Method for preparing 2'-deoxyuridine by chemical-biological enzyme method in combination

A biological enzyme method and deoxyuridine technology, which is applied in the field of chemical-biological enzyme method combined preparation of 2'-deoxyuridine, can solve the problems of inability to obtain a single isomer, high cost, insufficient raw materials, etc., and achieve environmental friendliness, The effect of strong specificity and high conversion rate

Active Publication Date: 2012-12-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0016] The problem to be solved by the present invention is that the chemical synthesis of 2'-deoxyuridine cannot obtain a single isomer, and the problem of insufficient raw materials and high cost in the biological enzymatic synthesis process

Method used

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  • Method for preparing 2'-deoxyuridine by chemical-biological enzyme method in combination
  • Method for preparing 2'-deoxyuridine by chemical-biological enzyme method in combination
  • Method for preparing 2'-deoxyuridine by chemical-biological enzyme method in combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Put 7.02g of orthophosphoric acid into the Erlenmeyer flask, add 100mL of acetonitrile, add 5.6mL of n-tributylamine, 10g of 4A molecular sieve (Shanghai Jialin Molecular Sieve Co., Ltd.) 8.5 g of bis(O-p-chlorobenzoyl)-2-deoxy-D-ribose was stirred and reacted for 13 hours under nitrogen protection, then 16.0 mL of n-tributylamine was added, and the reaction was continued for 8 hours to obtain a wine-red transparent liquid, which was removed by filtration. Molecular sieves, and the filtrate was evaporated to dryness to obtain a reddish-brown syrupy substance. After dissolving the syrupy substance in 120mL of tetramethyldipentanone, wash with 100mL of water three times, cool the obtained organic phase to 0°C, add 5.60mL of cyclohexylamine and stir for 0.5h to precipitate a white precipitate, filter it, and wash the filter cake with tetramethyldipentanone Wash with pentanone and acetone, and dry in vacuo to obtain 12.78 g of a white solid with a yield of 94.03% and a puri...

Embodiment 2

[0042] Dissolve 7.02g of 3,5-O-bis(4-benzoyl chloride)-2-deoxy-α-D-ribose-1-phosphate dicyclohexylamine salt in 120mL of methanol, add 3.0mL of cyclohexylamine, and stir at 40°C After reacting for 24 h, the reaction solution was concentrated, and the final precipitate was washed with ethanol and dried in vacuo to obtain a white solid (3.46 g, 89.4%). IR, 1 H NMR spectrum see image 3 , Figure 4 ,Data are as follows:

[0043] Melting point mp: 166-167°C; IR(KBr): 2939cm -1 , 2853cm -1 , 2238cm -1 , 1630cm -1 ;

[0044] 1 H NMR: 5.67 (1H, dd), 4.14-4.07 (2H, ddd), 3.60 (1H, dd), 3.52 (1H, dd), 3.07 (2H, m), 2.28-2.23 (1H, ddd), 1.99 (1H, ddd), 1.89 (4H, m), 1.71 (4H, m), 1.57 (2H, m), 1.29-1.21 (8H, m), 1.12 (2H, m).

Embodiment 3

[0046] Uracil 0.120g, 2-deoxy-α-D-ribose-1-phosphate 0.432g, Escherichia coli ATCC8379 wet bacterium 2.48g (the strain comes from the Guangdong Provincial Institute of Microbiology, and the freeze-dried tube strain is activated and cultivated on an inclined plane , inoculated into the enzyme production medium, the composition of the enzyme production medium is: peptone 10g / L; yeast extract powder 5g / L; sodium chloride 10g / L, pH 7.0, cultured in a shaker at 36°C and 250r / min for 24h, then centrifuged , collected wet cells and sonicated), dissolved in 50 mL of phosphate buffer solution with pH=7, reacted in a constant temperature water bath at 65°C for 6 hours, diluted the reaction solution by 100 times, and detected it by HPLC.

[0047] Liquid chromatography conditions: column, SB-C 18 column (5 μm, 4.6×250 mm); detection wavelength: 262; mobile phase: methanol: water = 9:1; flow rate: 0.8 mL / min; injection volume: 20 μL.

[0048] The isolated 2′-deoxyuridine 13 C NMR, 1 H ...

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Abstract

The invention provides a method for preparing 2'-deoxyuridine by a chemical-biological enzyme method in combination. A 'crystallization induction asymmetric transformation' technology is adopted to synthetize and obtain single alpha-configuration nucleoside analogues medicine intermediate 2-deoxy-alpha-D-ribose-1-phosphate; the intermediate is utilized as a substrate; uracil is added, and 2'-deoxyuridine is synthetized and obtained by biotransformation under the action of uridine phosphorylase. By applying the chemical-biological combination technology disclosed by the invention, the advantages of a chemical method and a biological method are combined; the defects of the chemical method and the biological method are avoided; the yield is high; the method is suitable for industrial production; enough supply can be obtained by the 'crystallization induction asymmetric transformation' technology; 2-deoxy-alpha-D-ribose-1-phosphate intermediate with single configuration does not need to be separated and purified; the biotransformation process is finished by catalysis with efficient and specific uridine phosphorylase in one step; the specificity is strong; the conversion rate is high; the condition is mild and the environment is friendly.

Description

(1) Technical field [0001] The invention relates to a method for preparing 2'-deoxyuridine in combination with a chemical-biological enzyme method. (2) Background technology [0002] 2′-Deoxyuridine belongs to nucleoside analogues. It has different degrees of similarities with natural nucleosides in chemical structure, and has the effect of falsehood in the body, thereby interfering with or directly acting on the metabolic process of nucleic acids and blocking proteins. , The biosynthesis of nucleic acid plays a very important role in antitumor and antiviral drugs. It is also an important intermediate of other nucleoside drugs. [0003] The method for synthesizing 2'-deoxyuridine mainly includes following three kinds: [0004] 1. Obtain nucleosides from natural products, and then obtain them through chemical structure modification. But there is the shortcoming of raw material shortage in this method, can't satisfy the demand of market. [0005] 2. The chemical total synt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/38
CPCY02P20/582
Inventor 应国清钱捷彭美红王鸿蒲通易喻梅建凤陈建澍
Owner ZHEJIANG UNIV OF TECH
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