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Synthesis method of S-nicotine

A synthesis method and nicotine technology, applied in the field of S-nicotine synthesis, can solve the problems that enzymes cannot be reused, harsh reaction conditions, and cannot be reused, and achieve convenient separation and purification of products, easy preparation, and stereoselectivity. control effect

Pending Publication Date: 2022-07-29
上海锐康生物技术研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, the patent CN111511726A reports the synthesis of racemic nicotine, and uses L-DBTA to carry out chiral resolution to obtain S-nicotine; but this method needs to split the mixture, the process is complicated, the reaction conditions are harsh, and the obtained product low yield
In the prior art, there is also the use of enzymes as biocatalysts to synthesize S-nicotine. For example, the patent CN112409327A reports a method for preparing nicotine with high optical purity. The method first uses nicotinic acid ester and vinylpyrrolidone to prepare mesmin. Mesmin is catalytically reduced under the biological enzyme system (including glucose dehydrogenase and imine reductase) to obtain an intermediate with high optical purity-nornicotine, and finally undergoes aminomethylation reaction to obtain S-nicotine, but the The method has the problem that the enzymes used are complex and diverse, and the enzymes cannot be reused after use
Patent CN113272289A discloses the use of imine reductase to reduce mesmin to generate S-nornicotine, and methylate S-nornicotine to form S-nicotine, but this method also requires the preparation of two enzymes and the enzymes cannot be repeated problem of use
[0005] Several known methods for the preparation of nicotine have not yet been used in commercial production, so that a synthetic method that can commercially prepare high-purity S-nicotine is very precious

Method used

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  • Synthesis method of S-nicotine
  • Synthesis method of S-nicotine
  • Synthesis method of S-nicotine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, the preparation of fusion enzyme IRED_Pa-GDH

[0081] (1) Design of fusion enzyme IRED_Pa-GDH

[0082] On the basis of imine reductase (amino acid sequence shown in SEQ ID.NO1., derived from Paenibacillus sp.IB182493, coded in NCBI database as WP_190863553.1), through linker (amino acid sequence shown in SEQ ID.NO3. ) to connect glucose dehydrogenase (amino acid sequence as shown in SEQ ID.NO2.), thereby forming a corresponding fusion enzyme (amino acid sequence as shown in SEQ ID.NO4.), and the gene nucleotide sequence of fusion enzyme is as shown in SEQ ID.NO5. shown.

[0083] SEQ ID. NO1.:

[0084] MENNNRSGNEQNGNSRPIGEETVAKQDSRMPVTIIGMGPMERALAGAFVKNGHLATVWNRSAGKTDALVKEGAVLADTIGGAVAASPLVIVCVLDYDAVQSILAPVGSELQGRTLVNLTADTPARAREMAAWAAGHGIDYLDGAIMTPTPSIGTPAASILYSGPESVYRNVQSALSSLGGTASYLGSDPGRAAAHDVALLDLFWTSMSGYVHALALAASEHISAKDFAVHARGIAAILPDIITGIADEADEGRYPGDASNLVSAEAGMAHIIHAAEHHGIDASVLSAARALARRAIDEGLGEDGFGRLAELLSRRSGA

[0085] SEQ ID. NO2.:

[0086] MYPDLK...

Embodiment 2

[0098] Embodiment 2, the preparation of immobilized fusion enzyme IRED_Pa-GDH

[0099] Take 15g of the fusion enzyme IRED_Pa-GDH fermentation broth centrifuged bacterial slurry, add 150mL of 100 mM KPB buffer (pH7.0), mix it with high pressure homogenization and crush it, collect the supernatant enzyme liquid by centrifugation, add 30g KPB 2 HPO 4 ·3H 2 O and 3g KH 2 PO 4 , stir to dissolve, add 10g of resin (see the following table 1 for resin model, resin LX series is purchased from Xi'an Lanxiao Technology New Materials Co., Ltd., resin ES series is purchased from Tianjin Nankai Synthetic Technology Co., Ltd.) for fixing, at 25 ℃, 200rpm shaker for 20-25h, suction filtration, washing with deionized water, suction filtration to obtain the immobilized enzyme. Using mesmin as the substrate, the activity of the immobilized enzyme was detected. The enzyme activity detection method was as follows:

[0100] First prepare the substrate solution: weigh 2g of mesmin, 20mg of NAD...

