WO2021180019A1 - 一种(s)-尼古丁的合成方法 - Google Patents

一种(s)-尼古丁的合成方法 Download PDF

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WO2021180019A1
WO2021180019A1 PCT/CN2021/079492 CN2021079492W WO2021180019A1 WO 2021180019 A1 WO2021180019 A1 WO 2021180019A1 CN 2021079492 W CN2021079492 W CN 2021079492W WO 2021180019 A1 WO2021180019 A1 WO 2021180019A1
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nicotine
synthesizing
nornicotine
coenzyme
buffer
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PCT/CN2021/079492
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French (fr)
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林文清
郑宏杰
陈泽聪
朱剑平
乐庸堂
胡集铖
王建冲
李凌宇
刘小波
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重庆博腾制药科技股份有限公司
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Priority to EP21768682.3A priority Critical patent/EP4119671A4/en
Publication of WO2021180019A1 publication Critical patent/WO2021180019A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)

Definitions

  • the invention belongs to the technical field of organic synthesis, and specifically relates to a method for synthesizing (S)-nicotine, in particular to a method for synthesizing (S)-nicotine with high chemical purity and high optical purity.
  • (S)-Nicotine (nicotine) is an alkaloid found in Solanaceae (Solanum) and an important component of tobacco. Tobacco leaves contain 1.5%-3.5% (S)-nicotine. Extracting nicotine from tobacco leaves is currently the most important method for obtaining nicotine. Although there have been reports on chemical synthesis methods, the chemical synthesis methods are not yet mature. 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 reagent methods.
  • Patent US20160326134 reports a method for obtaining (S)-nicotine by condensation with NMP, intramolecular rearrangement, reduction, and chiral resolution using methyl nicotinate as a raw material.
  • the synthesis route is shown below.
  • the asymmetric catalytic hydrogenation method requires the use of expensive chiral metal catalysts, so the cost is high, and the reaction needs to be carried out under high pressure, the equipment investment is large, and the use of hydrogen also has potential safety hazards.
  • the literature only 6-substituted substrates have better catalytic performance, and the direct hydrogenation of mesmine has not yet achieved a technological breakthrough.
  • the chiral auxiliary reagent method of this method requires the use of stoichiometric chiral auxiliary reagents, and cannot be recovered, resulting in high cost and no practical application value.
  • the purpose of the present invention is to provide a method for synthesizing (S)-nicotine, especially to provide a method for synthesizing (S)-nicotine with high chemical purity and high optical purity.
  • the present invention provides a method for synthesizing (S)-nicotine, which includes:
  • the coenzyme is used as the hydrogen donor, and the imine reductase is used as the catalyst to catalyze the reduction of mesmine to (S)-nornicotine, and (S)-nornicotine is then methylated Chemical reaction to obtain the (S)-nicotine.
  • the synthesis method is simple, safe and reliable, high yield, low cost, and has excellent chemical selectivity.
  • the purity of the product can be as high as 99.5% or more, and it also has excellent enantioselectivity, and the optical purity can reach 99.5%. above.
  • the chemical name of Mesmin involved in the present invention is 3-(3,4-dihydro-2H-pyrrol-5-yl)pyridine, and the chemical name of (S)-nornicotine is (S)-3-(pyrrole) Alk-2-yl)pyridine.
  • the coenzyme circulation system includes coenzyme, glucose and glucose dehydrogenase.
  • the coenzyme includes NADP salt and/or NAD salt, preferably NADP salt.
  • the glucose dehydrogenase includes any one or a combination of at least two of GDH101-GDH109, specifically, the glucose dehydrogenase includes GDH101, GDH102, GDH103, GDH104, GDH105, GDH106, GDH107, GDH108 Or any one or a combination of at least two of GDH109.
  • GDH109 (batch number: S20191121) is preferred.
  • the above-mentioned glucose dehydrogenases were purchased from Shanghai Shangke Biomedical Co., Ltd.
  • the imine reductase includes any one or a combination of at least two of IRED101-IRED112, IRED1321104, IRED1321108, IRED1321110 or IRED1321114, specifically, the imine reductase includes IRED101, IRED102, IRED103, IRED104 , IRED105, IRED106, IRED107, IRED108, IRED109, IRED110, IRED111, IRED112, IRED1321104, IRED1321108, IRED1321110 or IRED1321114 any one or a combination of at least two of them.
  • IRED103 batch number: S20191121
  • IRED1321110 any one or a combination of at least two of them.
  • IRED101-IRED112 was purchased from Shanghai Shangke Biomedical Co., Ltd., and IRED1321104, IRED1321108, IRED1321110 or IRED1321114 were independently mined from the gene bank.
