JP2001026599A - Production of nucleoside compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for enzymatically synthesizing a nucleoside compound. SOLUTION: In this method for producing a nucleoside compound from pentose-1-phosphate and a nucleic acid base by using a nucleoside phosphorylase or a microorganism having the nucleoside phosphorylase activity, a metal salt capable of forming a slightly water-soluble salt with phosphoric acid is added to a reaction system so that the phosphoric acid produced as a by-product is removed as precipitate from the reaction system and an equilibrium reaction is moved in a nucleoside synthesis direction to give the objective nucleoside compound in a high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a nucleoside compound using a nucleoside phosphorylase. Various nucleosides and their analog compounds,
It is used as a raw material or drug substance for antiviral drugs, anticancer drugs and antisense drugs.

[0002]

2. Description of the Related Art Nucleoside phosphorylase is a general term for enzymes that phosphorylate N-glycosidic bonds of nucleosides in the presence of phosphoric acid, and catalyzes a reaction represented by the following formula using ribonucleosides as an example.

[0003] These enzymes, which are roughly classified into ribonucleoside + phosphate (salt) → nucleobase + ribose 1-phosphate purine nucleoside phosphorylase and pyrimidine phosphorylase, are widely distributed in the living world,
Present in tissues such as mammals, birds and fish, yeast and bacteria. This enzymatic reaction is reversible, and the synthesis of various nucleoside compounds utilizing the reverse reaction has been known for some time.

For example, from 2′-deoxyribose 1-phosphate and a nucleobase (thymine, adenine or guanine) to thymidine (JP-A-01-104190), 2′-
Deoxyadenosine (JP-A-11-137290) or 2'-deoxyguanosine (JP-A-11-1137)
No. 37290) is known. Further, Agric. Biol. Chem., Vol. 50 (1), PP. 121-126,
In (1986), inosine was converted to Enterobacter a in the presence of phosphoric acid.
erogenes decomposes to ribose 1-phosphate and hypoxanthine by a reaction using purine nucleoside phosphorylase from
A technology for producing ribavirin, an antiviral agent, from phosphoric acid and 1,2,4-triazole-3-carboxamide by purine nucleoside phosphorylase also derived from Enterobacter aerogenes has been reported.

However, since the reaction for producing a nucleoside compound from pentose-1-phosphate or a salt thereof and a nucleic acid base by utilizing the reverse reaction of the enzyme is an equilibrium reaction, the technical disadvantage that the conversion is not improved. Had.

[0006]

When a nucleoside compound is produced industrially, it is essential to carry out the reaction at a high conversion in order to reduce the raw material cost and to improve the product purity.

That is, an object of the present invention is to provide a pentose
1-phosphate and a nucleobase or nucleobase analog,
An object of the present invention is to provide a method for producing a versatile nucleoside compound having a step of reacting in the presence of a nucleoside phosphorylase activity and having an improved conversion of the nucleoside compound in the reaction.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to solve these problems, and as a result, when a metal cation capable of forming a sparingly water-soluble salt with phosphate ions is present in the reaction solution, the reaction is stopped. It has been found that the by-product phosphate ion precipitates as a poorly water-soluble salt and is excluded from the reaction system, whereby the equilibrium of the reaction shifts toward the synthesis of the nucleoside compound, and the reaction conversion rate is improved.

Further, the present inventors have found that a metal cation capable of forming a poorly water-soluble salt with phosphoric acid is present in the reaction solution as a salt of pentose-1-phosphate, so that the above-mentioned salt is difficult to react with phosphoric acid. It has been found that the same effect can be obtained as when a metal cation capable of forming a water-soluble salt or a salt thereof is added.

The present invention has been made based on these findings by the present inventors.

That is, the process for producing a nucleoside compound of the present invention comprises reacting pentose-1-phosphate with a nucleobase or a nucleobase analog in an aqueous reaction medium in the presence of nucleoside phosphorylase activity. In the aqueous reaction medium, wherein a metal cation capable of forming a sparingly water-soluble salt with phosphate ions is present in the aqueous reaction medium.

As the pentose-1-phosphate in this production method, ribose-1-phosphate or 2-deoxyribose-1-phosphate can be used.

As the metal cation capable of forming a poorly water-soluble salt with phosphoric acid, at least one selected from calcium ions, barium ions and aluminum ions can be used.

