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WO2002012264A1 - Procede de production de 2'-o-alkylguanosine - Google Patents

Procede de production de 2'-o-alkylguanosine Download PDF

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Publication number
WO2002012264A1
WO2002012264A1 PCT/JP2001/006737 JP0106737W WO0212264A1 WO 2002012264 A1 WO2002012264 A1 WO 2002012264A1 JP 0106737 W JP0106737 W JP 0106737W WO 0212264 A1 WO0212264 A1 WO 0212264A1
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
alkylguanosine
phosphate
reaction
diaminopurine riboside
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2001/006737
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English (en)
Japanese (ja)
Inventor
Shinji Sakata
Toshio Yamada
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yamasa Corp
Original Assignee
Yamasa Corp
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 Yamasa Corp filed Critical Yamasa Corp
Priority to AU2001276736A priority Critical patent/AU2001276736A1/en
Publication of WO2002012264A1 publication Critical patent/WO2002012264A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • 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
    • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides

Definitions

  • the present invention provides a method for producing 2'-0-alkylguanosine from 2,6-diaminopurine riboside which is safe, excellent in operability and practicality.
  • methylazomethane or methyl iodide all of the above-mentioned conventional methods use methylazomethane or methyl iodide.
  • This process consists of a discontinuous process in which the hydrolysis reaction is carried out in a non-aqueous system such as an organic solvent containing no water, and the deamination reaction using adenosine deaminase is carried out in an aqueous system such as water.
  • a discontinuous process is performed as a series of processes, there is a problem that the handling of each process must be strictly distinguished and equipment for executing the process must be prepared, so that it can be performed easily by anyone. It wasn't a simple method.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems of the conventional method.
  • 2′-0-methylguanosine from 2,6-diaminopurine riboside
  • trimethyl phosphate as a methylating agent
  • the methylation reaction can be carried out in an aqueous solvent, and the 2′-hydroxyl group of 2,6-diaminopurine riboside can be selectively formed. It was found that 2′-10-methylguanosine could be efficiently obtained after deamination by adenosine deminase.
  • methylation of nucleosides using trimethyl phosphate has focused on methylation of nucleoside nucleobases, and no report has been made on methylation of sugar hydroxyl groups.
  • nucleobase methylation experiments in the case of nucleobase methylation experiments, in the case of inosine and adenosine, which were performed as comparative experiments, methylation of the sugar hydroxyl group slightly progressed with trimethyl phosphate.For example, inosine was methylated using trimethyl phosphate.
  • the present invention relates to a method for producing 2′-0-alkylguanosine from 2,6-diaminopurine riboside, wherein an aqueous solvent is used as a solvent, , 2,6-Diaminopurine liposide is treated with an alkylating agent of an alkyl phosphate or an alkyl sulfate to form 2'-0-alkyl-2,6-diaminopurine riboside, which is then formed.
  • a process for producing 2'-0-alkylguanosine which comprises deaminating the product to obtain 2'-0-alkylguanosine c
  • the starting compound, 2,6-diaminopurine riboside is a known compound having the following formula (I). Such a compound can be synthesized chemically or enzymatically, or a commercially available product may be used. In particular, when 2,6-diaminopurine riboside is synthesized enzymatically, it is not necessary to isolate the synthesized product, and the enzyme reaction solution itself or a crude product from the enzyme reaction solution is used directly as a raw material. No problem.
  • the method of the present invention comprises the steps of: first, in a water-based solvent, under basic conditions,
  • 6-di ⁇ amino riboside is treated with an alkylating agent, 2 7 -0- alkyl one 2 having the formula (II), 6 - to produce a di ⁇ amino riboside.
  • an alkyl phosphate or an alkyl sulfate can be used as the alkylating agent used here.
  • trialkyl phosphates such as trimethyl phosphate, triethyl phosphate, tripropyl phosphate, and ethyl dimethyl phosphate
  • Dialkyl phosphates such as dimethyl phosphate and getyl phosphate
  • dialkyl sulfates such as dimethyl sulfate and getyl sulfate.
  • trimethyl phosphate is preferred.
  • the alkylation reaction is carried out under basic conditions in an aqueous solvent such as water or a buffer solution, under basic conditions, with 1 to 100 mol of alkyl phosphate or alkyl sulfate per mol of 2,6-diaminopurine riboside.
  • the reaction can be carried out preferably by using 10 to 30 moles and reacting at 10 to 90 ° C, preferably 30 to 70 ° C for about 1 to 20 hours.
  • Basic conditions include, for example, pH 11 or higher, preferably pH 11 to 13. When the pH is less than 11, the alkylation reaction becomes slow, and when the pH exceeds 13, the selectivity to the 2'-hydroxyl group in the methylation reaction decreases. Not preferred. It is desirable that the above basic conditions be maintained or adjusted from the start to the end of the reaction, but it is important to adjust at least the pH at the beginning of the reaction to the basic conditions described above. It is.
  • the 2'-0-alkyl-1,2,6-diaminopurine riboside thus produced can be purified by a known purification method (eg, solvent extraction method; ion exchange resin, activated carbon, etc.) if necessary. And subject it to the next deamination reaction.
  • a known purification method eg, solvent extraction method; ion exchange resin, activated carbon, etc.
  • the deamination reaction can be carried out by a known method using adenosine deaminase.
  • adenosine deaminase any enzyme derived from animals and microorganisms can be used as long as it can deaminate the amino group at the 6-position of 2'-0-alkyl-1,2,6-diaminopurine riboside. can do.
  • E. coli-derived enzymes are advantageous in terms of reaction efficiency and price.
  • the deamination reaction varies depending on the enzyme used, an excess amount of adenosine deaminase is used in water or a buffer at a pH of 6 to 8 and at a temperature of 20 to 50 ° C. The reaction can be carried out for about 100 hours.
  • the resulting 2'-0-alkylguanosine (compound of the following formula (III)) is It can be isolated and purified by known methods (eg, crystallization method, solvent extraction method, chromatography method using an adsorption resin, etc.).
  • R means lower alkyl having 5 or less carbon atoms.
  • 2,6-Diaminopurine riboside (5.65 s 2 Ommo 1) is suspended in a mixture of trimethyl phosphate (40 ml) and deionized water (40 ml), heated to 50 ° C, The reaction was carried out with stirring while adjusting with an aqueous solution of sodium hydroxide so that the pH of the reaction solution became 12.0 to 12.5.
  • reaction solution was analyzed by HPLC, and as a result, 2'-0-methyl-2,6-diaminopurine riboside and 3'-0-methyl-1,2,6-diaminopurine riboside However, it was confirmed that 41% and 8.5% (HPLC: analyzed by UV260 nm) were generated respectively.
  • the adsorbate After filtering the reaction solution and passing the filtrate through an adsorption resin column, the adsorbate is eluted with deionized water, and the eluted fraction of 2'-0-methylguanosine thus obtained is decompressed to 5 Oml. Concentrate and cool overnight. The resulting precipitate was collected by filtration and dried to obtain 1.72 g of a crystal of —0-methylguanosine.
  • Perisine (14.6 g, 60 mmo 1) and 2,6-diaminopurine (6 g, 40 mmo 1) are suspended in 25 mM phosphate buffer (pH 7.0, 1000 ml), and then suspended.
  • Pyrimidine nucleoside phosphorylase (2100 units) and purine nucleoside phosphorylase (5400 units) were added to the suspension, followed by stirring at 50 ° C for 100 minutes. The reaction was boiled for 10 minutes to stop the reaction.
  • reaction solution was concentrated to 20 Oml, trimethyl phosphate (15 Oml) was added, and the mixture was reacted at 50 ° C for 7 hours while adjusting the pH to 12 to 12.5 with an aqueous solution of sodium hydroxide.
  • -Analysis of the reaction mixture obtained by HP LC shows that 2'- ⁇ -methyl-2,6-diaminopurine riboside is produced from 2,6-diaminopurine riboside at a conversion of 47%. was confirmed.
  • 2′-0-alkylguanosine can be efficiently produced from 2,6-diaminopurine riboside using an aqueous solvent without using any flammable or toxic chemical. Since it is an extremely useful method and does not require special equipment or work management, it is an extremely practical method for mass synthesis of 2'-0-alkylguanosine.