  • IRED1321104 is from Streptomyces Kanamyceticus
  • IRED1321108 is from Mesorhizobium sp.L2C084
  • IRED1321110 is from Salinispora pacifica
  • IRED1321114 is from Mesorhizobium ciceri.
  • the above-selected imine reductase was sent to the gene synthesis company for synthesis and loaded into the plasmid pET28(a), induced to be introduced into E. coli BL21 to construct a recombinant strain of Escherichia coli.
  • the above recombinant strains were inoculated into autoclaved LB medium (sodium chloride 10g/L, tryptone 10g/L, yeast powder 5g/L), and cultivated at 37°C and 220RPM to OD600 to 2-3. Add 0.1M IPTG and induce at 25°C for 16h. The cells were collected by centrifugation, and the cell debris was removed by centrifugation after sonication to obtain the enzyme solution. Homemade enzyme powder is obtained after freeze-drying.
  • the temperature of the catalytic reduction reaction is 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 the range can be selected, I will not repeat them here. It is preferably 25-30°C.
  • the time of the catalytic reduction reaction is 8-72h, such as 8h, 10h, 12h, 16h, 24h, 30h, 48h, 56h, 60h or 72h, etc., and other specific points within the range can be selected, here I will not repeat them one by one.
  • the catalytic reduction reaction is carried out under stirring conditions, and the stirring speed is 150-400r/min, such as 150r/min, 200r/min, 250r/min, 300r/min, 350r/min or 400r/min Etc., other specific point values within the range can be selected, so I won’t repeat them here.
  • the stirring speed is 150-400r/min, such as 150r/min, 200r/min, 250r/min, 300r/min, 350r/min or 400r/min Etc., other specific point values within the range can be selected, so I won’t repeat them here.
  • the catalytic reduction reaction is performed in a buffer system
  • the buffer includes a phosphate buffer, a trimethylolamine-hydrochloric acid buffer or a triethanolamine-hydrochloric acid buffer.
  • other specific point values within the range can be selected, so I won’t repeat them here.
  • Preferably 5.8-6.5 Preferably 5.8-6.5.
  • the methylation reaction includes: mixing and reacting (S)-nornicotine with formaldehyde and formic acid to obtain the (S)-nicotine.
  • the temperature of the methylation reaction is 55-80°C, such as 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, etc., and other specific points within the range can be selected. I will not repeat them one by one.
  • the time of the methylation reaction is 8-48h, such as 9h, 12h, 18h, 26h, 32h, 38h, 46h or 48h, etc., and other specific points within the range can be selected. Let me repeat them one by one.
  • the synthesis method of (S)-nicotine includes:
  • the mass ratio of mesmine, coenzyme and imine reductase is 1:(0.001-0.05):(0.05-0.1).
  • the mass ratio of coenzyme, glucose and glucose dehydrogenase is (0.001-0.05):(1.2-2.4):(0.01-0.05).
  • the preparation method has better chemical selectivity.
  • the present invention has the following beneficial effects:
  • the synthetic method of (S)-nicotine involved in the present invention is simple to operate, safe and reliable, high yield, low cost, and has excellent chemical selectivity, the purity of the product can be as high as 99.5% or more, and it also has excellent resistance. Enantioselectivity and optical purity can reach more than 99.5%.
  • Figure 1 is a hydrogen NMR spectrum characterization diagram of the Mesmin sample in Example 1;
  • Figure 2 is a liquid chromatogram of the chemical purity of the Mesmin sample in Example 1;
  • Figure 3 is a hydrogen nuclear magnetic spectrum characterization diagram of the (S)-nornicotine sample in Example 2;
  • Example 4 is a liquid chromatogram of the chemical purity of the (S)-nornicotine sample in Example 2;
  • Figure 5 is a chiral chromatogram of a nornicotine racemate sample
  • Figure 6 is a chiral chromatogram of the (S)-nornicotine sample in Example 2.
  • Figure 7 is a liquid chromatogram of the chemical purity of the (S)-nornicotine sample in Example 3;
  • Figure 8 is a chiral chromatogram of the (S)-nornicotine sample in Example 3.
  • Figure 9 is a liquid chromatogram of the chemical purity of the (S)-nornicotine sample in Example 6;
  • Figure 10 is a chiral chromatogram of the (S)-nornicotine sample in Example 6;
  • Example 11 is a hydrogen nuclear magnetic spectrum characterization diagram of the (S)-nicotine sample in Example 7;
  • Figure 12 is a liquid chromatogram of the chemical purity of the (S)-nicotine sample in Example 7;
  • Figure 13 is a chiral chromatogram of a nicotine racemate sample
  • Figure 14 is a chiral chromatogram of the (S)-nicotine sample in Example 7.