The metal cation capable of forming a sparingly water-soluble salt with phosphoric acid is a metal salt with one or more anions selected from chloride ion, nitrate ion, carbonate ion, sulfate ion and acetate ion. It can be added to the reaction medium.

The metal cation capable of forming a poorly water-soluble salt with phosphoric acid can be present in the reaction aqueous medium as a salt of pentose-1-phosphate.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Pentose-1- in the present invention
Phosphoric acid refers to polyhydroxyaldehyde or polyhydroxyketone and derivatives thereof in which phosphoric acid is ester-bonded to position 1.

A typical example is ribose 1
-Phosphate, 2'-deoxyribose 1-phosphate, 2 ',
3'-dideoxyribose 1-phosphate, arabinose 1
-Phosphoric acid and the like, but are not limited thereto.

The polyhydroxy aldehyde or polyhydroxy ketone as used herein includes those derived from natural products such as aldopentoses such as D-arabinose, L-arabinose, D-xylose, L-lyxose and D-ribose; -Xylulose, L-xylulose, D-
Examples include, but are not limited to, ketopentoses such as ribulose, deoxysaccharides such as D-2-deoxyribose, and D-2,3-dideoxyribose.

These pentose-1-phosphates are produced by a phosphorolysis reaction of a nucleoside compound by the action of nucleoside phosphorylase (J. Biol. C
hem. Vol. 184, 437, 1950) or an anomer-selective chemical synthesis method.

The nucleobases and nucleobase analogs used in the present invention are heterocyclic compounds containing a nitrogen atom which is a chemical structural element of DNA or RNA, and include the group consisting of pyrimidines, purines and nucleobase analogs. And natural or unnatural nucleobases and nucleobase analogs selected from:

The nucleobases and nucleobase analogs are
Halogen atom, alkyl group, haloalkyl group, alkenyl group, haloalkenyl group, alkynyl group, amino group, alkylamino group, hydroxyl group, hydroxyamino group, aminooxy group, alkoxy group, mercapto group, alkylmercapto group, aryl group, aryl It may be substituted by an oxy group or a cyano group.

Examples of the halogen atom as a substituent include chlorine, fluorine, iodine and bromine.

Examples of the alkyl group include an alkyl group having 1 to 7 carbon atoms such as a methyl group, an ethyl group and a propyl group.

Examples of the haloalkyl group include haloalkyl groups having 1 to 7 carbon atoms such as fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl and bromoethyl.

Examples of the alkenyl group include alkenyl groups having 2 to 7 carbon atoms such as vinyl and allyl.

Examples of the haloalkenyl group include haloalkenyl groups having 2 to 7 carbon atoms such as bromovinyl and chlorovinyl.

Examples of the alkynyl group include alkynyl groups having 2 to 7 carbon atoms such as ethynyl and propynyl.

Examples of the alkylamino group include alkylamino groups having 1 to 7 carbon atoms such as methylamino and ethylamino.

Examples of the alkoxy group include C1-C7 alkoxy groups such as methoxy and ethoxy.

Examples of the alkyl mercapto group include alkyl mercapto groups having 1 to 7 carbon atoms, such as methyl mercapto and ethyl mercapto.

Examples of the aryl group include a phenyl group; an alkylphenyl group having 1 to 5 carbon atoms such as methylphenyl and ethylphenyl; an alkoxyphenyl group having 1 to 5 carbon atoms such as methoxyphenyl and ethoxyphenyl. An alkylaminophenyl group having 1 to 5 carbon atoms such as dimethylaminophenyl and diethylaminophenyl; and a halogenophenyl group such as chlorophenyl and bromophenyl.

Specific examples of pyrimidines include cytosine, uracil, 5-fluorocytosine, 5-fluorouracil, 5-chlorocytosine, 5-chlorouracil and 5
-Bromocytosine, 5-bromouracil, 5-iodocytosine, 5-iodouracil, 5-methylcytosine, 5
-Methyluracil (thymine), 5-ethylcytosine, 5
-Ethyluracil, 5-fluoromethylcytosine, 5-
Fluorouracil, 5-trifluorocytosine, 5-trifluorouracil, 5-vinyluracil, 5-bromovinyluracil, 5-chlorovinyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-propynyluracil, pyrimidin-2-one , 4-hydroxyaminopyrimidin-2-one, 4-aminooxypyrimidin-2-one, 4-methoxypyrimidin-2-one, 4-
Acetoxypyrimidin-2-one, 4-fluoropyrimidin-2-one, 5-fluoropyrimidin-2-one and the like can be mentioned.