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

Abstract

Procédé de production de 2'-O-alkylguanosine à partir de 2,6-diaminopurine riboside, ce procédé se caractérisant par le fait que l'on utilise un milieu aqueux pour traiter le 2,6-diaminopurine riboside, avec un phosphate alkylique ou un sulfate alkylique en tant qu'agent d'alkylation, dans des conditions basiques, pour produire un 2'-O-alkyl-2,6-diaminopurine riboside, et que l'on désamine ensuite le composé diamino.
PCT/JP2001/006737 2000-08-08 2001-08-06 Procede de production de 2'-o-alkylguanosine Ceased WO2002012264A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001276736A AU2001276736A1 (en) 2000-08-08 2001-08-06 Process for producing 2'-o-alkylguanosine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000-239447 2000-08-08
JP2000239447A JP3596669B2 (ja) 2000-08-08 2000-08-08 ２’−ｏ−アルキルグアノシンの製造法

Publications (1)

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WO2002012264A1 true WO2002012264A1 (fr) 2002-02-14

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/006737 Ceased WO2002012264A1 (fr) 2000-08-08 2001-08-06 Procede de production de 2'-o-alkylguanosine

Country Status (3)

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JP (1) JP3596669B2 (fr)
AU (1) AU2001276736A1 (fr)
WO (1) WO2002012264A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002501A1 (fr) * 1992-07-23 1994-02-03 Isis Pharmaceuticals, Inc. Nouveaux 2'-o-alkyle nucleosides et phosporamidites, leurs procedes de preparation et d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002501A1 (fr) * 1992-07-23 1994-02-03 Isis Pharmaceuticals, Inc. Nouveaux 2'-o-alkyle nucleosides et phosporamidites, leurs procedes de preparation et d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YORISATO HISANAGA ET AL.: "The methylation of ribonucleosides by trimethyl phosphate or dimethyl sulfate in the presence of boric acid", BULL. CHEM. SOC. JPN., vol. 54, 1981, pages 1569 - 1570, XP002945943 *

Also Published As

Publication number Publication date
JP3596669B2 (ja) 2004-12-02
AU2001276736A1 (en) 2002-02-18
JP2002053591A (ja) 2002-02-19

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