  • Example 15 is a liquid chromatogram of the chemical purity of the (S)-nicotine sample in Example 8.
  • Figure 16 is a chiral chromatogram of the (S)-nicotine sample in Example 8.
  • the compound mesmine was prepared, and the synthesis method was carried out with reference to the method in patent document EP2487172A1.
  • the resulting product was characterized by hydrogen nuclear magnetic spectroscopy.
  • the characteristic diagram is shown in Figure 1.
  • the nuclear magnetic data results are: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.97 (1H, d), 8.64 (1H, dd), 8.17 (1H, dt), 7.29-7.34 (m, 1H), 4.06 (2H), 2.91-2.96 (m, 2H), 2.01 -2.09 (m, 2H). Shows that Mesmine was successfully synthesized.
  • the sample was analyzed and tested by high performance liquid chromatography, and its purity was 99.3%.
  • the chromatographic characterization chart is shown in Figure 2.
  • This embodiment provides a method for synthesizing (S)-nornicotine, and the operation method is as follows:
  • the prepared (S)-nornicotine was characterized by hydrogen NMR spectroscopy.
  • the characterization diagram is shown in Figure 3.
  • the nuclear magnetic data results are: 1 H-NMR (400MHz, CDCl 3 ): ⁇ ppm 8.56(1H,d), 8.44 (1H, dd), 7.68 (1H, dt), 7.19-7.22 (m, 1H), 4.12 (1H, t), 3.13-3.19 (1H, m), 2.98-3.00 (1H, m), 2.15 2.23 (1H, m), 2.02 (1H, s), 1.80-1.93 (2H, m), 1.58-1.67 (1H, m). It shows that (S)-nornicotine was successfully synthesized.
  • the chemical purity of the prepared (S)-nornicotine was detected by a high performance liquid chromatograph. As shown in Figure 4, the purity was 93.6%.
  • the optical purity of the sample was detected by high performance liquid chromatography and a chiral chromatography column.
  • Figure 5 is the chiral separation spectrum of the nornicotine racemate.
  • the peak time of (R)-nornicotine is At 20.08min, the peak time of (S)-nornicotine was 21.79min, and the peak areas were 49.9% and 50.1% respectively;
  • Figure 6 is the chiral chromatogram of the product, and no (R)-nornicotine was detected. Alkali, only the (S)-nornicotine peak at 21.6 min is detected, that is, the optical purity of the sample is close to 100%.
  • This embodiment provides a method for synthesizing (S)-nornicotine, and the operation method is as follows:
  • the chemical purity and optical purity of the prepared (S)-nornicotine were determined using high performance liquid chromatography.
  • the specific operation method is the same as that in Example 2.
  • the chromatogram is shown in Figure 7.
  • the calculated chemical purity is 97.2%; the chiral chromatogram is shown in Figure 8, and the optical purity is close to 100%.
  • This embodiment provides a method for synthesizing (S)-nornicotine, and the operation method is as follows:
  • This embodiment provides a method for synthesizing (S)-nornicotine, and the operation method is as follows:
  • This embodiment provides a method for synthesizing (S)-nornicotine, and the operation method is as follows:
  • the chemical purity and optical purity of the prepared (S)-nornicotine were determined by high performance liquid chromatography.
  • the specific operation method is the same as that in Example 2.
  • the chromatogram is shown in Figure 9.
  • the calculated chemical purity is 95.9%; the chiral chromatogram is shown in Figure 10, and the optical purity is 99.9%.
  • This embodiment provides a method for synthesizing (S)-nicotine, and the operation method is as follows:
  • the prepared (S)-nicotine was characterized by hydrogen NMR spectroscopy.
  • the characteristic diagram is shown in Figure 11.
  • the NMR data results are: 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). Shows that (S)-nicotine was successfully synthesized.
  • This embodiment provides a method for synthesizing (S)-nicotine, and the operation method is as follows:
  • the chemical purity and optical purity of the prepared (S)-nicotine were determined using a high performance liquid chromatograph. The specific operation is the same as that in Example 5.
  • the chromatogram is shown in Figure 15, and the chemical purity is calculated to be 99.7%;
  • the sex chromatogram is shown in Figure 16, with an optical purity of 99.87%.
  • IRED103 and IRED1321110 are more preferred types of imine reductase.
  • glucose dehydrogenase In this example, the preferred types of glucose dehydrogenase are discussed, and the operation method is as follows:
  • Glucose dehydrogenase Conv.% Glucose dehydrogenase Conv.% GDH101 55 GDH106 20 GDH102 96.6 GDH107 twenty one GDH103 2.7 GDH108 0 GDH104 16 GDH109 100 GDH105 27 To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To
  • GDH109 is a more preferred type of glucose dehydrogenase.
