89.5 85.5 S
6 88.6 95.3 S
7 0 / /
8 56.4 88.1 S
9 83.2 99.2 R
10 77.9 87.6 R
11 23.5 99.5 S
13 99.5 99.9 S
14 99.5 99.5 S
15 99.8 99.8 S
16 99.5 99.7 S
17 99.5 99.7 S
18 99.8 99.5 S
19 99.5 99.3 S
20 99.3 99.5 S
21 99.5 99.3 S
22 99.9 99.9 S
23 99.3 99.2 S
由表2数据可知：相比于其他酶，酶1，酶3，酶4，酶13-18，酶22的催化效果最好，转化率能达到94.6％以上。
实施例9
本实施例采用生物酶催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000032
向50mL三口圆底烧瓶中加入4.5g制备例4-1制得的化合物C，加入20mL 0.1M的磷酸盐缓冲液，调节pH至7.0。再向反应瓶中加入4.8g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入10mL 0.1M的磷酸盐缓冲液，0.3g制备例5-2制得的酶1，0.04g制备例5-1制得的酶2和0.008g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中的溶液缓慢加入第一个烧瓶中，升温至30℃，以300r/min搅拌反应16h。过滤，滤液用氢氧化钠溶液调节至pH＝10后用甲基叔丁基醚萃取，无水硫酸钠干燥，浓缩后得(S)-尼古丁2.6g。纯度99％，光学纯度100％，收率89.5％。
对制备得到的(S)-尼古丁进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例10
本实施例采用生物酶催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000033
向50mL三口圆底烧瓶中加入4.5g制备例3-1制得的化合物C，加入20mL 0.1M的磷酸盐缓冲液，调节pH至6.0。再向反应瓶中加入4.8g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入10mL 0.1M的磷酸盐缓冲液，0.4g制备例5-2制得的酶3，0.04g制备例5-1制得的酶2和0.008g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中的溶液缓慢加入第一个烧瓶中，升温至35℃，以300r/min搅拌反应16h。过滤，滤液用氢氧化钠溶液调节至pH＝10后用乙酸乙酯萃取，无水硫酸钠干燥，浓缩后得(S)-尼古丁2.4g。纯度99％，光学纯度99.4％，收率82.6％。
对制备得到的(S)-尼古丁进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
实施例11
本实施例采用生物酶催化还原制备(S)-尼古丁：
Figure PCTCN2022095423-appb-000034
向50mL三口圆底烧瓶中加入4.5g制备例3-1制得的化合物C，加入20mL 0.1M的磷酸盐缓冲液，调节pH至6.0。再向反应瓶中加入4.8g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入10mL 0.1M的磷酸盐缓冲液，0.4g制备例5-3制得的酶12和0.008g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中的溶液缓慢加入第一个烧瓶中，升温至35℃，以300r/min搅拌反应16h。过滤，滤液用氢氧化钠溶液调节至pH＝10后用乙酸乙酯萃取，无水硫酸钠干燥，浓缩后得(S)-尼古丁2.3g。纯度99％，光学纯度99.6％，收率79.2％。
对制备得到的(S)-尼古丁进行核磁氢谱表征，数据为： 1H-NMR(400MHz,CDCl 3):δppm 8.54(1H,d),8.50(1H,dd),7.70(1H,dt),7.24-7.27(1H,m),3.22-3.27(1H,m),3.08(1H,t),2.27-2.34(1H,m),2.17-2.24(1H,m),2.16(3H,m),1.91-2.02(1H,m),1.79-1.87(1H,m),1.68-1.76(1H,m)。表明(S)-尼古丁被成功合成。
申请人声明，本申请通过上述实施例来说明本申请的一种利用还原方式制备(S)-尼古丁的方法，但本申请并不局限于上述实施例，即不意味着本申请必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。
以上详细描述了本申请的优选实施方式，但是，本申请并不限于上述实施方式中的具体细节，在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单 变型均属于本申请的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。

Claims (12)

