Process for preparing (S)-nicotine from myosmine

Abstract

The present application provides a method for producing (S)-nicotine ([(S)-3-(1-methylpyrrolidin-2-yl)pyridine]) by a synthetic method.

Description

[0001] This application is a divisional application of the invention patent application with the Chinese patent application number 201980058561.8 and the invention title "Method for Preparing (S)-Nicotine from Myosmine", which is the Chinese national phase entry of the PCT international application PCT / EP2019 / 056194 filed on March 12, 2019 and entered the Chinese national phase on March 8, 2021. Technical Field

[0002] The present invention relates to a method for producing (S)-nicotine ([(S)-3-(1-methylpyrrolidin-2-yl)pyridine]) by a synthetic method. Background Art

[0003] Nicotine (3-[1-methylpyrrolidin-2-yl]pyridine) is a natural product that can be obtained from the leaves of the genus Nicotiana (i.e., tobacco plants). There is a significant demand for nicotine products throughout the tobacco industry and throughout the pharmaceutical field. For example, there is still a need for traditional tobacco products, such as traditional cigarettes, perhaps due to the addictive nature of nicotine. However, due to the increasing concern about the harmful effects of traditional cigarette products on consumer health, there is a growing demand for nicotine-containing tobacco replacement products, such as e-cigarette devices, patches, lozenges, nasal sprays, and chewing gums. Tobacco replacement products can be provided as alternatives to traditional tobacco products that can cause harmful carcinogenic effects (e.g., due to the presence of pyridine alkaloids, polycyclic aromatic compounds, phenols, and N-nitrosamines). Tobacco replacement products can be particularly useful for treating nicotine dependence. In the pharmaceutical field, there is also interest in the possible therapeutic applications of nicotine.

[0004] There are challenges in obtaining nicotine with an appropriate level of enantiomeric purity and chemical purity. Nicotine is optically active, i.e., it can exist in one of two possible enantiomeric forms: (R)-nicotine or (S)-nicotine. There are methods for obtaining a racemic mixture of nicotine (e.g., WO2016065209). However, it is recognized that (S)-nicotine (i.e., [(S)-3-(1-methylpyrrolidin-2-yl)pyridine]) has significantly higher activity than (R)-nicotine. Thus, in the tobacco industry and the pharmaceutical field, what is needed is nicotine with a high level of enantiomeric purity for the (S) enantiomer. The pharmaceutical industry has strict regulations on the required level of enantiomeric purity in new drug products, and the currently required level of nicotine enantiomeric purity may increase. In addition to the requirement for the enantiomeric purity of nicotine, in the pharmaceutical and tobacco industries, obtaining a high level of chemical purity is also important - chemical purity refers to the amount of nicotine (i.e., both (R) and (S) enantiomeric forms) compared to non-nicotine impurities. The pharmaceutical industry has very strict regulations on the required level of chemical purity of nicotine relative to non-nicotine impurities. In fact, for the chemical purity of nicotine, the current reference standard in the United States Pharmacopeia is at least 99% and the content of any one impurity does not exceed 0.5%. High chemical purity is also very important for the tobacco industry because the above-mentioned harmful carcinogenic effects can be caused by impurities that can exert carcinogenic effects.

[0005] (S)-Nicotine can be obtained by extracting the leaves of tobacco plants. However, when nicotine is obtained in this way, its chemical purity is usually lower than 95% due to the presence of related alkaloid impurities. The typical composition of a nicotine sample obtained by extracting from tobacco leaves includes 93% (S)-nicotine, 2.4% (S)-nornicotine, 3.9% (S)-anatabine, and 0.5% (S)-anabasine (E. Leete and M. Mueller, J. Am. Chem., Soc., 1982, 104, 6440 - 44). The chemical structures of the alkaloid impurities are similar to that of nicotine, so it is difficult to remove them. The actual composition of nicotine also depends on factors such as geographical origin and harvest season.

[0006] (S)-Nicotine can also be obtained by synthetic methods. There are various examples in the prior art for the production of (S)-nicotine by synthetic methods. For example, the prior art methods are such methods: wherein a racemic (i.e., equal amounts) mixture of (R)-nicotine and (S)-nicotine is prepared, and this racemic mixture is subsequently resolved to obtain the (S) enantiomer (US 8,389,733, US2014 / 0031554 and US 8,378,111). There are also examples in the prior art of synthetic methods for the production of (S)-nicotine using enzymes as biocatalysts (WO 2014 / 174505); outside the field of nicotine, the use of biocatalysts in enantioselective methods is generally known (LS Bleicher et al., J. Org. Chem., 1998, 63, 1109-18, WO2013 / 170050, WO2015 / 073555, PN Scheller et al., Chembiochem, 2014, 15, 2201-4, Gan et al., J Mol. Cat. B, Enzymatic, 2014, 110, 126-32). However, selectively synthesizing (S)-nicotine with high enantioselectivity (preferably over the (R) enantiomer), while also achieving high chemical purity, remains a challenge. Summary of the Invention

[0007] In a first aspect, the present invention provides a method for preparing (S)-nicotine, which comprises the following steps:

[0008] (i) reducing myosmine with an enzyme having imine reductase activity to form (S)-nornicotine; and

[0009] (ii) methylating the (S)-nornicotine formed in step (i) to form (S)-nicotine.