  • This embodiment discusses the preferred types of coenzymes, and the operation method is as follows:
  • NADP sodium salt is a more preferred type of coenzyme.
  • the present invention uses the above-mentioned embodiments to illustrate a method for synthesizing (S)-nicotine of the present invention, but the present invention is not limited to the above-mentioned embodiments, which does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. .
  • Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. fall within the scope of protection and disclosure of the present invention.

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Abstract

提供了一种(S)-尼古丁的合成方法，包括：在辅酶循环系统的条件下，以辅酶为供氢体，以亚胺还原酶为催化剂，催化还原麦斯明为(S)-去甲烟碱，(S)-去甲烟碱再经甲基化反应得到所述(S)-尼古丁。

Description

一种(S)-尼古丁的合成方法 技术领域
本发明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，尤其涉及一种高化学纯度和高光学纯度的(S)-尼古丁的合成方法。
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US20160326134报道了以烟酸甲酯为原料，经过与NMP缩合，分子内重排，还原，手性拆分得到(S)-尼古丁的方法，其合成线路如下所示。
Figure PCTCN2021079492-appb-000001
文献(Peyton Jacob,Resolution of(±)-5-Bromonornicotine.Synthesis of(R)-and(S)-Nornicotine of High Enantiomeric Purity,The Journal of Organic Chemistry 1982,4165-4167)报道了利用手性酸为拆分剂，通过拆分、还原脱溴等过程得到(S)-去甲烟碱的方法，(S)-去甲烟碱可进一步方便地通过甲基化反应得到(S)-尼古丁，其合成路线如下所示。但化学拆分法的不足是收率低，成本高。
Figure PCTCN2021079492-appb-000002
文献(Gui Guo,Dong-Wei Sun,Shuang Yang,et al,Iridium-Catalyzed Asymmetric Hydrogenation of 2-Pyridyl Cyclic Imines:A Highly Enantioselective Approach to Nicotine Derivatives,Journal of the American Chemical Society,2015,90-93)报道了利用不对称催化氢化方法合成尼古丁的线路，其合成路线如下所示。
Figure PCTCN2021079492-appb-000003
但不对称催化氢化法需使用昂贵的手性金属催化剂，因而成本高，且反应需要在高压下进行，设备投入大，氢气的使用也存在着安全隐患。另外，从文献看，仅对6-取代底物的催化性能较好，而对麦斯明的直接氢化还未实现技术突破。
文献(Teck-Peng Loh,Jian-Rong Zhou,Xu-Ran Li,Keng-Yeow Sim,A novel reductive aminocyclization for the syntheses of chiral pyrrolidines:stereoselective syntheses of(S)-nornicotine and 2-(2’-pyrrolidl)-pyridines,Tetrahedron Letters,1999,7847-7850)报道了以氨基半乳糖为手性辅助试剂合成(S)-去甲烟碱的方法，它可进一步反应得到(S)-尼古丁，其合成路线如下所示。
Figure PCTCN2021079492-appb-000004
但该方法的手性辅助试剂法需要用化学计量的手性辅助试剂，且不能回收，导致成本高昂，没有实际应用价值。
综上所述，现有技术中各种(S)-尼古丁的化学合成方法均存在着不足，需要开发更经济、有效、安全的合成方法。
发明内容
