Pyrimidine nucleosides preparation method and novel pyrimidine nucleoside

A technology for pyrimidine nucleoside compound and cytidine nucleoside phosphorylase activity, applied in the field of preparation of pyrimidine nucleoside compound

Inactive Publication Date: 2005-02-09
MITSUI CHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In the method for preparing pyrimidine nucleoside compounds from phosphate sugar and pyrimidine base derivatives by using cytosine phosphorylase, EP1254959A2 (Patent

Method used

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  • Pyrimidine nucleosides preparation method and novel pyrimidine nucleoside
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  • Pyrimidine nucleosides preparation method and novel pyrimidine nucleoside

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Embodiment

[0053] The following examples illustrate the present invention, but the enumerated examples do not limit the scope of protection of the present invention.

[0054] The identification of the generated pyrimidine nucleoside compound was carried out as follows: the reaction solution was ultrafiltered and purified with a silica gel column, the product was extracted and vacuum-dried, and the obtained product was analyzed by C13-NMR and H1-NMR.

[0055] In addition, the generated pyrimidine nucleoside compounds were quantitatively analyzed by high performance liquid chromatography (HPLC). The analysis conditions are as follows:

[0056] Chromatographic column: Develosil ODS-MG-5, 4.6×250mm (Nomura Chemical)

[0057] Column temperature: 40°C

[0058] Pump flow rate: 1.0ml / min

[0059] Detection: UV 254nm

[0060] Eluent: potassium dihydrogen phosphate (50mM): methanol = 8: 1 (V / V)

reference example 1

[0061] Reference Example 1 (Preparation of cells producing cytidine phosphorylase)

[0062] Escherichia coli chromosomal DNA was prepared as follows:

[0063] Escherichia coli K-12 / XL-10 strain (Stratagene Company) was implanted into 50 ml of LB culture medium, cultivated overnight at 37° C., and then the colony was collected and treated with a lysozyme-containing 1 mg / ml lysozyme. After treating the lysate with phenol, the DNA was precipitated with ethanol by a usual method. The resulting DNA precipitate was wound and precipitated along the glass rod, recovered and washed, and used for PCR.

[0064] Oligonucleotides (synthesized by Hokkaido System Science Co., Ltd.) having base sequences in SED ID NOs: 1, 2, respectively, were used as primers for PCR. These nucleotide sequences were designed based on the nucleotide sequence of the deoD gene encoding the known purine nucleoside phosphorylase of Escherichia coli (the number in the gene bank is AE000508 (the base number of the...

reference example 2

[0069] Reference Example 2 (Preparation of Purified Cytidine Phosphorylase)

[0070]The bacteria in the suspension prepared in Reference Example 1 were destroyed with an ultrasonic crusher. Heat treatment at 70°C for 10 minutes, and then centrifuge to obtain a crude enzyme solution, which is then injected into a DEAE-Toyopearl (3cm×3cm) chromatographic column manufactured by Toso, which has been washed with 50mM Tris-HCl buffer (pH 7.5) balanced. Then it was eluted with a linear gradient with 50mM-500mM NaCl solution to collect active fractions. The eluate was saturated with 70% aqueous ammonium sulfate to form a precipitate for recovery. The precipitate was dialyzed against 10 mM phosphate buffer (pH 7.5). The dialyzed solution was injected into a chromatographic column loaded with hydroxyapatite (3 cm×15 cm), which had been equilibrated with 10 mM phosphate buffer (pH 7.5). It was then eluted with 10mM-50mM 10mM phosphate buffer (pH 7.5) to collect active fractions. The...

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Abstract

A method for producing a pyrimidine nucleoside compound includes reacting a sugar phosphate and pyrimidine base derivative using an enzyme having cytosine nucleoside phosphorylase activity, the pyrimidine base derivative being represented by general formula (I): (wherein R1 represents an amino group that may be replaced with an acyl group having an alkyl group of 1 to 3 carbon atoms or an alkyl group of 1 to 3 carbon atoms, an alkyl group of 1 to 3 carbon atoms, or a thiol group; R2 represents an amino group, a thiol group, a hydroxyl group, or a hydrogen atom; R3 represents an alkyl group of 1 to 3 carbon atoms that may be replaced with a hydroxyl group, an amino group, or a hydrogen atom; R4 represents a hydroxyl group or a hydrogen atom; when R1 is an amino group, when R2 is a hydroxyl group, and when R4 is a hydrogen atom; R3 is neither an alkyl group of 1 to 3 carbon atoms nor a hydrogen atom).

Description

technical field [0001] The present invention relates to a preparation method of a pyrimidine nucleoside compound, which is suitable as a synthetic raw material for medicines and the like, and a new pyrimidine nucleoside compound. Background technique [0002] Japanese Unexamined Patent Publication No. 59-213397 (Patent Document 1) and Japanese Unexamined Publication No. 5-49493 (Patent Document 2) etc. disclose methods for synthesizing pyrimidine nucleoside compounds from pyrimidine base derivatives using nucleoside phosphorylase, wherein The nucleic acid bases are all derivatives of uracil base. The aforementioned uracil derivatives are compounds having a carbonyl structure at the 4-position of the pyrimidine base, and methods for preparing corresponding nucleoside compounds from uracil, 5-halogenated uracil, and thymine are known. [0003] In the method for preparing pyrimidine nucleoside compounds from phosphate sugar and pyrimidine base derivatives by using cytosine pho...

Claims

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Application Information

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IPC IPC(8): C12P19/40C07H17/02C07H19/067C07H19/073
CPCC07H19/073C07H17/02C07H19/067C12P19/40
Inventor 安乐城正三宅仁基及川利洋
Owner MITSUI CHEM INC
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