[0010] Surprisingly, it has been found that by steps (i) and (ii) of this method (wherein myosmine is used as the starting material), a very high enantiomeric purity and chemical purity of (S)-nicotine are achieved. This indicates that step (i) is a highly enantioselective synthesis step that prefers the (S) isomer, and step (ii) retains this preference in the final nicotine product while also maintaining a high chemical purity. This allows the production of (S)-nicotine without resorting to the resolution of racemic mixtures. High chemical purity is particularly advantageous; the reduction in the levels of unwanted impurities typically associated with nicotine results in a reduced risk of potential adverse effects related to the impurities. In addition, steps (i) and (ii) provide a convenient preparation method for preparing (S)-nicotine.

[0011] In a second aspect, the present invention provides a method for producing a pharmaceutical composition, which comprises forming (S)-nicotine using the method of the first aspect and including (S)-nicotine in the pharmaceutical composition together with one or more pharmaceutical excipients.

[0012] In a third aspect, the present invention provides a method for producing a formulation for an e-cigarette device, which comprises forming (S)-nicotine using the method of the first aspect and including (S)-nicotine in a solvent together with one or more additives.

[0013] In a fourth aspect, the present invention provides the use of myosmine and an enzyme having imine reductase activity in a method for forming (S)-nicotine.

[0014] In a fifth aspect, the present invention provides a kit comprising myosmine and an enzyme having imine reductase activity, which is used in the above method for forming (S)-nicotine. Detailed Description

[0015] As understood by those skilled in the art, myosmine, (S)-nornicotine, and (S)-nicotine have the following structures:

[0016]

[0017]

[0018] Those skilled in the art are familiar with suitable reaction schemes for preparing myosmine.

[0019] As used herein, "an enzyme having imine reductase activity" refers to an enzyme that is capable of asymmetrically reducing an imino group (especially a secondary imino group) to the corresponding amino group (especially a secondary amino group). In particular, the enzyme having imine reductase activity used in the methods disclosed herein is an enzyme that can catalyze the conversion of myosmine to (S)-nornicotine. Those skilled in the art are familiar with such enzymes. The enzyme can be added to the reaction mixture in various forms, such as in the form of spray-dried cells.

[0020] Preferably, the method uses an enzyme that can convert myosmine to (S)-nornicotine such that (S)-nornicotine is obtained with an enantiomeric excess of at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%. The enantiomeric excess is measured in the manner given in the examples. In the methods disclosed herein, such a high enantiomeric excess is also achieved for the (S)-nicotine finally obtained as the end product.

[0021] As understood by those skilled in the art, enzymes with imine reductase activity generally include NADH / NADPH-dependent oxidoreductases, such as NADH / NADPH-dependent dehydrogenases and NADH / NADPH-dependent imine reductases. NADH / NADPH-dependent dehydrogenases include those mentioned by the enzyme classification number E.C.1.1.1, and particularly include 6-phosphogluconate dehydrogenase mentioned by the enzyme classification number E.C.1.1.1.44. Imine reductases include those mentioned by the enzyme classification number E.C.1.5.1, especially those mentioned by the enzyme classification number E.C 1.5.1.48.

[0022] Examples of different types of imine reductases include thiazoline imine reductase, dihydrofolate reductase, Δ 1 -pyrroline-2-carboxylic acid reductase, Δ 1 -piperidine-2-carboxylic acid reductase, sanguinarine reductase, and 1,2-reticuline reductase. Such enzymes can be isolated from or derived from the following sources: such as Streptomyces, Verrucosispora, Mesorhizobium, Yersinia, Pseudomonas, Candida albicans, Eschscholzia, and Papaver.

[0023] Examples of possible enzymes also include those disclosed in WO2013170050 (the content of which is incorporated into this specification by reference).

[0024] The enzyme can be IRED_A, IRED_B, IRED_C, IRED_D, IRED_E, IRED_F, IRED_P, IRED_X, IRED_AB, IRED-20, or its homolog. IRED_A, IRED_B, IRED_C, IRED_D, IRED_E, IRED_F, IRED_P, IRED_X, and IRED_AB are purchased from Enzymicals; IRED-20 is purchased from Almac Group. For example, in one embodiment, the enzyme is IRED_A, IRED_B, IRED_C, IRED_D, IRED_E, IRED-20, or its homolog.

[0025] The enzymes disclosed herein may include an amino acid sequence according to any one of SEQ I.D.NO:1, SEQ I.D.NO:2, SEQ I.D.NO:3, SEQ I.D.NO:4 or its homologs. In another embodiment, the enzyme includes an amino acid sequence according to any one of SEQ I.D.NO:1, SEQ I.D.NO:2, SEQ I.D.NO:3 or SEQ I.D.NO:4.

[0026] As used herein, "its homologs" refers to such enzymes that contain an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the enzymes disclosed herein. For example, "its homologs" may mean such enzymes that contain an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence according to any one of SEQ I.D.NO:1, SEQ I.D.NO:2, SEQ I.D.NO:3 or SEQ I.D.NO:4.