针对现有技术的不足，本发明的目的在于提供一种(S)-尼古丁的合成方法，尤其提供一种高化学纯度和高光学纯度的(S)-尼古丁的合成方法。
为达到此发明目的，本发明采用以下技术方案：
本发明提供一种(S)-尼古丁的合成方法，所述合成方法包括：
在辅酶循环系统的条件下，以辅酶为供氢体，以亚胺还原酶为催化剂，催化还原麦斯明为(S)-去甲烟碱，(S)-去甲烟碱再经甲基化反应得到所述(S)-尼古丁。
本发明所涉及的合成方法可以由如下反应式表示：
Figure PCTCN2021079492-appb-000005
该合成方法操作简单、安全可靠，收率高，成本低，且具有极好的化学选 择性，产品的纯度可高达99.5％以上，还具有极好的对映选择性，光学纯度可达到99.5％以上。
本发明所涉及的麦斯明化学名称为3-(3,4-二氢-2H-吡咯-5-基)吡啶，(S)-去甲烟碱化学名称为(S)-3-(吡咯烷-2-基)吡啶。
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GDH101-GDH109中的任意一种或至少两种的组合，具体地，所述葡萄糖脱氢酶包括GDH101、GDH102、GDH103、GDH104、GDH105、GDH106、GDH107、GDH108或GDH109中的任意一种或至少两种的组合。优选GDH109(批号：S20191121)。上述这些葡萄糖脱氢酶购于上海尚科生物医药有限公司。
优选地，所述亚胺还原酶包括IRED101-IRED112、IRED1321104、IRED1321108、IRED1321110或IRED1321114中的任意一种或至少两种的组合，具体地，所述亚胺还原酶包括IRED101、IRED102、IRED103、IRED104、IRED105、IRED106、IRED107、IRED108、IRED109、IRED110、IRED111、IRED112、IRED1321104、IRED1321108、IRED1321110或IRED1321114中的任意一种或至少两种的组合。优选IRED103(批号：S20191121)或IRED1321110。其中IRED101-IRED112购自于上海尚科生物医药有限公司，IRED1321104、IRED1321108、IRED1321110或IRED1321114为自主从基因库中挖掘得到。
具体地，IRED1321104来自Streptomyces Kanamyceticus，IRED1321108来自Mesorhizobium sp.L2C084，IRED1321110来自Salinispora pacifica，IRED1321114来自Mesorhizobium ciceri。以上被选择的亚胺还原酶送至基因合成公司进行合成并装载在质粒pET28(a)中，诱导导入至E.coli BL21中构建大肠 杆菌重组菌种。取上述重组菌种接种于高压灭菌的LB培养基(氯化钠10g/L、胰蛋白胨10g/L、酵母粉5g/L)中，于37℃，220RPM培养至OD600至2-3。加入0.1M IPTG于25℃诱导16h。离心收集细胞，超声破碎后离心去除细胞碎片后得酶液。冷冻干燥后得自制酶粉。
优选地，所述催化还原反应的温度为15-45℃，例如15℃、20℃、25℃、30℃、35℃、40℃或45℃等，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25-30℃。
优选地，所述催化还原反应的时间为8-72h，例如8h、10h、12h、16h、24h、30h、48h、56h、60h或72h等，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搅拌条件下进行，所述搅拌的转速为150-400r/min，例如150r/min、200r/min、250r/min、300r/min、350r/min或400r/min等，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，所述缓冲液的pH值为5.0-9.0，例如pH＝5.0、pH＝5.5、pH＝6.0、pH＝6.5、pH＝7.0、pH＝7.5、pH＝8.0、pH＝8.5或pH＝9.0等，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5.8-6.5。
优选地，所述甲基化反应包括：将(S)-去甲烟碱与甲醛和甲酸混合反应，得到所述(S)-尼古丁。
优选地，所述甲基化反应的温度为55-80℃，例如55℃、60℃、65℃、70℃、75℃或80℃等，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，所述甲基化反应的时间为8-48h，例如9h、12h、18h、26h、32 h、38h、46h或48h等，范围内的其他具体点值均可选择，在此便不再一一赘述。
作为本发明的优选技术方案，所述(S)-尼古丁的合成方法包括：
将麦斯明、辅酶、葡萄糖、葡萄糖脱氢酶、缓冲液与亚胺还原酶混合后，在15-45℃下反应8-72h，得到(S)-去甲烟碱，然后将(S)-去甲烟碱与甲醛和甲酸混合后在55-80℃下反应8-48h，得到所述(S)-尼古丁。
