WO2018196235A1 - PROCÉDÉ DESTINÉ À ÊTRE UTILISÉ DANS LA SYNTHÈSE DE β-THYMIDINE - Google Patents

PROCÉDÉ DESTINÉ À ÊTRE UTILISÉ DANS LA SYNTHÈSE DE β-THYMIDINE Download PDF

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Publication number
WO2018196235A1
WO2018196235A1 PCT/CN2017/097784 CN2017097784W WO2018196235A1 WO 2018196235 A1 WO2018196235 A1 WO 2018196235A1 CN 2017097784 W CN2017097784 W CN 2017097784W WO 2018196235 A1 WO2018196235 A1 WO 2018196235A1
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WO
WIPO (PCT)
Prior art keywords
thymidine
kinase
methyluridine
synthesizing
diphosphate
Prior art date
Application number
PCT/CN2017/097784
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English (en)
Chinese (zh)
Inventor
陈车翘
Original Assignee
苏州笃美生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州笃美生物科技有限公司 filed Critical 苏州笃美生物科技有限公司
Publication of WO2018196235A1 publication Critical patent/WO2018196235A1/fr

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Classifications

    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/385—Pyrimidine nucleosides

Definitions

  • the invention relates to the technical field of chemical synthesis, in particular to a method for synthesizing ⁇ -thymidine.
  • ⁇ -Thymidine plays an important role in the production of antiviral drugs and biomedical applications.
  • ⁇ -thymidine is a synthetic anti-AIDS drug (AIDS) Zidovudine and Stavudine.
  • AIDS anti-AIDS drug
  • Stavudine Stavudine
  • the synthesis method of ⁇ -thymidine is mainly prepared by chemical synthesis method through bromination reaction, reduction reaction and saponification reaction, and the synthesis process is as follows:
  • the present invention proposes a method for synthesizing ⁇ -thymidine, which can effectively shorten the chemical reaction step and remove the high pressure by constructing a thymidine-specific enzyme catalyst and reversely synthesizing the design of the thymidine route.
  • Dangerous processes such as hydrogenation, reducing the use of more than 5 solvents.
  • the method steps are as follows:
  • thymidine diphosphate forms thymidine monophosphate under the action of thymidylate kinase
  • Thymidine monophosphate forms ⁇ -thymidine under the action of thymidine kinase.
  • the invention provides a method for synthesizing ⁇ -thymidine, which uses five enzymes in one reaction to promote the conversion of 5-methyluridine to thymidine, and studies the use of two phosphorylation steps to generate nucleotides, and then uses ribose.
  • Nucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and dephosphorylation in two steps to produce thymidine.
  • RNR Nucleotide reductase
  • the invention adopts enzymatic engineering to increase the stability and activity of reverse synthesis of thymidine, and the reaction is pushed
  • the power is oxidized from glucose to gluconolactone and consumes ADP.
  • Two ATP consumption steps (S1 & S2) are combined with two ATP generation steps (S4 & S5).
  • the overall response is ATP neutral - no consumption of ATP, since the ATP/ADP step is regenerative, and glucose dehydrogenase (GDH) catalyzes circulating NADH.
  • GDH glucose dehydrogenase
  • the present invention has the beneficial effects of:
  • a method for synthesizing ⁇ -thymidine provided by the present invention, which completes all reactions in one pot end in the same reactor, and is simple to operate; most of the cost required is enzyme, glucose and 5-methyluridine; and the impurity is phosphoric acid Compounds, easy to separate, greatly reduce costs, shorten production steps, reduce industrial waste, non-toxic and environmentally friendly.
  • thymidine-specific enzyme catalyst and reverse engineering synthesis of thymidine route, it can effectively shorten the chemical reaction step, remove dangerous processes such as high-pressure hydrogenation, reduce the use of more than 5 solvents, and reduce the wastewater generated by the reaction process by 30%. the above.
  • Source of raw materials 5-methyluridine, Lianyungang Haoxiang Chemical Co., Ltd.; 80-90% purity adenosine triphosphate (ATP), 80-90% purity flavin adenine dinucleotide (FAD), MgSO 4 , glucose (Glucose ), HCl, NaOH, NaCl, phosphate buffer, from any commercial source.
  • ATP adenosine triphosphate
  • FAD flavin adenine dinucleotide
  • MgSO 4 glucose (Glucose ), HCl, NaOH, NaCl, phosphate buffer, from any commercial source.
  • the wastewater produced by the reaction process is reduced by 33% compared to conventional chemical synthesis.
  • the wastewater produced by the reaction process is reduced by 31% compared to conventional chemical synthesis.
  • the wastewater produced by the reaction process is reduced by 38% compared to conventional chemical synthesis.
  • the wastewater produced by the reaction process is reduced by 42% compared to conventional chemical synthesis.
  • the wastewater produced by the reaction process is reduced by 36% compared to conventional chemical synthesis.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé à utiliser dans la synthèse de β-thymidine : l'ingénierie d'enzyme est utilisée pour synthétiser de manière inverse la thymidine ; la glucose déshydrogénase, l'uridine kinase, l'acide kinase de calmar, la ribonucléoside diphosphate réductase, la thymidylate kinase et la thymidine kinase sont utilisées, tandis que la 5-méthyluridine est utilisée comme matière première et subit deux étapes de phosphorylation pour générer des nucléotides, lesquels sont ensuite convertis en désoxynucléotides, deux étapes de déphosphorylation étant réalisées pour générer la thymidine.
PCT/CN2017/097784 2017-04-25 2017-08-17 PROCÉDÉ DESTINÉ À ÊTRE UTILISÉ DANS LA SYNTHÈSE DE β-THYMIDINE WO2018196235A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710273108.1 2017-04-25
CN201710273108.1A CN106967767B (zh) 2017-04-25 2017-04-25 一种β-胸苷的合成方法