[0027] As used herein, the term "sequence identity" refers to the relationship between two or more amino acid sequences. When a position in one sequence is occupied by the same amino acid residue in the corresponding position of the comparison sequence, the sequences are said to be "identical" at that position. The "sequence identity" percentage is calculated by determining the number of positions at which the same amino acid residue appears in both sequences to obtain the number of "identical" positions. Then, the number of "identical" positions is divided by the total number of positions in the comparison window and multiplied by 100 to obtain the "sequence identity" percentage. The "sequence identity" percentage is determined by comparing the two best-aligned sequences in the comparison window. To best-align the sequences for comparison, a portion of the polypeptide sequence in the comparison window may contain additions or deletions called gaps, while the reference sequence remains unchanged. The best alignment is such an alignment that produces the maximum possible number of "identical" positions between the reference sequence and the comparison sequence even with gaps. Known methods can be used to calculate the sequence identity level between coding sequences.

[0028] Sequence identity can be calculated using publicly available computer-based methods for determining sequence identity, including BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol., 215: 403-410, (1990)), the BLASTX program available from NCBI, and the Gap program available from Genetics Computer Group (Madison WI). The level of sequence identity is obtained using the Gap program, where the Gap penalty for amino acid sequence comparison is 50 and the Gap length penalty is 3.

[0029] Typically, step (i) involves reducing mescaline enzymatically in the presence of a suitable cofactor, particularly NADH or NADPH. As will be understood by those skilled in the art, the enzyme and cofactor can be introduced into the reaction mixture as separate components, or they can be introduced as part of the same component (e.g., in the form of whole microbial cells containing both the enzyme and the appropriate cofactor). A suitable cofactor recycling system can be present to convert the cofactor from its oxidized form (NAD+ or NADP+) to its reduced form (NADH or NADPH). Those skilled in the art are familiar with suitable cofactor recycling systems, which include glucose (monohydrate) / glucose dehydrogenase, formic acid / formic acid dehydrogenase, and isopropanol / alcohol dehydrogenase. When a cofactor recycling system is present, the cofactor can be added to the reaction mixture in its oxidized form (i.e., in the form of NAD+ or NADP+).

[0030] The cofactor itself can be present in the range of 0.02 parts by weight to 10 parts by weight per 100 parts of mescaline. Preferably, the cofactor can be present in the range of 0.05 parts by weight to 5 parts by weight per 100 parts of mescaline. More preferably, the cofactor can be present in the range of 0.5 parts by weight to 2 parts by weight per 100 parts of mescaline.

[0031] The amount of enzyme present in step (i) can be present in an amount of 0.1 parts by weight to 30 parts by weight per 100 parts of mescaline. Preferably, the amount of enzyme present in step (i) can be present in an amount of 0.5 parts by weight to 10 parts by weight of mescaline. Those skilled in the art will understand that the amount of enzyme present in step (i) can be adjusted according to the desired time of the reaction in step (i), where more enzyme can be used for a shorter reaction time and vice versa.

[0032] Step (i) can be carried out in the presence of an ion exchange resin, but preferably step (i) is carried out in the absence of an ion exchange resin. When an ion exchange resin is present, the ion exchange resin is an Amberlite resin, an Amberlyst resin, an Amberjet resin (such as Amberlite IR-120), or a Dowex resin, and these ion exchange resins are all commercially available from Aldrich.

[0033] The possible pH of step (i) can be in the range of pH 5 - 9.

[0034] (S)-Nornicotine is converted to (S)-nicotine in another step:

[0035] (ii) Methylating the (S)-nornicotine formed in step (i) to form (S)-nicotine.

[0036] Surprisingly, it was found that after step (ii), (S)-nicotine with particularly high chemical purity and particularly high enantiomeric excess was obtained.

[0037] The methylation step (i.e., step (ii)) can be carried out by a multi-step method. For example, step (ii) may include forming a compound (such as N-formyl-(S)-nornicotine), and then reducing the compound to obtain the methylation product, i.e., (S)-nicotine. However, preferably, step (ii) is carried out by a one-step method (such as reductive methylation). As understood by those skilled in the art, the term "reductive methylation" refers to the process of forming and reducing a substance in a single step to obtain the methylation product (i.e., (S)-nicotine).

[0038] Preferably, (S)-nornicotine is reductively methylated using formaldehyde or a formaldehyde-based compound. When such a reagent is used, step (ii) is particularly effective.

[0039] As used herein, a formaldehyde-based compound refers to a compound that can generate formaldehyde in situ during a chemical reaction. Those skilled in the art should understand that this means that the formaldehyde-based compound is added to the reaction mixture and then decomposed to release formaldehyde (and other related compounds), which can then react with (S)-nornicotine to form (S)-nicotine. In the case of adding a formaldehyde-based compound, those skilled in the art are familiar with how to adjust the appropriate amount of the added formaldehyde-based compound to achieve the in situ release of a specific amount of formaldehyde.

[0040] The chemical formula of formaldehyde itself is HC(O)H, and it is usually introduced in liquid or gaseous form. Formaldehyde can be introduced into the reaction mixture as part of an aqueous formaldehyde solution (such an aqueous solution can be called formalin).