  1. 一种利用还原方式制备(S)-尼古丁的方法，其包括：
    将式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物进行还原反应，得到(S)-尼古丁：
    Figure PCTCN2022095423-appb-100001
  2. 如权利要求1所述的方法，其中，所述还原反应采用生物酶催化方法，包括：
    在辅酶循环系统的条件下，以亚胺还原酶为催化剂进行催化还原反应，催化还原式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物，得到(S)-尼古丁。
  3. 如权利要求2所述的方法，其中，所述辅酶循环系统包括辅酶、葡萄糖和葡萄糖脱氢酶；
    优选地，所述辅酶包括NADP盐和/或NAD盐，优选NADP盐；
    优选地，所述葡萄糖脱氢酶包括SEQ ID No.1所示的氨基酸序列。
  4. 如权利要求2所述的方法，其中，所述亚胺还原酶包括SEQ ID No.2-SEQ ID No.6、SEQ ID No.8、SEQ ID No.11、SEQ ID No.12、或具有与SEQ ID No.12至少95％同一性的氨基酸序列，优选SEQ ID No.2-SEQ ID No.4、SEQ ID No.12、或具有与SEQ ID No.12至少95％同一性的氨基酸序列。
  5. 如权利要求2所述的方法，其中，所述催化还原反应在15-45℃下进行；
    优选地，所述催化还原反应在缓冲液体系中进行，所述缓冲液包括磷酸盐缓冲液、三羟甲基甲胺-盐酸缓冲液或三乙醇胺-盐酸缓冲液；
    优选地，所述催化还原反应在pH＝6.0-8.0下进行。
  6. 如权利要求1所述的方法，其中，所述还原反应采用手性金属催化剂催化方法，包括：
    在氢气气氛中，式Ⅰ所示的烯胺化合物和/或式Ⅱ所示的亚胺正离子化合物被手性金属催化剂催化还原为(S)-尼古丁。
  7. 如权利要求6所述的方法，其中，所述手性金属催化剂包括手性铱催化剂、手性钌催化剂或手性铑催化剂，优选手性铱催化剂；
    优选地，在所述催化还原体系中还添加配体；
    优选地，所述催化还原反应在50-100℃下进行；
    优选地，所述催化还原反应体系中的氢气维持在1.0-6.0MPa；
    优选地，所述催化还原反应在pH＝4.0-13.0下进行。
  8. 如权利要求1-7中任一项所述的方法，其中，所述式Ⅰ所示的烯胺化合物或式Ⅱ所示的亚胺正离子化合物是由式Ⅲ所示的化合物或其盐经过脱盐和/或环化得到的：
    Figure PCTCN2022095423-appb-100002
    优选地，所述盐包括盐酸盐、二盐酸盐、氢溴酸盐、二氢溴酸盐、硫酸盐或硫酸氢盐。
  9. 如权利要求8所述的方法，其中，式Ⅲ所示化合物的盐的合成方法包括：
    将化合物A与N-甲基吡咯烷酮、有机碱混合反应，得到化合物D；化合物D再与酸混合反应，得到式Ⅲ所示化合物的盐；其反应式如下所示：
    Figure PCTCN2022095423-appb-100003
  10. 如权利要求8所述的方法，其中，式Ⅲ所示化合物的盐的合成方法包括：
    将化合物A与N-甲基吡咯烷酮、有机碱混合反应后，加入酸中和至pH＝7-8，得到化合物B；化合物B再与酸混合反应，得到式Ⅲ所示化合物的盐；其反应式如下所示：
    Figure PCTCN2022095423-appb-100004
  11. 如权利要求9或10所述的方法，其中，所述化合物A的合成方法包括：将烟酸与甲醇混合，在强酸性环境中进行酯化反应，即得。
  12. 如权利要求1-11中任一项所述的方法，其中，所述方法包括：
    将烟酸与甲醇混合，在强酸性环境中进行酯化反应，得到化合物A；化合物A与N-甲基吡咯烷酮、有机碱混合反应后，可选地加入酸进行中和，得到化合物B或D；然后再与酸混合反应，得到式Ⅲ所示化合物的盐；脱盐及发生环化，最后采用生物酶催化方法或手性金属催化剂催化方法进行还原反应，得到(S)-尼古丁；其反应式如下所示：
    Figure PCTCN2022095423-appb-100005
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014174505A2 (en) 2013-04-22 2014-10-30 Perrigo Api Ltd. A process for the preparation of nicotine comprising the enzymatic reduction of 4- (methylamino) -1- (pyridin-3- yl) butan-1-one
US20180030028A1 (en) * 2014-12-09 2018-02-01 Njoy, Llc Synthesis and resolution of nicotine
CN110256403A (zh) * 2019-07-02 2019-09-20 深圳市馨艺坊生物科技有限公司 一种人工合成尼古丁的制备方法
WO2020098978A1 (en) * 2018-11-16 2020-05-22 Zanoprima Lifesciences Limited Process for the preparation of (s)-nicotin from myosmine
US10913962B2 (en) 2018-11-16 2021-02-09 Zanoprima Lifesciences Limited Process of making (S)-nicotine
CN112795603A (zh) * 2020-12-14 2021-05-14 山东金城医药化工有限公司 一种制备(s)-2-(3-吡啶)-吡咯烷的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636146B (zh) * 2017-02-16 2019-09-13 云南省烟草农业科学研究院 一种降低尼古丁转化率的cyp82e5基因突变体及其应用
CN110627769A (zh) * 2019-09-27 2019-12-31 深圳黑尔格科技有限公司 亚胺盐衍生物、其制备方法及尼古丁的制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014174505A2 (en) 2013-04-22 2014-10-30 Perrigo Api Ltd. A process for the preparation of nicotine comprising the enzymatic reduction of 4- (methylamino) -1- (pyridin-3- yl) butan-1-one
US20180030028A1 (en) * 2014-12-09 2018-02-01 Njoy, Llc Synthesis and resolution of nicotine
WO2020098978A1 (en) * 2018-11-16 2020-05-22 Zanoprima Lifesciences Limited Process for the preparation of (s)-nicotin from myosmine
US10913962B2 (en) 2018-11-16 2021-02-09 Zanoprima Lifesciences Limited Process of making (S)-nicotine
CN110256403A (zh) * 2019-07-02 2019-09-20 深圳市馨艺坊生物科技有限公司 一种人工合成尼古丁的制备方法
CN112795603A (zh) * 2020-12-14 2021-05-14 山东金城医药化工有限公司 一种制备(s)-2-(3-吡啶)-吡咯烷的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. AUO12718.1
SVANTE BRANDANGE , LARS LINDBLOM , AKE PILOTTI , BENITO RODERIQUEZ: "Ring-Chain Tautomerism of Pseudooxynicotine and Some Other Iminium Compounds", ACTA CHEMICA SCANDINAVICA B, vol. 37, no. 7, 31 December 1983 (1983-12-31), pages 617 - 622, XP093011792 *

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