Embodiment 3

[0105] Embodiment 3, immobilized fusion enzyme catalyzes the synthesis of S-nornicotine

[0106] 20mL 100mM KPB buffer (pH 7.0), 100mg / mL immobilized fusion enzyme IRED_Pa-GDH (ES-108 or LXTE-604 resin immobilized enzyme), 2eq glucose, 0.1~0.5mg / mL NADP + , the substrate mesmin concentration was 100 mg / mL, and the reaction was carried out at 30 °C. During the reaction, the pH was adjusted to 6.9-7.1 with a concentration of 2M sodium carbonate solution, and the reaction process was monitored by TLC dot plate. After 24 hours, the filtrate was collected by suction filtration, the immobilized enzyme was recovered and applied mechanically, the filtrate was extracted with ethyl acetate, the ethyl acetate phase was added with anhydrous sodium sulfate to dry, filtered, and the ethyl acetate was recovered by rotary evaporation to obtain S-nornicotine. 20 batches of immobilized enzymes were applied, and the results are shown in Table 2 below.

[0107] Table 2. Results of the synthesis ...

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Abstract

The invention provides a synthesis method of S-nicotine, and belongs to the technical field of biochemical engineering. The synthesis method of the S-nicotine comprises the following steps: (1) taking myosmin 4 as a raw material, in a solvent containing a coenzyme circulation system, utilizing a fusion enzyme formed by enzyme connection of imine reductase and the coenzyme circulation system as a biocatalyst, and reacting to prepare a compound 5S-nornicotine; and (2) carrying out methylation reaction on the compound 5 and a methylation reagent to obtain a compound 6S-nicotine. According to the synthesis method, the S-nicotine is synthesized by adopting the immobilized fusion enzyme and utilizing the synergistic effect of a chemical method and a biological enzyme method, raw materials are easy to obtain, operation is easy and convenient, reaction conditions are mild, product separation and purification are convenient, and cost is low; meanwhile, the synthesized S-nicotine is high in yield, the chiral purity is as high as 99%, the stereoselectivity is controllable, and the synthesized product is excellent in quality. The synthesis method has the possibility of large-scale production, is expected to realize industrial and commercial production, and has a good application prospect and a desirable economic benefit.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a method for synthesizing S-nicotine. Background technique [0002] Nicotine, also known as nicotine, is usually found in natural tobacco leaves. High-purity nicotine is an organic intermediate with important potential value. In the field of medicine, it can be used to treat Parkinson's syndrome, Alzheimer's disease, ADHD, arthritis, and it can also help improve memory and relieve depression; in agriculture, nicotine is used as a pesticide and herbicide. It is a broad-spectrum pesticide with high efficiency and low toxicity; in addition, nicotine can also be used to synthesize functional proteins of medicine and health care, and is also an important raw material for daily cosmetics (Nature. 2004, 430, 980; Br. J. Addict. 1991, 86, 571-575). Therefore, it has a huge market demand. [0003] Nicotine is optically active and exists in two enantiomers, R-ni...

Claims

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Application Information

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IPC IPC(8): C12P17/16C12N11/089C12N9/06C12N9/04C07K19/00C12N15/62C07D401/04
CPCC12P17/165C12N11/089C12N9/0028C12N9/0006C12Y101/9901C07D401/04C07K2319/00
Inventor 刘涵李正华张磊王圣克赵华
Owner 上海锐康生物技术研发有限公司
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