[0041] Formaldehyde-based compounds are typically introduced in solid or liquid form. The formaldehyde-based compound can be a dimer of formaldehyde, a polymer of formaldehyde, or an acetal of formaldehyde. Preferably, the formaldehyde-based compound is a polymer of formaldehyde.

[0042] As understood by those skilled in the art, the term "polymer of formaldehyde" refers to a compound having three or more polymerized formaldehyde repeating units. Preferably, the polymer of formaldehyde is paraformaldehyde. As used herein, the term "paraformaldehyde" refers to a formaldehyde polymer having a degree of polymerization of 8 to 100 units.

[0043] When formaldehyde or a formaldehyde-based compound is used for the reductive methylation of (S)-nornicotine, the formaldehyde or formaldehyde-based compound can be added in an amount of 50 to 110 parts by weight (preferably 60 to 90 parts by weight) per 100 parts of (S)-nornicotine. Such an amount refers to the actual amounts of formaldehyde, formaldehyde-based compound, and (S)-nornicotine present. Thus, if, for example, (S)-nornicotine is formed as part of a solution (such as an aqueous solution) and / or when formaldehyde or a formaldehyde-based compound is introduced into the reaction mixture as part of a solution (such as an aqueous solution), the parts by weight disclosed herein refer to the actual amounts of formaldehyde, formaldehyde-based compound, and (S)-nornicotine contained in each solution.

[0044] In the case where the methylation step is a reductive methylation step, the reducing agent can be formic acid, sodium cyanoborohydride, or palladium / hydrogen, preferably formic acid. As understood by those skilled in the art, the appropriate amount of the reducing agent will depend on the specific reducing agent used. For example, when the reducing agent is formic acid, the reducing agent can be present in an amount of 40 - 110 parts by weight per 100 parts of (S)-nornicotine, preferably 40 - 100 parts by weight, more preferably 50 to 70 parts by weight. Such an amount refers to the actual amounts of the reducing agent and (S)-nornicotine present.

[0045] Preferably, steps (i) and (ii) can be carried out without separating the (S)-nornicotine formed in step (i). This allows for the formation of (S)-nicotine with high enantiomeric excess and high chemical purity while using a particularly convenient synthetic route. The need to separate (S)-nornicotine from the reaction mixture formed in step (i) and then convert it to (S)-nicotine is avoided, which has the advantage of providing a particularly convenient synthetic route because the separation of (S)-nornicotine is process-intensive due to expensive plant time and energy (e.g., due to the need for large amounts of solvent for extraction and / or concentrating solutions). For example, in step (i), (S)-nornicotine can be formed as part of an aqueous solution, and the aqueous solution containing (S)-nornicotine is then directly used in step (ii). Thus, the methylation step (step (ii)) is carried out on the aqueous solution of (S)-nornicotine formed in step (i). When the method is carried out in this way, reductive methylation of (S)-nornicotine is preferably carried out by using paraformaldehyde or by using formaldehyde introduced into the reaction mixture as part of an aqueous solution. When the method is carried out in this way, reductive methylation of (S)-nornicotine by using formaldehyde introduced into the reaction mixture as part of an aqueous solution is more preferred because it has been found that this reduces the unwanted foaming of the reaction mixture as the process proceeds.

[0046] The (S)-nicotine produced using the method disclosed herein has an enantiomeric excess of at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%. Those skilled in the art are familiar with how to measure enantiomeric excess. Enantiomeric excess can be measured, for example, in the manner given in the examples.

[0047] The (S)-nicotine produced using the method disclosed herein has a chemical purity of at least 98%, preferably at least 99%. Those skilled in the art are familiar with how to measure chemical purity. Chemical purity can be measured, for example, in the manner given in the examples. The chemical purity levels obtained through the examples are particularly high.

[0048] The (S)-nicotine produced using the above method steps can be included in a pharmaceutical composition together with one or more pharmaceutical excipients. Preferably, the pharmaceutical composition is a transdermal patch, lozenge, or inhalation preparation.

[0049] The (S)-nicotine prepared using the above method steps can also be included in a formulation for an e-cigarette device. The formulation includes (S)-nicotine in a solvent having one or more additives. The solvent can include glycerol, propylene glycol, water, or a mixture thereof. Preferably, the solvent includes glycerol and propylene glycol, where the ratio of glycerol to propylene glycol ranges from 80:20 to 20:80 (by volume). One or more additives can include one or more flavoring agents.

[0050] The present invention also provides a kit for a method for forming (S)-nicotine, which comprises myosmine and an enzyme having imine reductase activity.

[0051] A particularly preferred reaction scheme is shown below as Scheme 1:

[0052]

[0053] The present invention will be illustrated by the following non-limiting examples.

[0054] Examples

[0055] The following examples demonstrate the results related to the methods disclosed herein. Various reagents have been used to illustrate the method.

[0056] The enzymes used include the following enzymes:

[0057] IRED_A from Verrucosispora maris (strain AB-18-032, Uniprot: F4F8G5_VERMA), which has the amino acid sequence (a) or (b) given below - sequence (a) corresponds to SEQ I.D. NO:1 and sequence (b) corresponds to SEQ I.D. NO:2.