Specific examples of pudding include pudding, 6
-Aminopurine (adenine), 6-hydroxypurine,
6-fluoropurine, 6-chloropurine, 6-methylaminopurine, 6-dimethylaminopurine, 6-trifluoromethylaminopurine, 6-benzoylaminopurine, 6-acetylaminopurine, 6-hydroxyaminopurine, 6- Aminooxypurine, 6-methoxypurine, 6-acetoxypurine, 6-benzoyloxypurine, 6-methylpurine, 6-ethylpurine, 6-trifluoromethylpurine, 6-phenylpurine, 6-mercaptopurine, 6-methyl Mercaptopurine, 6-aminopurine-1-oxide, 6-hydroxypurine-1-oxide, 2-amino-6-hydroxypurine (guanine), 2,6-diaminopurine, 2-amino-6-chloropurine, 2 -Amino-6-iodopurine, 2-aminopurine, 2-amino-6 Mercaptopurine, 2-amino-6-methylmercaptopurine, 2-amino-6-hydroxyaminopurine, 2-amino-6-methoxypurine, 2-amino-6-benzoyloxypurine, 2-amino-6-acetoxypurine , 2-amino-6-methylpurine, 2-amino-6-cyclopropylaminomethylpurine, 2-amino-6-phenylpurine, 2-amino-8-bromopurine, 6-cyanopurine, 6-amino-2 -Chloropurine (2-chloroadenine), 6-amino-2-fluoropurine (2-fluoroadenine) and the like.

Specific examples of nucleobase analogs include:
6-amino-3-deazapurine, 6-amino-8-azapurine, 2-amino-6-hydroxy-8-azapurine, 6-amino-7-deazapurine, 6-amino-1-
Deazapurine, 6-amino-2-azapurine and the like.

In the present invention, the nucleoside compound is a compound in which a pyrimidine, a purine, or a nucleobase analog is N-glucoside-linked at the 1-position of the above-mentioned polyhydroxyaldehyde or polyhydroxyketone and derivatives thereof. Deoxycytidine, deoxyuridine, deoxyguanosine, deoxyadenosine, dideoxyadenosine, trifluorothymidine, ribavirin, orotidine, uracil arabinoside, adenine arabinoside, 2-methyl-adenine arabinoside,
2-chloro-hypoxanthine arabinoside, thioguanine arabinoside, 2,6-diaminopurine arabinoside, cytosine arabinoside, guanine arabinoside, thymine arabinoside, enocitabine, gemcitabine, azidothymidine, idoxuridine , Dideoxyadenosine, dideoxyinosine, dideoxycytidine, didehydrodeoxythymidine, thiadedeoxycytidine, sorivudine, 5-methyluridine, virazole, thioinosine, tegafur, doxyfluridine, bredinin, nebulin, allopurinol uracil, 5-fluorouracil, 2'-aminouridine 2,2′-aminoadenosine, 2′-aminoguanosine, 2-chloro-2′-aminoinosine and the like.

The term "nucleoside phosphorylase" as used in the present invention is a general term for enzymes which decompose N-glycoside bonds of nucleoside compounds in the presence of phosphoric acid. In the present invention, a reverse reaction of a reaction utilizing this enzyme activity is utilized. Can be. The enzyme used in the reaction may be of any type and origin as long as it has an activity capable of producing a target nucleoside compound from the corresponding pentose-1-phosphate and a nucleobase or nucleobase analog.
The enzymes are broadly classified into purine-type and pyrimidine-type. Known examples include, for example, purine-type purine nucleoside phosphorylase (EC 2.4.2.1), guanosine phosphorylase (EC 2.4.2.15), and pyrimidine. Pyrimidine nucleoside phosphorylase (EC
2.4.2.2), uridine phosphorylase (EC2.
4.2.3), thymidine phosphorylase (EC 2.4.
2.4), deoxyuridine phosphorylase (EC2.
4.2.23).

The nucleoside phosphorylase activity in the present invention can also be caused to act on the reaction system by using a microorganism having the enzyme activity in an aqueous reaction medium or in contact with the aqueous reaction medium. Such microorganisms
Purine nucleoside phosphorylase (EC 2.4.2.
1), guanosine phosphorylase (EC 2.4.2.1)
5), pyrimidine nucleoside phosphorylase (EC
2.4.2.2), uridine phosphorylase (EC2.
4.2.3), thymidine phosphorylase (EC 2.4.
2.4), deoxyuridine phosphorylase (EC2.
The microorganism is not particularly limited as long as it is a microorganism expressing one or more nucleoside phosphorylases selected from the group consisting of 4.2.23).