优选地，麦斯明、辅酶和亚胺还原酶的质量比为1:(0.001-0.05):(0.05-0.1)。
优选地，辅酶、葡萄糖、葡萄糖脱氢酶的质量比为(0.001-0.05):(1.2-2.4):(0.01-0.05)。在上述质量关系下，该制备方法具有更好的化学选择性。
相对于现有技术，本发明具有以下有益效果：
本发明所涉及的(S)-尼古丁的合成方法操作简单、安全可靠，收率高，成本低，且具有极好的化学选择性，产品的纯度可高达99.5％以上，还具有极好的对映选择性，光学纯度可达到99.5％以上。
附图说明
图1是实施例1中麦斯明样品的核磁氢谱表征图；
图2是实施例1中麦斯明样品的化学纯度的液相色谱图；
图3是实施例2中(S)-去甲烟碱样品的核磁氢谱表征图；
图4是实施例2中(S)-去甲烟碱样品的化学纯度的液相色谱图；
图5是去甲烟碱外消旋体样品的手性色谱图；
图6是实施例2中(S)-去甲烟碱样品的手性色谱图；
图7是实施例3中(S)-去甲烟碱样品的化学纯度的液相色谱图；
图8是实施例3中(S)-去甲烟碱样品的手性色谱图；
图9是实施例6中(S)-去甲烟碱样品的化学纯度的液相色谱图；
图10是实施例6中(S)-去甲烟碱样品的手性色谱图；
图11是实施例7中(S)-尼古丁样品的核磁氢谱表征图；
图12是实施例7中(S)-尼古丁样品化学纯度的液相色谱图；
图13是尼古丁外消旋体样品的手性色谱图；
图14是实施例7中(S)-尼古丁样品的手性色谱图；
图15是实施例8中(S)-尼古丁样品化学纯度的液相色谱图；
图16是实施例8中(S)-尼古丁样品的手性色谱图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1
本实施例制备化合物麦斯明，其合成方法参照专利文献EP2487172A1中的方法进行，对所得产物进行核磁氢谱表征，表征图如图1所示，核磁数据结果为： 1H-NMR(400MHz,CDCl 3):δppm 8.97(1H,d),8.64(1H,dd),8.17(1H,dt),7.29-7.34(m,1H),4.06(2H),2.91-2.96(m,2H),2.01-2.09(m,2H)。表明麦斯明被成功合成。样品经高效液相色谱分析检测，其纯度为99.3％，色谱表征图如图2所示。
实施例2
本实施例提供一种(S)-去甲烟碱的合成方法，操作方法如下：
向50mL三口圆底烧瓶中加入3g麦斯明，加入10mL 0.1M的磷酸盐缓冲 液，调节pH至6.0。再向反应瓶中加入5.5g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入0.2g亚胺还原酶IRED103，0.04g葡萄糖脱氢酶GDH109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25℃，以300r/min搅拌反应24h。过滤，滤液用氯仿萃取，无水硫酸钠干燥，浓缩后得(S)-去甲烟碱2.2g。
对制备得到的(S)-去甲烟碱进行核磁氢谱表征，表征图如图3所示，核磁数据结果为： 1H-NMR(400MHz,CDCl 3):δppm 8.56(1H,d),8.44(1H,dd),7.68(1H,dt),7.19-7.22(m,1H),4.12(1H,t),3.13-3.19(1H,m),2.98-3.00(1H,m),2.15-2.23(1H,m),2.02(1H,s),1.80-1.93(2H,m),1.58-1.67(1H,m)。表明(S)-去甲烟碱被成功合成。
利用高效液相色谱仪对制得的(S)-去甲烟碱分别进行化学纯度检测，如图4所示，纯度为93.6％。利用高效液相色谱仪和手性色谱柱对样品的光学纯度进行了检测，图5是去甲烟碱外消旋体的手性分离谱图，(R)-去甲烟碱出峰时间是20.08min,(S)-去甲烟碱出峰时间是21.79min，其峰面积分别是49.9％和50.1％；图6是产品的手性色谱图，未检测到有(R)-去甲烟碱，只检测到21.6min的(S)-去甲烟碱峰，即样品的光学纯度接近100％。
实施例3
本实施例提供一种(S)-去甲烟碱的合成方法，操作方法如下：
向50mL三口圆底烧瓶中加入3g麦斯明，加入15mL 0.1M磷酸盐缓冲液，调节pH至5.8。再向反应瓶中加入5.5g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入0.3g亚胺还原酶IRED103，0.04g葡萄糖脱氢酶GDH102和0.01g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中溶液缓慢加入第一个烧 瓶中，升温至37℃，搅拌24h。过滤，滤液用氯仿萃取，无水硫酸钠干燥，浓缩后得(S)-去甲烟碱2.4g。
利用高效液相色谱仪对制得的(S)-去甲烟碱分别进行化学纯度和光学纯度的测定，具体操作方法同实施例2，色谱图如图7所示，计算得到其化学纯度为97.2％；手性色谱图如图8所示，光学纯度接近100％。
实施例4