[0058] (a) When used with a hexahistidine tag, there are a total of 302 amino acid residues:

[0059] MHHHHHHAADSRAPVTVIGLGAMGSALARAFLAAGHPTTVWNRSPDKADDLVGQGAVRAATVADAMSAGNLIVICVLDYRAMREIIDSTGHSPADRVIVNLTSGTPGDARATAAWAQEQGMEYIDGAIMATPSMIGSEETLIFYGGPQEVYDAHADTLRSIAGAGTYLGEEPGLPSLYDVALLGLMWTTWAGFMHSAALLASEKVPAAAFLPYAQAWFEYVISPEVPNLATQVDTGAYPDNDSTLGMQTVAIEHLVEASRTQGVDPTLPEFLHARAEQAIRRGHAGDGFGAVFEVLRAPAAQ

[0060] (b) The native enzyme, with a total of 296 amino acid residues:

[0061] MAADSRAPVTVIGLGAMGSALARAFLAAGHPTTVWNRSPDKADDLVGQGAVRAATVADAMSAGNLIVICVLDYRAMREIIDSTGHSPADRVIVNLTSGTPGDARATAAWAQEQGMEYIDGAIMATPSMIGSEETLIFYGGPQEVYDAHADTLRSIAGAGTYLGEEPGLPSLYDVALLGLMWTTWAGFMHSAALLASEKVPAAAFLPYAQAWFEYVISPEVPNLATQVDTGAYPDNDSTLGMQTVAIEHLVEASRTQGVDPTLPEFLHARAEQAIRRGHAGDGFGAVFEVLRAPAAQ

[0062] IRED_B from Mesorhizobium sp. L48C026A00, also known as 6-phosphogluconate dehydrogenase, which has the amino acid sequence (a) or (b) given below - sequence (a) corresponds to SEQ I.D. NO:3 and sequence (b) corresponds to SEQ I.D. NO:4.

[0063] (a) When used with a hexahistidine tag, it has a total of 310 amino acid residues:

[0064] MHHHHHHASNVCVLGAGRMGSSIARTLLDRGYPTWVWNRTAAKCEPLAALGAKVASSVQEGIQAAEVVIINVLDYAASDALLKRDGIASALAGKAVVQLTSGSPRLAREEARWVEAHGAGYLDGAIMATPDFIGKPETAMLYSGSRDVYEKHKPLLFALGGGTNYVGELPGQASALDTALLTQMWGGLFGALQGMAVAEAEGLDLETFRNHLSAFKPVVDASLFDLVDRTNARRFAGDDATLASLGAHYSAFQHLLEACEERGLDAAMPRAMDMIFRQALSLGSMEDDLASLALLFRNGSPRQSREPANA

[0065] (b) The native enzyme, which has a total of 304 amino acid residues

[0066] MASNVCVLGAGRMGSSIARTLLDRGYPTWVWNRTAAKCEPLAALGAKVASSVQEGIQAAEVVIINVLDYAASDALLKRDGIASALAGKAVVQLTSGSPRLAREEARWVEAHGAGYLDGAIMATPDFIGKPETAMLYSGSRDVYEKHKPLLFALGGGTNYVGELPGQASALDTALLTQMWGGLFGALQGMAVAEAEGLDLETFRNHLSAFKPVVDASLFDLVDRTNARRFAGDDATLASLGAHYSAFQHLLEACEERGLDAAMPRAMDMIFRQALSLGSMEDDLASLALLFRNGSPRQSREPANA

[0067] Example 1

[0068] Bioconversion was carried out on a 0.5 mL scale using a solution of 10 mM mesopramine and NADP+ (0.5 mM), glucose (25 mM), glucose dehydrogenase (10 U / mL), and an enzyme with imine reductase activity. The enzymes used are detailed in Table 1 and are available from Enzymicals. The amount of each enzyme used was 9 mg / mL of cell-free extract (estimated to contain approximately 0.9 mg / mL of the enzyme). In particular for IRED_B and IRED_C, additional tests were carried out using 0.9 mg / mL of cell-free extract.

[0069] The enantiomeric excess of (S)-nornicotine obtained from the bioconversion was determined as follows: using a Chiralpak AD-H column (250 x 4.6 mm id (inner diameter)), eluting with a mixture of hexane:ethanol:diethylamine 74.9:25.0:0.1 (v / v / v) at 1 mL / min for 18 minutes at 30 °C. This method was also used to measure the conversion of mesopramine to nornicotine, and the relative response factor detected by uv absorption at 254 nm was determined to be 2.18:1.

[0070] The results are shown in Table 1 below.

[0071]

[0072]

[0073] Table 1

[0074] The enantiomeric excess % of (S)-nicotine is determined according to the formula [(S)-(R)] / ((S)+(R)]×100, where (S) and (R) are the amounts of the (S) enantiomer and the (R) enantiomer present, respectively. The conversion % is determined based on the consumption of myosmine, i.e., according to the formula 100 - (final amount of myosmine) / (starting amount of myosmine)×100.

[0075] Example 2

[0076] The reaction was carried out in a similar manner to Example 1, except that 1.5 equivalents of glucose and 1 mol% of NADP+ were used relative to the myosmine substrate, and a reaction time of 24 hours was used. The enzymes used are detailed in Tables 2, 3, and 4 (available from Enzymicals).

