Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-03-14
2003-09-16
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S089000, C435S087000, C435S193000, C435S252500, C536S124000, C536S027100, C536S027300
Reexamination Certificate
active
06620596
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-087302, filed Mar. 27, 2000, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
The present invention relates to a method of preparing a guanosine-group compound, which is used as a raw material of an anti-virus agent, antisense medicine and the like, and a method of preparing a glyoxal-guanosine-group compound as an intermediate of the guanosine-group compound.
Guanosine or 2′-deoxyguanosine is industrially produced mainly by extraction/separation of hydrolysate of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), because the yield is extremely low when guanosine or 2′-deoxyguanosine is chemically synthesized. However, the hydrolysate of DNA contains 2′-deoxyadenosine, 2′-deoxycytidine and thymidine other than the targeted 2′-deoxyguanosine. The hydrolysate of RNA contains adenosine, cytidine and uridine other than the targeted guanosine. Accordingly, in order to collect only guanosine or 2′-deoxyguanosine, it is necessary to perform complicated processes of extraction/separation, thereby inevitably raising the production cost.
On the other hand, a method has been reported in which nucleoside (or deoxynucleoside) and nucleic acid base, which are the raw materials, are subjected to a base-exchange reaction by nucleoside phosphorylase, whereby the aimed nucleoside (or the aimed deoxynucleoside) is obtained (Hori, N., Watanabe, M., Yamazaki, Y., Mikami, Y., Agric. Biol. Chem., 53, 197-202 (1989)). By this method, adenosine and 2′-deoxyadenosine can be easily prepared (Jpn. Pat. Appln. KOKAI Publication No. 11-46790 “Method of preparing a purine nucleoside compound”). In order to obtain guanosine and 2′-deoxyguanosine by this method of enzymatic synthesis reaction, guanine must be soluble to water at least in a range of pH where the enzymes necessary for the reaction can function. In general, it is considered that the solubility of the reaction substrate is preferably at least equal to the concentration of Michaelis constant (Km) of the enzyme or higher, in order to carry out the enzyme reaction smoothly. However, as the solubility of guanine to water does not exceed a few ppm, the above method of enzymatic synthesis reaction cannot be employed in practice. Due to such an impasse-like situation, it has been demanded to develop a method of efficiently preparing guanosine or 2′-deoxyguanosine.
BRIEF SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of efficiently preparing guanosine or 2′-deoxyguanosine (i.e., a guanosine-group compound) at a high yield, as well as a method of efficiently preparing a glyoxal-guanosine or glyoxal-2′-deoxyguanosine (i.e., glyoxal-guanosine-group compound) as an intermediate of the guanosine-group compound at a high yield.
The inventors of the present invention have assiduously studied for solving the aforementioned problems. As a result, the inventors have discovered the following 1)-6).
1) When guanine, whose solubility to water does not exceed a few ppm, is reacted with glyoxal, glyoxal-guanine represented by the following formula (1) is obtained, and glyoxal-guanine is soluble to water.
(Glyoxal-guanine is also referred to as 6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one.)
2) When microorganism itself which contains purine nucleoside phosphorylase (EC 2.4.2.1) or this enzyme derived from the microorganism are applied, for a reaction, to glyoxal-guanine and any one of ribose-1-phosphate and 2-deoxyribose-1-phosphate, a glyoxal-guanosine-group compound (i.e., glyoxal-guanosine or glyoxal-2′-deoxyguanosine) represented by the following formula (2) is obtained. Alternatively, when microorganism itself which contains purine nucleoside phosphorylase (EC 2.4.2.1) and pyrimidine nucleoside phosphorylase (EC 2.4.2.2) or these enzymes derived from the microorganism are applied, for a reaction, to glyoxal-guanine and any one selected from the group consisting of uridine, 2′-deoxyuridine and thymidine under the presence of phosphate ion, a glyoxal-guanosine-group compound (i.e., glyoxal-guanosine or glyoxal-2′-deoxyguanosine) represented by the following formula (2) is obtained. In other words, glyoxal-guanine can be a substrate of purine nucleoside phosphorylase.
(wherein R represents a hydrogen atom or a hydroxyl group.)
(Glyoxal-guanosine is also referred to as 3-(&bgr;-D-erythropentofuranosyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one, and glyoxal-2′-deoxyguanosine is also referred to as 3-(2-deoxy-&bgr;-D-erythropentofuranosyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one.)
3) In the method of preparing a glyoxal-guanosine-group compound described in 2) above, when at least one compound selected from the group consisting of glycine, iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid (which will be referred to as “EDTA” hereinafter), ethylene glycol bis(&bgr;-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (which will be referred to as “EGTA” hereinafter) and salts of these substances is added, or the above at least one compound is added in combination with boric acid or a salt thereof, the reaction yield is significantly improved. The above compound is added as a stabilizer of ribose-1-phosphate or 2-deoxyribose-1-phosphate, which is the substrate or an intermediate produced during base-exchange reaction.
4) As a principle, nucleoside phosphorylase derived from various organism can be efficient as the enzyme to be used in the preparation method of 2) above. However, the enzyme derived from microorganism of Bacillus genus, Escherichia genus or Klebsiella genus is suitable, and the thermotolerant enzyme derived from
Bacillus stearothermophilus
JTS 859 (FERM BP-6885) is especially preferable.
5) When an alkali is applied, for a reaction, to the glyoxal-guanosine-group compound obtained by the method of 2) above, the glyoxal portion of the glyoxal-guanosine-group compound is easily released, whereby a guanosine-group compound (i.e., guanosine or 2′-deoxyguanosine) can be obtained.
6) The preparation processes of the aforementioned 1)-5) can be carried out by one-pot reaction.
On the basis of the aforementioned discoveries, the present invention has been completed.
In summary, the present invention relates to the following methods (1)-(16).
(1) A method of preparing a glyoxal-guanosine-group compound represented by the formula (2):
wherein R represents a hydrogen atom or a hydroxyl group, which comprises the step of:
reacting glyoxal-guanine represented by the formula (1):
with ribose-1-phosphate or 2-deoxyribose-1-phosphate in the presence of purine nucleoside phosphorylase, thereby obtaining a glyoxal-guanosine or glyoxal-2′-deoxyguanosine.
(2) A method of preparing a glyoxal-guanosine-group compound represented by the formula (2):
wherein R represents a hydrogen atom or a hydroxyl group, which comprises the step of:
reacting glyoxal-guanine represented by the formula (1):
with any one selected from the group consisting of uridine, 2′-deoxyuridine and thymidine, together with phosphate ion, in the presence of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, thereby obtaining a glyoxal-guanosine or glyoxal-2′-deoxyguanosine.
(3) A method of preparing a guanosine-group compound, which comprises the steps of:
reacting glyoxal-guanine represented by the formula (1):
with ribose-1-phosphate or 2-deoxyribose-1-phosphate in the presence of purine nucleoside phosphorylase, thereby obtaining a compound represented by the formula (2):
wherein R represents a hydrogen atom or a hydroxyl group; and
decomposing, by alkali, the compound represented by the formula (2), thereby obtaining guanosine or 2′-deoxyguanosine.
(4) A method of preparing a guanosine-group comp
Hayashi Yoshinori
Matsumoto Seiichiro
Mikami Yoichi
Sato Toyoki
Marx Irene
Pennie & Edmonds LLP
Yuki Gosei Kogyo Co. Ltd.
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