Specific examples of such microorganisms include the genus Nocardia and Microbacteria.
erium), Corynebacterium (Corynebacterium),
Brevibacterium genus, Cellulomonas genus, Flavobacterium
terium, genus Kluyvere, genus Mycobacterium, Haemophilus
Genus, Micoplana, Protaminobacterium (Pro
genus taminobacter, genus Candida, genus Saccharomyces, bacillus
Genus, thermophilic Bacillus, Pseudomonas
s), Micrococcus, Hafnia
nia), Proteus, Vibrio
Genus, Staphyrococcus, Propionibacterium, Sartina (Sa
genus rtina), genus Planococcus, genus Escherichia, genus Kurthia, genus Rhodococcus, Acinetobacter (Acinetob)
acter), Xanthobacter, Streptomyces, Rhizobiu
m), Salmonella, Klebs
iella), Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter
genus, Agrobacterium genus,
A preferred example is a microorganism strain included in the genus Arthrobacter or the genus Pseudonocardia.

With the recent advances in molecular biology and genetic engineering, the molecular genes and amino acid sequences of the nucleoside phosphorylases of the above-mentioned microorganism strains are analyzed to obtain the gene for the protein from the microorganism strain. Constructing a recombinant plasmid into which the gene and the control region necessary for expression have been inserted, introducing this into any host, and making it possible to produce a genetically modified bacterium expressing the protein, and It's also relatively easy. In view of the state of the art, genetically modified bacteria having such a nucleoside phosphorylase gene introduced into an arbitrary host are also included in the microorganism expressing the nucleoside phosphorylase of the present invention.

The control region required for expression herein includes a promoter sequence (including an operator sequence for controlling transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence, and the like. Specific examples of the promoter sequence include:
Trp promoter of tryptophan operon derived from Escherichia coli, lac promoter of lactose operon, and P _L promoter and P _R promoter derived from lambda phage, derived from Bacillus subtilis gluconate synthase promoter (g
nt), alkaline protease promoter (ap
r), neutral protease promoter (npr), α-
Amylase promoter (amy) and the like. In addition, sequences that have been independently modified and designed, such as the tac promoter, can also be used. The ribosome binding sequence includes a sequence derived from Escherichia coli or Bacillus subtilis, but is not particularly limited as long as it functions in a desired host such as Escherichia coli or Bacillus subtilis. For example, a consensus sequence in which a sequence complementary to the 3 ′ terminal region of 16S ribosomal RNA is continuous for 4 bases or more may be prepared by DNA synthesis and used. Although a transcription termination sequence is not necessarily required, a p-factor independent one such as a lipoprotein terminator or a trp operon terminator can be used. The sequence of these control regions on the recombinant plasmid is preferably arranged in the order of the promoter sequence, the ribosome binding sequence, the gene encoding nucleoside phosphorylase, and the transcription termination sequence from the 5 'terminal side upstream.

Specific examples of the plasmid referred to herein include:
PBR3 having an autonomously replicable region in Escherichia coli
22, pUC18, Bluescript II SK
(+), PKK223-3, pSC101, and pUB110, pSC110 which has a region capable of autonomous replication in Bacillus subtilis.
TZ4, pC194, ρ11, φ1, φ105 and the like can be used as vectors. Examples of plasmids capable of autonomous replication in two or more hosts include p
HV14, TRp7, YEp7 and pBS7 can be used as vectors.

As an optional host, Escherichia coli can be mentioned as a representative example as described in Examples below, but it is not particularly limited to Escherichia coli but may be Bacillus subtilis or the like. Other microbial strains such as Bacillus, yeast and actinomycetes are also included.

As the nucleoside phosphorylase or the microorganism having the enzymatic activity in the present invention, commercially available enzymes, microbial cells having the enzymatic activity, treated cells, or immobilized products thereof can be used. The treated bacterial cells are, for example, acetone-dried bacterial cells and bacteria prepared by mechanical disruption, ultrasonic crushing, freeze-thaw treatment, pressurized and depressurized treatment, osmotic treatment, autolysis, cell wall decomposition treatment, surfactant treatment, etc. It may be a crushed body or the like, and may be used after repeated purification by ammonium sulfate precipitation, acetone precipitation, or column chromatography, if necessary.