[0077] At a myosmine concentration of 100 mM, using 0.9 mg / mL of enzyme cell-free extract, the results are shown in the following table:

[0078] Enzyme Conversion [%] Enantiomeric excess [%S] i IRED_A 63.6 99.8 ii IRED_B 99.9 98.7 iii IRED_C 99.9 99.8 iv IRED_D 99.0 99.9 v IRED_E 99.9 99.9

[0079] Table 2

[0080] At a myosmine concentration of 100 mM, using 9 mg / mL of enzyme cell-free extract, the results are shown in the following table:

[0081] Enzyme Conversion [%] Enantiomeric excess [%S] i IRED_A 99.9 99.8 ii IRED_B 99.8 98.8 iii IRED_C 99.8 99.9 iv IRED_D 99.9 100.0 v IRED_E 99.9 99.9

[0082] Table 3

[0083] At a myosmine concentration of 250 mM, using 9 mg / mL of enzyme cell-free extract, the results are shown in the following table:

[0084] Enzyme Conversion [%] Enantiomeric excess [%S] i IRED_A 100.0 99.7 ii IRED_B 99.9 98.6 iii IRED_C 100.0 99.9 iv IRED_D 100.0 99.9 v IRED_E 99.9 99.9

[0085] Table 4

[0086] Example 3

[0087] A solution of the following substances in 100 mM sodium phosphate buffer (200 mL) at pH 7.5 was mixed at 30 °C at 200 rpm for 24 h using an overhead stirrer: mescaline (20 mmol, 2.924 g), D-glucose (30 mmol, 5.405 g), nicotinamide adenine dinucleotide phosphate sodium salt (0.2 mmol, 157 mg), a lyophilized cell-free extract of enzyme IRED_A (available from Enzymicals) (1.0 g), glucose dehydrogenase (2000 U, 40 mg). During the reaction, the nicotine in the solution was analyzed by HPLC, showing a conversion of 77% after 8 h and more than 99% after 24 h, with (S)-nicotine being 99.7% e.e. The solution was then treated at 80 °C with 37% formaldehyde solution (8.1 g) and formic acid (2.8 g) for 4 h, and the reaction was completed after 2 h. After cooling, 6 g of solid sodium hydroxide (pH 12.7) was added, and the mixture was extracted with 2 × 75 ml of MTBE. After drying with sodium sulfate, the solvent was removed to obtain 2.25 g of crude (S)-nicotine, which had a purity >99% as measured by HPLC (area % at 260 nm) and an enantiomeric excess of 98.7%.

[0088] Example 4

[0089] A solution of the following substances in 100 mM sodium phosphate buffer (200 mL) at pH 7.5 was mixed at 30 °C at 200 rpm for 24 h using an overhead stirrer: mescaline (20 mmol, 2.924 g), D-glucose (30 mmol, 5.405 g), nicotinamide adenine dinucleotide phosphate sodium salt (0.2 mmol, 157 mg), a lyophilized cell-free extract of enzyme IRED_B (purchased from Enzymicals) (0.5 g), glucose dehydrogenase (2000 U, 40 mg). During the reaction, the nicotine in the solution was analyzed by HPLC, showing a conversion of 91% after 4 h and more than 99% after 6 h. After 24 h, (S)-nicotine was 98.2% e.e. The solution was then treated at 80 °C with paraformaldehyde (3 g) and formic acid (2.8 g) for 6 h, and the reaction was completed after 4 h. After cooling, 6 g of solid sodium hydroxide (pH 12.7) was added, and the mixture was extracted with 2 × 75 ml of MTBE. After drying with sodium sulfate, the solvent was removed to obtain 2.31 g of crude (S)-nicotine, which had a purity >99% as measured by HPLC (area % at 260 nm) and an enantiomeric excess of 98.3%.

[0090] Example 5

[0091] This example confirmed the enantioselectivity and conversion rate at high substrate concentrations. This example was carried out in a similar manner to Example 1, except that all reactions were carried out over a 24-hour period using 1.5 equivalents of glucose, NADP (1%, relative to mescaline), imine reductase (specifically IRED_C (4.5 mg / ml cell-free extract) available from Enzymicals), GDH (10 U / ml for every 250 mM mescaline concentration), and 100 mM sodium phosphate buffer pH 7.5. The results are shown below.

[0092] Concentration of starting material myosmine Conversion [%] Enantiomeric excess [%S] i 250 mM 99.9 99.7 ii 400 mM 99.6 99.8 iii 600 mM 68.8 99.8 iv 800 mM 56.5 99.7 v 1000 mM 52.4 99.6

[0093] Table 5

[0094] Example 6

[0095] This example demonstrated the enantioselectivity and conversion rate on a larger scale.

[0096] The following substances were mixed in a solution of 100 mM sodium phosphate buffer (1000 mL) at pH 7.5 at 30 °C at 200 rpm for 24 hours using an overhead stirrer: mescaline (400 mmol, 58.5 g), D-glucose (600 mmol, 118.9 g), nicotinamide adenine dinucleotide phosphate sodium salt (4 mmol, 3.15 g), enzyme IRED_C (lyophilized cell-free extract available from enzymicals) (10.0 g), and glucose dehydrogenase CFE (0.32 g). After 24 hours, the nicotine in the solution was analyzed by HPLC and showed a conversion of over 98%.