Publications (1)

Publication Number Publication Date
WO2018196235A1 true WO2018196235A1 (fr) 2018-11-01

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PCT/CN2017/097784 WO2018196235A1 (fr) 2017-04-25 2017-08-17 PROCÉDÉ DESTINÉ À ÊTRE UTILISÉ DANS LA SYNTHÈSE DE β-THYMIDINE

Country Status (2)

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CN (1) CN106967767B (fr)
WO (1) WO2018196235A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410561A (zh) * 2022-01-28 2022-04-29 天津科技大学 一种生产胸苷的基因工程菌株及其构建方法与应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110408667A (zh) * 2019-06-29 2019-11-05 赤峰蒙广生物科技有限公司 一种提高β-胸苷产量的发酵工艺

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5672698A (en) * 1993-11-15 1997-09-30 Bristol-Myers Squibb Co. Preparation of 2',3'-didehydro-3'-deoxythymidine from 5-methyluridine
CN102086222A (zh) * 2010-12-28 2011-06-08 浙江先锋科技有限公司 一种β-胸苷的制备方法
CN102863493A (zh) * 2012-10-12 2013-01-09 安徽金太阳生化药业有限公司 一种β-胸苷的制备方法
CN104725452A (zh) * 2013-12-20 2015-06-24 上海迪赛诺化学制药有限公司 一种制备β-胸苷的方法
CN105566421A (zh) * 2014-10-09 2016-05-11 江苏笃诚医药科技股份有限公司 一种β-胸苷的制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5672698A (en) * 1993-11-15 1997-09-30 Bristol-Myers Squibb Co. Preparation of 2',3'-didehydro-3'-deoxythymidine from 5-methyluridine
CN102086222A (zh) * 2010-12-28 2011-06-08 浙江先锋科技有限公司 一种β-胸苷的制备方法
CN102863493A (zh) * 2012-10-12 2013-01-09 安徽金太阳生化药业有限公司 一种β-胸苷的制备方法
CN104725452A (zh) * 2013-12-20 2015-06-24 上海迪赛诺化学制药有限公司 一种制备β-胸苷的方法
CN105566421A (zh) * 2014-10-09 2016-05-11 江苏笃诚医药科技股份有限公司 一种β-胸苷的制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DENG, WEIRAN ; SHEN, RONGKUN ET AL.: "Enzymatic Synthesis of Thymidine", CHINESE JOURNAL OF PHARMACEUTICALS, vol. 26, no. 9, 21 September 1995 (1995-09-21), pages 424 *
HIROSHI, SHIRAGAMI ET AL.: "Synthesis of 1-(2, 3-Dideoxy- beta -d-glycero-pent-2-enofuranosyl) thymine (d4T; Stavudine) from 5-Methyluridine", NUCLEOSIDES AND NUCLEOTIDES, vol. 15, no. 1-3, 31 December 1996 (1996-12-31), pages 47 - 58, XP002169116 *
SHEN, RONGKUN ET AL.: "Enzymatic Synthesis of Thymidine by Nucleoside Phosphorylases in Escherihia Coli", JOURNAL OF EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY, vol. 22, no. 6, 31 December 1996 (1996-12-31), pages 701 - 704 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410561A (zh) * 2022-01-28 2022-04-29 天津科技大学 一种生产胸苷的基因工程菌株及其构建方法与应用
CN114410561B (zh) * 2022-01-28 2023-09-01 天津科技大学 一种生产胸苷的基因工程菌株及其构建方法与应用

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CN106967767A (zh) 2017-07-21
CN106967767B (zh) 2019-09-24

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