In the present invention, the metal cation capable of forming a poorly water-soluble salt with a phosphate ion forms a poorly water-soluble salt with a phosphate ion by-produced in the reaction, and can precipitate in a reaction solution. If it is a thing, it will not be limited. Such include metal cations such as calcium, magnesium, barium, iron, cobalt, nickel, copper, silver, molybdenum, lead, zinc, lithium, and the like. Among them, metal salts which are industrially high in versatility and safety and do not affect the reaction are particularly preferable, and examples of such metal salts include calcium ion, barium ion and aluminum ion.

In the present invention, the metal cation capable of forming a sparingly water-soluble salt with phosphate ion includes a metal ion capable of forming a sparingly water-soluble salt with phosphoric acid, such as chloride ion, nitrate ion, carbonate ion, and sulfate ion. It can be added to the reaction solution as a metal salt with one or more anions selected from ions and acetate ions. Specifically, calcium chloride, calcium nitrate, calcium carbonate, calcium sulfate, calcium acetate, barium chloride, barium nitrate, barium carbonate, barium sulfate, barium acetate, aluminum chloride, aluminum nitrate, aluminum carbonate, aluminum sulfate, aluminum acetate, etc. Is exemplified.

The metal cation in the present invention may be present in the reaction solution as a salt of pentose-1-phosphate. Specifically, ribose-1-phosphate / calcium salt, 2-deoxyribose-1-phosphate / calcium salt, 2,3-dideoxyribose-1-phosphate / calcium salt, arabinose-1-phosphate / calcium salt Calcium salt, ribose-1-phosphate / barium salt, 2-deoxyribose-1-phosphate / barium salt, 2,3-dideoxyribose-1-phosphate / barium salt, arabinose-1-phosphate / barium salt , Ribose-1-phosphate aluminum salt, 2-deoxyribose-1-phosphate aluminum salt, 2,3-dideoxyribose-1-phosphate aluminum salt, arabinose-1-phosphate aluminum salt, etc. Is mentioned.

The aqueous reaction medium constituting the reaction solution used in the method of the present invention is a reaction medium mainly composed of water,
The reaction utilizing the enzyme activity intended in the present invention proceeds,
Any substance can be used as long as it can be removed from the reaction system by reacting the phosphate ion with the metal cation to precipitate as a hardly soluble salt. Such aqueous reaction media include
Examples thereof include an aqueous reaction medium in which water or a water-soluble organic solvent such as lower alcohol, acetone, and dimethyl sulfoxide is added to water. When a water-soluble organic solvent is used, the mixing ratio of the water-soluble organic solvent to water is, for example, 0.1.
It can be 1 to 70% by weight. Further, in order to ensure the stability of the reaction, the reaction solution using the aqueous reaction medium can be prepared as a buffer. As this buffer, a known buffer that can be used for the enzyme reaction according to the present invention can be used.

In the synthesis reaction of the nucleoside compound according to the present invention, the target nucleoside compound, pentose-1-phosphate as a substrate and a nucleobase or nucleobase analog, nucleoside phosphorylase as a reaction catalyst or a microorganism having the enzyme activity Alternatively, reaction conditions such as appropriate pH and temperature may be selected depending on various preparations from the microorganism and the type and characteristics of a metal salt for excluding phosphoric acid from the reaction system.
0, the temperature can be in the range of 10 to 60 ° C. pH
If the control range is out of the control range, the reaction conversion rate may decrease due to the stability of the target substance or substrate, the decrease in enzyme activity, the formation of poorly water-soluble salts with phosphoric acid, and the like. If the pH changes during the reaction, an acid such as hydrochloric acid or sulfuric acid or an alkali such as sodium hydroxide or potassium hydroxide may be added as needed. The concentration of pentose-1-phosphate and the nucleobase or nucleobase analog used in the reaction is suitably about 0.1 to 1000 mM, and the molar ratio of the two is such that the ratio of nucleobase or nucleobase analog to be added is pentose-phosphate. The reaction can be carried out in a molar amount of 0.1 to 10 times relative to 1-phosphoric acid or a salt thereof. Considering the reaction conversion, the molar amount is preferably 0.95 times or less. When two or more nucleobases or nucleobase analogs are used as a mixture, the above concentrations and molar ratios can be adopted for the total amount of the nucleobases or nucleobase analogs.