[0097] The post-treatment was as follows: The biocatalytic reaction mixture was acidified to pH 1-2 with concentrated sulfuric acid and then heated to 90 °C for 20 minutes to precipitate all proteins. The proteins were filtered out of the mixture with diatomaceous earth. The resulting clarified solution was basified to pH > 11 with 40% NaOH solution and extracted four times with 500 mL of methyl tert-butyl ether (MTBE). The combined MTBE phases were dried over anhydrous magnesium sulfate and the solvent was evaporated. The yield of the separated nicotine as a pale yellow liquid was 41.1 g (70%).

[0098] Before post-treatment and separation, a separate sample of the nicotine reaction mixture was subjected to a methylation step. Specifically, paraformaldehyde (60 g) and formic acid (49.2 g) were added to the biocatalytic reaction mixture without separating the nicotine. The reaction was heated to 85 °C and stirred vigorously to form (S)-nicotine.

[0099] Example 7

[0100] The general experimental method for forming (S)-nicotine is as follows. At a concentration of 400 mM of myosmine, biocatalysis of myosmine to (S)-nornicotine is carried out using IRED_C (available from Enzymicals). (S)-Nornicotine is separated by extraction with methyl tert-butyl ether and removal of the solvent. Alternatively, the aqueous solution from the biocatalysis is heated at 90 °C for 15 minutes to precipitate the protein, and then the cooled mixture is acidified to pH 1-2 with sulfuric acid. The precipitated protein is removed by filtration through diatomaceous earth, and then the solution is neutralized to about pH 7 with aqueous sodium hydroxide solution.

[0101] Example 7a

[0102] The crude nornicotine isolated from the enzymatic reduction of myosmine (92 g) was added to 800 ml of water. Paraformaldehyde (74 g, 4 eq) and formic acid (58 g, 2 eq) were added. The mixture was gradually warmed to 80-85 °C. HPLC analysis after 2 h showed that the reaction was complete. The mixture was kept at the same temperature for another 2 h and then cooled to room temperature. 50% Sodium hydroxide solution was added to obtain a pH of about 13. The mixture was extracted with 2 × 500 ml of MTBE and dried over sodium sulfate. The solvent was removed and the crude (S)-nicotine was distilled under vacuum. After about 4 g of fore-run, 87 g of pure nicotine was obtained (measured by HPLC >99%, measured by chiral HPLC >99.6% ee).

[0103] Example 7b

[0104] Paraformaldehyde (112.5 g, 4 eq) and formic acid (88 g, 2 eq) were added to 2.5 L of an aqueous solution of nornicotine (5.63 g / 100 ml) from the same biocatalysis used in Example 1. The mixture was gradually heated to 80-85 °C, where the reaction started at about 70 °C and foamed somewhat due to the evolution of gas. After 1 h at 80-85 °C, HPLC indicated that the reaction was complete. The reaction was heated for a total of 4 h and then cooled. The mixture was basified with 50% sodium hydroxide solution and extracted with MTBE (800 ml, then 500 ml). After drying, the crude mixture was distilled to give 118.7 g of (S)-nicotine (measured by HPLC >99%, measured by HPLC >99.5% ee).

[0105] Example 7c

[0106] To a 2.5 L aqueous nicotine solution (5.63 g / 100 ml) from the same biocatalysis used in Example 1, 37% formaldehyde solution (290 ml, ~4 eq) and formic acid (88 g, 2 eq) were added. The mixture was gradually heated to 80 - 85 °C, where the reaction started at about 60 °C and foamed somewhat due to the evolution of gas. After 1 h at 80 - 85 °C, HPLC indicated that the reaction was complete. The reaction was heated for a total of 4 h and then cooled. The mixture was basified with 50% sodium hydroxide solution and extracted with MTBE (800 ml, then 500 ml). After drying, the crude mixture was distilled to give 119.1 g of (S)-nicotine (>99% by HPLC, >99.5% ee by HPLC).