本实施例提供一种(S)-去甲烟碱的合成方法，操作方法如下：
向50mL三口圆底烧瓶中加入3g麦斯明，加入15mL 0.1M磷酸盐缓冲液，调节pH至5.8。再向反应瓶中加入9g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入0.3g亚胺还原酶IRED1321110，0.03g葡萄糖脱氢酶GDH109和0.01g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中溶液缓慢加入第一个烧瓶中，升温至37℃，搅拌24h。过滤，滤液用氯仿萃取，无水硫酸钠干燥，浓缩后得(S)-去甲烟碱2.2g。光学纯度99.5％。
实施例5
本实施例提供一种(S)-去甲烟碱的合成方法，操作方法如下：
向50mL三口圆底烧瓶中加入3g麦斯明，加入15mL 0.1M磷酸盐缓冲液，调节pH至5.8。再向反应瓶中加入5.5g葡萄糖，搅拌至完全溶解。在另一个50mL烧瓶中加入0.3g亚胺还原酶IRED13121110，0.06g葡萄糖脱氢酶GDH102和0.006g NADP盐，搅拌至完全溶解。然后将第二个烧瓶中溶液缓慢加入第一个烧瓶中，升温至37℃，搅拌24h。过滤，滤液用氯仿萃取，无水硫酸钠干燥，浓缩后得(S)-去甲烟碱2.3g。光学纯度99.5％。
实施例6
本实施例提供一种(S)-去甲烟碱的合成方法，操作方法如下：
向1000mL三口圆底烧瓶中加入100g麦斯明，加入650mL 0.1M的磷酸盐缓冲液，调节pH至6.2。再向反应瓶中加入180g葡萄糖，搅拌至溶液清澈。在500mL烧瓶中加入100g浓度为10％的亚胺还原酶液IRED103，50g浓度为10％的GDH102葡萄糖脱氢酶液和5g NADP盐，搅拌至溶液清澈。然后将瓶中溶液缓慢加入1000mL三口圆底烧瓶中，升温至37℃，搅拌36h。过滤，滤液用氯仿萃取，无水硫酸钠干燥，浓缩后得(S)-去甲烟碱75g。
利用高效液相色谱仪对制得的(S)-去甲烟碱分别进行化学纯度和光学纯度的测定，具体操作方法同实施例2，色谱图如图9所示，计算得到其化学纯度为95.9％；手性色谱图如图10所示，光学纯度99.9％。
实施例7
本实施例提供一种(S)-尼古丁的合成方法，操作方法如下：
向三个500mL三口瓶中均加入40g(S)-去甲烟碱，32g 37％甲醛溶液，25g 85％甲酸溶液，升温到60℃反应24h。反应完成后，加入氢氧化钠，调节pH＝12。水相用甲叔醚萃取，合并萃取液，浓缩，减压蒸馏，得无色液体29g，即(S)-尼古丁。
对制备得到的(S)-尼古丁进行核磁氢谱表征，表征图如图11所示，核磁数据结果为： 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。
利用高效液相色谱仪对制得的(S)-尼古丁样品的化学纯度进行了检测，结果 如图12所示，检测纯度为99.9％。利用高效液相色谱仪和手性色谱柱对样品的光学纯度进行了检测，图13是尼古丁外消旋体的手性谱图，5.43min是(S)-尼古丁，6.17min是(R)-尼古丁，其峰面积比例分别是50.1％和49.9％；图14是样品的手性色谱图，从检测谱图可见5.43min峰的面积是99.65％，即(S)-尼古丁的纯度是99.65％。
实施例8
本实施例提供一种(S)-尼古丁的合成方法，操作方法如下：
向三个500mL三口瓶中均加入107g(S)-去甲烟碱，80g 37％甲醛溶液，升温到75℃。滴加60g 85％甲酸溶液，滴加完毕后，保温反应24h。反应完成后，加入氢氧化钠，调节pH＝12。水相用甲叔醚萃取，合并萃取液，浓缩，减压蒸馏，得无色液体80g，即(S)-尼古丁。
利用高效液相色谱仪对制得的(S)-尼古丁分别进行化学纯度和光学纯度的测定，具体操作同实施例5，色谱图如图15所示，计算得到其化学纯度为99.7％；手性色谱图如图16所示，光学纯度99.87％。
实施例9
本实施例对亚胺还原酶的优选类型进行讨论，操作方法如下：
分别称取10mg IRED酶粉于2mL反应瓶中，每个小瓶中分别加入9.2mg葡萄糖，1mg NAD钠盐，1mg NADP钠盐，2mg葡萄糖脱氢酶GDH105和900μL 0.1M pH7.0的磷酸盐缓冲液，充分震荡至溶清。然后向每个小瓶中加入5mg麦斯明和100μL DMSO。将上述小瓶盖紧置于30℃恒温摇床中震荡过夜。取样经HPLC分析转化率及手性，结果如表1所示：
表1
亚胺还原酶 Conv.％ e.e％ 亚胺还原酶 Conv.％ e.e％
IRED101 90.6 100 IRED109 36.4 -89.4
IRED102 0 0 IRED110 0 0
IRED103 100 100 IRED111 99.8 -27
IRED104 51.1 100 IRED112 99.8 -43.4
IRED105 4.1 0 IRED1321104 100 93.4
IRED106 29.2 83.8 IRED1321108 100 65
IRED107 84.7 100 IRED1321110 99.6 99.9
IRED108 99.2 38 IRED1321114 100 95.4