The metal cation which can form a sparingly water-soluble salt with phosphoric acid to be added depends on the pentose used in the reaction.
It is preferred to add 0.1 to 10 times mol amount, more preferably 0.5 to 5 times mol amount to 1-phosphoric acid. In high-concentration reactions, the nucleobase or nucleobase analog of the substrate or the nucleoside compound of the product may be present in the reaction solution without being completely dissolved, but the present invention is also applied to such a case. You. When a mixture of two or more metal cations is used, the above molar ratio can be adopted for the total amount of the metal cations used.

To isolate the nucleoside compound thus produced from the reaction solution, conventional methods such as concentration, crystallization, dissolution, electrodialysis, and adsorption / desorption with an ion exchange resin or activated carbon can be applied.

[0051]

The present invention will be described below with reference to examples and comparative examples, but the present invention is not limited to these examples.

Analytical Method All the generated nucleoside compounds were quantified by high performance liquid chromatography. The analysis conditions are as follows. Column: YMC-Pack ODS-A312 150
× 6.0 mmI. D. (YMC Co., Ltd.) Column temperature; 40 ° C. Pump flow rate; 0.75 ml / min detection; UV 260 nm, eluent; 10 mM phosphoric acid: acetonitrile = 95: 5
(V / V) Example 1 2.5 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA),
2.5 mM adenine (Wako Pure Chemical Industries, special grade), 12un
1 ml of a reaction solution consisting of pursine nucleoside phosphorylase (its fungus), 10 mM calcium chloride (Wako Pure Chemical Industries, special grade), and 10 mM Tris-HCl buffer (pH 7.4) was reacted at 30 ° C. for 24 hours. After the completion of the reaction, a white precipitate was formed. When the reaction solution was diluted and analyzed, 2.40 mM deoxyadenosine was produced. At that time, the reaction yield was 96.0%.

Comparative Example 1 Example 1 except that no calcium chloride was added.
The reaction was carried out under the same procedure and conditions as described above. After completion of the reaction, no precipitate was formed. When the reaction solution was diluted and analyzed, 2.01 mM deoxyadenosine was generated. At that time, the reaction yield was 80.4%.

Example 2 A reaction was carried out under the same procedure and conditions as in Example 1 except that the concentration of calcium chloride was changed to 2.5 mM. After the reaction,
A white precipitate had formed. When the reaction solution was diluted and analyzed, 2.27 mM deoxyadenosine was produced. At that time, the reaction yield was 90.8%.

Example 3 A reaction was carried out in the same procedure and under the same conditions as in Example 1 except that aluminum chloride was added instead of calcium chloride. After the completion of the reaction, a white precipitate was formed. When the reaction solution was diluted and analyzed, 2.31 mM of deoxyadenosine was produced. At that time, the reaction yield was 93.
3%.

Example 4 A reaction was performed in the same procedure and under the same conditions as in Example 1 except that barium chloride was added instead of calcium chloride. After the completion of the reaction, a white precipitate was formed. When the reaction solution was diluted and analyzed, 2.31 mM of deoxyadenosine was produced. At that time, the reaction yield was 92.4%.

Example 5 A reaction was carried out under the same procedure and conditions as in Example 4 except that the barium chloride concentration was changed to 2.5 mM. After the completion of the reaction, a white precipitate was formed. When the reaction solution was diluted and analyzed, 2.27 mM deoxyadenosine was produced. At that time, the reaction yield was 90.2%.

Example 6 Journal of Biologcal Chemistry, Vol. 184, pp. 449-45
9, 1950, 2-deoxyribose 1-barium phosphate salt was prepared. 2.5 mM 2-deoxyribose-1-barium phosphate, 2.5 mM adenine (manufactured by Wako Pure Chemical Industries, special grade), 12 units / ml purine nucleoside phosphorylase (manufactured by Funakoshi), 10 m
M calcium chloride (Wako Pure Chemical Industries, special grade), 1 ml of a reaction solution containing 10 mM Tris-HCl buffer (pH 7.4)
The reaction was performed at 0 ° C. for 24 hours. After the completion of the reaction, a white precipitate was formed. After diluting the reaction solution and analyzing it,
2.40 mM deoxyadenosine was produced. At that time, the reaction yield was 91.1%.