[0107] Example 8

[0108] A solution of nicotine (298 g) and glucose monohydrate (505 g) was prepared in 0.1 M dipotassium hydrogen phosphate buffer (6 L). Amberlite IR-120 resin (2 kg, wet) was added as an ion exchange resin, and the solution was adjusted to pH 7 with 12 M sodium hydroxide (ca. 0.3 L), then stirred overnight at 25 °C to ensure a stable pH. Glucose dehydrogenase GDH-102 (6 g), β-NADP+ (6 g), and enzyme IRED-20 (30 g) commercially available from Almac Group were added, and the mixture was stirred at 150 rpm while maintaining at 25 °C, and the pH was maintained in the range of 6.8 - 7.0 by adding 4 M potassium hydroxide. After 72 h, the solution was decanted, and the Amberlite resin was washed with deionized water (3 × 3 L). The Amberlite resin was then transferred to a column, washed again with deionized water (4 L), then shaken with 2 M ammonia solution (4 L) for 3 h and washed with 2 M ammonia water (10 L). The combined solution was concentrated to dryness under reduced pressure to give (S)-nornicotine (131.2 g) as a yellow liquid. To further recover nornicotine from the reaction mixture, reactivated Amberlite resin (2 kg) was added thereto, and the mixture was stirred overnight at room temperature. The same treatment as above was repeated to further recover (S)-nornicotine (59.8 g), giving a total yield of 191.0 g. The above two batches were separately converted to (S)-nicotine. For the larger batch, (S)-nornicotine (126.2 g) was mixed with paraformaldehyde (154.5 g) and formic acid (118 g) dissolved in water (1 L), and the resulting stirred mixture was heated to 85 °C overnight. The mixture was then cooled to 0 °C and adjusted to pH 14 with 12 M sodium hydroxide. The mixture was extracted with methyl tert-butyl ether (3 × 8 volumes). The organic phase was dried over anhydrous magnesium sulfate and concentrated to dryness to give crude (S)-nicotine (131.2 g) as a yellow liquid. Similarly, the second batch of (S)-nornicotine (59.8 g) was converted to crude (S)-nicotine (60.7 g) in the same manner, giving a total yield of crude nicotine of 191.9 g. They were combined and distilled under reduced pressure (at 0.53 - 0.67 mbar, boiling point 70 - 77 °C) to give (S)-nicotine (174.5 g) as a colorless liquid, with an enantiomeric excess of 99.38% determined by HPLC and a chemical purity of 99.96% determined by HPLC. More details of the methods for measuring enantiomeric excess and chemical purity are given below.

[0109] Enantiomeric purity determined by HPLC: Using a Chiracel OD-H column, eluted with a mixture of 95:5 n-hexane and 1-butanol containing 0.1% diethylamine. The (R)-enantiomer was eluted at 6.1 minutes and the (S)-enantiomer was eluted at 5.6 minutes. The enantiomeric excess was determined by the peak areas according to the formula [(S)-(R)] / ((S)+(R)]. The enantiomeric excess thus determined was 99.38%.

[0110] Chemical purity determined by HPLC: Using an X-Bridge C18 column, the eluent contained a mixture of (i) 20 mM aqueous ammonium bicarbonate (pH = 8.7) and (ii) acetonitrile, with a gradient program of: 0 - 10 minutes, ratio 95:5; 10 - 13 minutes, ratio 70:30; 13 - 16 minutes, ratio 10:90; then 95:5. The temperature was 35 °C. The detector conditions were UV absorption at a wavelength of 260 nm. A single impurity with an area of 0.04% was found at 12.132 minutes, and nicotine was found at 9.925 minutes. With a single impurity at 0.04% area, the purity was considered 99.96%. In contrast, before distillation, the weighted average of the two batches used was 99.70%.

Claims

1. A method for preparing (S)-nicotine, which comprises the following steps: (i) Reducing myosmine with an NADH / NADPH-dependent enzyme having imine reductase activity in the presence of a cofactor and a cofactor recycling system to form (S)-nornicotine; wherein the cofactor is NADH or NADPH; wherein the cofactor recycling system is glucose / glucose dehydrogenase or glucose monohydrate / glucose dehydrogenase; wherein the cofactor is present in the range of 0.02 parts by weight to 10 parts by weight per 100 parts of myosmine; wherein the enzyme having imine reductase activity is present in the range of 0.1 parts by weight to 30 parts by weight per 100 parts of myosmine; and (ii) Methylating the (S)-nornicotine formed in step (i) to form (S)-nicotine; wherein steps (i) and (ii) are carried out without isolating the (S)-nornicotine formed in step (i).

2. The method according to claim 1, wherein step (ii) is carried out by reductive methylation.

3. The method according to claim 2, wherein in step (ii), (S)-nornicotine is subjected to reductive methylation using formaldehyde or a formaldehyde-based compound in the presence of a reducing agent.

4. The method according to claim 3, wherein formaldehyde is introduced as part of an aqueous solution.

5. The method according to claim 3, wherein the formaldehyde-based compound is a dimer of formaldehyde, a polymer of formaldehyde, or an acetal of formaldehyde.

6. The method according to any one of claims 3-5, wherein the reducing agent is formic acid, sodium cyanoborohydride, or palladium / hydrogen.

7. The method according to any one of claims 3-5, wherein the reducing agent is formic acid.

8. The method according to any one of claims 1-5, wherein in step (i), (S)-nornicotine is formed as part of an aqueous solution, and wherein step (ii) comprises methylating the (S)-nornicotine contained in the aqueous solution.

9. The method according to claim 7, wherein In step (ii), the (S)-nornicotine is reductively methylated using formaldehyde introduced as part of an aqueous solution.

10. The method according to any one of claims 1-5, wherein (S)-nicotine is obtained with an enantiomeric excess of at least 90%.

11. A method for producing a pharmaceutical composition, comprising forming (S)-nicotine according to any one of the preceding claims and including (S)-nicotine together with one or more pharmaceutical excipients in the pharmaceutical composition.

12. The method according to claim 11, wherein the pharmaceutical composition is a transdermal patch, a lozenge, or an inhalation preparation.

13. A method for producing a formulation for an electronic cigarette device, which comprises forming (S)-nicotine according to any one of claims 1-10 and including (S)-nicotine together with one or more additives in a solvent.

14. Use of myosmine and an enzyme having imine reductase activity in a method for forming (S)-nicotine, wherein the method comprises the features described in any one of claims 1-10.

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