由表1数据可知：IRED103和IRED1321110是更优选的亚胺还原酶类型。
实施例10
本实施例对葡萄糖脱氢酶的优选类型进行讨论，操作方法如下：
分别称取2mg GDH酶粉于2mL反应瓶中，每个小瓶中分别加入9.2mg葡萄糖，1mg NAD钠盐，1mg NADP钠盐，10mg IRED103和900μL 0.1M pH7.0的磷酸盐缓冲液，充分震荡溶清。然后向每个小瓶中加入150mg麦斯明和100μL DMSO。将上述小瓶盖紧置于30℃恒温摇床中震荡过夜。取样经HPLC分析转化率，结果如表2所示下：
表2
葡萄糖脱氢酶 Conv.％ 葡萄糖脱氢酶 Conv.％
GDH101 55 GDH106 20
GDH102 96.6 GDH107 21
GDH103 2.7 GDH108 0
GDH104 16 GDH109 100
GDH105 27    
由表2数据可知：GDH109是更优选的葡萄糖脱氢酶类型。
实施例11
本实施例对辅酶的优选类型进行讨论，操作方法如下：
分别称取1mg NAD钠盐，1mg NADP钠盐于两个2mL反应瓶中，每个小瓶中分别加入9.2mg葡萄糖，2mg GDH109酶粉，10mg IRED103和900μL 0.1M pH7.0的磷酸盐缓冲液，充分震荡溶清。然后向每个小瓶中加入150mg麦斯明和100μL DMSO。将上述小瓶盖紧置于30℃恒温摇床中震荡过夜。取样经HPLC分析转化率，结果如表3所示：
表3
辅酶 Conv.％
NAD钠盐 25.9
NADP钠盐 100
由表3数据可知：NADP钠盐是更优选的辅酶类型。
申请人声明，本发明通过上述实施例来说明本发明的一种(S)-尼古丁的合成方法，但本发明并不局限于上述实施例，即不意味着本发明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，对本发明的任何改进，对本发明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，均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
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，本发明对各种可能的组合方式不再另行说明。

Claims (10)

  1. 一种(S)-尼古丁的合成方法，其特征在于，所述合成方法包括：
    在辅酶循环系统的条件下，以辅酶为供氢体，以亚胺还原酶为催化剂，催化还原麦斯明为(S)-去甲烟碱，(S)-去甲烟碱再经甲基化反应得到所述(S)-尼古丁。
  2. 如权利要求1所述的(S)-尼古丁的合成方法，其特征在于，所述辅酶循环系统包括辅酶、葡萄糖和葡萄糖脱氢酶。
  3. 如权利要求1或2所述的(S)-尼古丁的合成方法，其特征在于，所述辅酶包括NADP盐和/或NAD盐，优选NADP盐。
  4. 如权利要求2或3所述的(S)-尼古丁的合成方法，其特征在于，所述葡萄糖脱氢酶包括GDH101-GDH109中的任意一种或至少两种的组合，优选GDH109。
  5. 如权利要求1-4中任一项所述的(S)-尼古丁的合成方法，其特征在于，所述亚胺还原酶包括IRED101-IRED112、IRED1321104、IRED1321108、IRED1321110或IRED1321114中的任意一种或至少两种的组合，优选IRED103或IRED1321110。
  6. 如权利要求1-5中任一项所述的(S)-尼古丁的合成方法，其特征在于，所述催化还原反应的温度为15-45℃，优选25-30℃；
    优选地，所述催化还原反应的时间为8-72h；
    优选地，所述催化还原反应在搅拌条件下进行，所述搅拌的转速为150-400r/min。
  7. 如权利要求1-6中任一项所述的(S)-尼古丁的合成方法，其特征在于，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，所述缓冲液的pH值为5.0-9.0，优选5.8-6.5。
  8. 如权利要求1-7中任一项所述的(S)-尼古丁的合成方法，其特征在于，所述甲基化反应包括：将(S)-去甲烟碱与甲醛和甲酸混合反应，得到所述(S)-尼古丁。
  9. 如权利要求1-8中任一项所述的(S)-尼古丁的合成方法，其特征在于，所述甲基化反应的温度为55-80℃；
    优选地，所述甲基化反应的时间为8-48h。
  10. 如权利要求1-9中任一项所述的(S)-尼古丁的合成方法，其特征在于，所述合成方法包括：
    将麦斯明、辅酶、葡萄糖、葡萄糖脱氢酶、缓冲液与亚胺还原酶混合后，在15-45℃下反应8-72h，得到(S)-去甲烟碱，其中麦斯明、辅酶和亚胺还原酶的质量比为1:(0.001-0.05):(0.05-0.1)；然后将(S)-去甲烟碱与甲醛和甲酸混合后在55-80℃下反应8-48h，得到所述(S)-尼古丁。
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