Example 7 2.5 mM 2-deoxyribose-1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA)
2.5 mM thymine (manufactured by Wako Pure Chemical Industries, special grade), 12 uni
ts / ml thymidine phosphorylase (SIGMA
Manufactured), 10 mM calcium nitrate (Wako Pure Chemical, special grade),
One ml of a reaction solution containing 10 mM Tris-HCl buffer (pH 7.4) was reacted at 30 ° C. for 24 hours. After the reaction,
A white precipitate had formed. When the reaction solution was diluted and analyzed, 2.28 mM of thymidine was produced.
At that time, the reaction yield was 91.2%.

Comparative Example 2 Example 7 except that no calcium nitrate was added.
The reaction was carried out under the same procedure and conditions as described above. After completion of the reaction, no precipitate was formed. When the reaction solution was diluted and analyzed, 1.88 mM of thymidine was produced. At that time, the reaction yield was 75.2%.

Example 8 2.5 mM ribose-1-cyclohexylammonium phosphate (SIGMA), 2.5 mM 2,6-diaminopurine (Aldrich), 12 units / m
l purine nucleoside phosphorylase (Funakoshi), 10 mM calcium chloride (Wako Pure Chemical, special grade),
One ml of a reaction solution containing 10 mM Tris-HCl buffer (pH 7.4) was reacted at 30 ° C. for 24 hours. After the reaction,
A white precipitate had formed. When the reaction solution was diluted and analyzed, 1.94 mM of 2,6-diaminopurine riboside was produced. At that time, the reaction yield was 77.6%.
Met.

Comparative Example 3 Example 8 except that no calcium chloride was added.
The reaction was carried out under the same procedure and conditions as described above. After completion of the reaction, no precipitate was formed. When the reaction solution was diluted and analyzed, 1.54 mM of 2,6-diaminopurine riboside was generated. At that time, the reaction yield was 61.6%.

Example 9 Escherichia coli chromosomal DNA was prepared as follows.

Escherichia coli K-12 / XL-10
The strain (Stratagene) was inoculated into 50 ml of LB medium,
After overnight culturing at 7 ° C, the cells are collected and lysozyme 1 mg / ml
Was lysed with a lysis solution containing After the lysate was treated with phenol, DNA was precipitated by ethanol precipitation according to a conventional method. The resulting precipitate of DNA was collected by winding it around a glass rod, washed, and used for PCR.

The primers for PCR include the base sequence of known deoD gene of Escherichia coli (GenBank ac
cession No. AE000508 (coding region is base number 11531-12
Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 (Hokkaido System
(Synthesis Co., Ltd.) was used.
These primers have EcoRI and HindIII restriction enzyme recognition sequences near the 5 ′ end and near the 3 ′ end, respectively.

Using a 0.1 ml PCR reaction solution containing 6 ng / μl of the above-mentioned E. coli chromosomal DNA and 3 μM of each primer completely digested with the restriction enzyme HindIII, denaturation: 96 ° C. for 1 minute, annealing: 55 ° C. for 1 minute ,
Extension reaction: A reaction cycle consisting of 74 ° C. for 1 minute
PCR was performed under the condition of 0 cycle.

The above reaction product and plasmid pUC18
(Takara Shuzo Co., Ltd.) was digested with EcoRI and HindIII, ligated using Ligation High (Toyobo Co., Ltd.), and transformed into Escherichia coli DH5α using the obtained recombinant plasmid. The transformant was treated with 50 μg / ml ampicillin (Am) and X-Ga
l (5-bromo-4-chloro-3-indolyl-β-D
-Galactoside) to obtain a transformant which became Am-resistant and a white colony.

A plasmid was extracted from the thus obtained transformant, and the plasmid into which the desired DNA fragment was inserted was named pUC-PNP73. The thus obtained transformant was used to transform Escherichia coli MT-10.
905.

Escherichia coli MT-10905 strain was transformed into 100 m of LB medium containing 50 μg / ml of ampicillin.
The cells were cultured at 37 ° C. overnight with shaking. The obtained culture solution is
The cells were centrifuged at 3000 rpm for 10 minutes to collect the cells. Bacterial cells were washed with 10 mM Tris-HCl buffer (pH 8.0) 1
A suspension suspended in 0 mL and crushed by ultrasonic waves was used as an enzyme source.

10 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (SIG
MA), 10 mM adenine (Wako Pure Chemical Industries, special grade),
A reaction solution 1.0 containing 20 mM calcium chloride, 100 mM Tris-HCl buffer (pH 8.0), and 20 units / ml of the purine nucleoside phosphorylase described above.
ml was reacted at 50 ° C. for 20 hours.

After the completion of the reaction, a white precipitate was formed. When the reaction solution was diluted and analyzed, 9.6 mM deoxyadenosine was produced. At that time, the reaction yield was 96.0%.

Comparative Example 4 Example 9 except that no calcium chloride was added.
The reaction was carried out under the same procedure and conditions as described above. After completion of the reaction, no precipitate was formed. When the reaction solution was diluted and analyzed, 7.9 mM deoxyadenosine was produced.
At that time, the reaction yield was 79%.

Example 10 10 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 1
0 mM guanine (manufactured by Wako Pure Chemical Industries, special grade), 20 mM calcium chloride, 100 mM Tris-HCl buffer (pH
8.0), a reaction solution comprising purine nucleoside phosphorylase prepared in Example 9 at 20 units / ml.
0 ml was reacted at 50 ° C. for 20 hours.

After the completion of the reaction, a white precipitate was formed. When the reaction solution was diluted and analyzed, 8.0 mM deoxyguanosine was generated. At that time, the reaction yield was 80.0%.

Comparative Example 5 Example 1 was repeated except that no calcium chloride was added.
The reaction was performed in the same procedure and under the same conditions as in Example 1. After completion of the reaction, no precipitate was formed. When the reaction solution was diluted and analyzed, 5.1 mM deoxyguanosine had been produced.
At that time, the reaction yield was 51%.

Example 11 10 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 1 mM
0 mM thymine (manufactured by Wako Pure Chemical Industries, special grade), 20 mM calcium chloride, 100 mM Tris-HCl buffer (pH 8.
0), 1.0 ml of a reaction solution containing 20 units / ml thymidine phosphorylase (manufactured by SIGMA) was reacted at 50 ° C. for 20 hours.

After the completion of the reaction, a white precipitate was formed. When the reaction solution was diluted and analyzed, 9.0 mM thymidine was produced. The reaction yield at that time was 90.0
%Met.

Comparative Example 6 Example 1 except that no calcium chloride was added.
The reaction was carried out under the same procedure and conditions as in 1. After completion of the reaction, no precipitate was formed. When the reaction solution was diluted and analyzed, 7.0 mM deoxyguanosine was produced.
At that time, the reaction yield was 70%.

[0079]

According to the present invention, in the production of a nucleoside compound from pentose-1-phosphate and a nucleobase using a nucleoside phosphorylase or a microorganism having the enzyme activity, a poorly water-soluble salt is formed with phosphoric acid. By adding a metal salt, phosphoric acid by-produced is excluded from the reaction system, and an improvement in the reaction yield, which has not been achieved by the prior art, is achieved. From an industrial point of view, the present invention leads to significant cost reduction, and its significance is significant.

[0080]

[Sequence List] SEQUENCE LISTING <110> MITSUI CHEMICALS, INC. <120> <160> 2 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide to act as a PCR primer for cloning of purine nucleo side phosphorylase of E. coli K-12 <400> 1 gtgaattcac aaaaaggata aaacaatggc 30 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide to act as a PCR primer for cloning of purine nucleo side phosphorylase of E. coli K-12 <400> 2 tcgaagcttg cgaaacacaa ttactcttt 29

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Claims (5)

[Claims] 1. A method according to claim 1, wherein said pentose-1-phosphate is present in an aqueous reaction medium in the presence of nucleoside phosphorylase activity.
A method for producing a nucleoside compound, comprising reacting a nucleobase or a nucleobase analog to form a nucleoside compound, wherein a metal cation capable of forming a poorly water-soluble salt with phosphate ions is present in the aqueous reaction medium. A method for producing a nucleoside compound. 2. Pentose-1-phosphate is converted to ribose-
The method for producing a nucleoside compound according to claim 1, which is 1-phosphate or 2-deoxyribose-1-phosphate. 3. The nucleoside according to claim 1, wherein the metal cation capable of forming a poorly water-soluble salt with phosphoric acid is at least one selected from calcium ions, barium ions and aluminum ions. A method for producing a compound. 4. A metal cation capable of forming a poorly water-soluble salt with phosphoric acid as a metal salt with one or more anions selected from chloride, nitrate, carbonate, sulfate and acetate ions. The method for producing a nucleoside compound according to claim 3, wherein the nucleoside compound is present in an aqueous reaction medium. 5. The process for producing a nucleoside compound according to claim 3, wherein the metal cation capable of forming a poorly water-soluble salt with phosphoric acid is present in the aqueous reaction medium as a salt of pentose-1-phosphate.