JPH0335915B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [] BACKGROUND TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for producing triazole deoxyribonucleosides using enzymes.

In the present invention, triazole deoxyribonucleoside has the structural formula [] The chemical name is 1-
(2-deoxy-β-D-erythro-pentofuranosyl)-1,2,4-triazole-3-carboxamide. This compound is suitable for DNA and RNA
It is an analog of Ribavirin, a compound that exhibits strong antiviral effects over a wide range of viruses, and is itself a promising compound as an antiviral agent.

Prior Art Conventionally, as a method for producing triazole deoxyribonucleoside, after condensing 3-methoxycarbonyl-1,2,4-triazole or 3-cyano-1,2,4-triazole with a deoxyribonucleoside derivative, methoxycarbonyl A method of converting a cyano group or a cyano group into a carboxamide group is known (J. Carbohydrates.
Nucleosides・Nucleotides, 2(1), 1-36
(1975)). Such synthetic methods require complicated reaction operations and have poor yields.

In addition, methods for producing triazole ribonucleosides such as ribavirin enzymatically or using microorganisms are known (Japanese Patent Application Laid-Open No. 1989-1999-1).
No. 29720, JP-A-50-123882 and JP-A-57-
(See No. 146593). However, no enzymatic method or method using microorganisms has been adopted for the production of triazole deoxyribonucleosides.

[] Summary of the Invention The present inventors have succeeded for the first time in producing triazole deoxyribonucleosides by the action of enzymes using 1,2,4-triazole derivatives and 2-deoxyribose donors as substrates, and have succeeded in producing triazole deoxyribonucleosides by the action of enzymes using 1,2,4-triazole derivatives and 2-deoxyribose donors as substrates. The present invention was completed based on this.

The present invention comprises reacting 1,2,4-triazole-3-carboxamide (hereinafter sometimes abbreviated as "triazole compound") with a 2-deoxyribose donor in the presence of a nucleoside phosphorylase source, and The present invention provides a method for producing triazole deoxyribonucleosides, which is characterized by obtaining triazole deoxyribonucleosides represented by the structural formula [].

[] Detailed description of the invention Source of nucleoside phosphorylase In the present invention, the term "source of nucleoside phosphorylase" refers to a source of nucleoside phosphorylase that acts on 1,2,4-triazole-3-carboxamide and a 2-deoxyribose donor defined later, A substance that contains a single enzyme or a group of enzymes that give nucleoside phosphorylase. In addition, "nucleoside phosphorylase" here refers to the action of phosphorolyzing 2'-deoxyribonucleoside to give 2-deoxyribose-1-phosphate and nucleobase, as well as the action of giving 2-deoxyribose-1-phosphate and nucleobase. An enzyme responsible for synthesizing triazole deoxyribonucleosides from triazole compounds.

The nucleoside phosphorylase source may also include enzymes other than nucleoside phosphorylase. These enzymes are enzymes that actively participate in the reaction of the present invention, for example, by acting on the raw material compound to produce a substrate for nucleoside phosphorylase, or are enzymes that actively participate in the reaction of the present invention under the reaction conditions of the present invention. It may be an enzyme that does not inhibit the reaction. Enzymes that actively participate in reactions include:
An example is a phosphatase using a deoxyribonucleotide as a 2-deoxyribose donor.

The source of the above-mentioned nucleoside phosphorylase can be used regardless of its origin and form during the reaction (soluble enzyme, immobilized enzyme, culture, live bacterial cells, processed bacterial cells, etc.), as long as it meets the purpose of the present invention.

That is, the above-mentioned enzyme-containing substances derived from microorganisms (fungi) such as bacteria, yeast, actinomycetes, filamentous fungi, and basidiomycetes, animal organs or tissues, or plants can be used. preferable. Brevibacterium as the most preferred nucleoside phosphorylase source
Microorganisms belonging to the genus are exemplified.

A specific example of a strain with particularly strong enzyme activity, which is the object of the present invention, is the AT-6-7 strain isolated from the sand of Koshien Stadium in Nishinomiya City, Hyogo Prefecture. The mycological properties of this strain are described below.

A Morphology (1) Cell morphology and size: short rod-shaped, 0.8~
1.0 x 1.0 - 1.2 μm (2) Spore formation: None (3) Gram staining: Positive B Growth status in various media (1) Internal juice agar plate culture (28℃, 48 hours) Colony shape: Circular ) Ridges on colony surface: Flat, smooth Size: 2 to 4 mm Color: Yellow to pinkish yellow (2) Juicy agar slant culture (28℃, 48 hours) Growth: Good Growth shape: Warts Echinulate (3) Liquid culture in broth (28℃, 48 hours) Growth: Forms a bacterial ring on the surface and produces a slight sediment.

(4) Meat juice Seratin puncture culture (20℃, 6 days):
Liquefies in Straitiform.

(5) Litmus milk medium (28°C, 4 days): Slight coagulation and peptonization is also observed.

C Physiological properties (1) Reduction of nitrate (28°C, 5 days): No reducibility.

(2) Hydrogen sulfide generation (28℃, 5 days): Not generated.

(3) Starch hydrolysis: Degradable.

(4) Catalase: positive (5) Indole generation: Not generated.

(6) Formation of ammonia from peptone and arginine: negative (7) Methylred test: negative (8) V-P test: positive (9) Attitude towards oxygen: aerobic (10) O-F test (Hugh (by Leifson method):
Type F (Fermentation) (11) Production of acid from sugars Positive: glucose, mannose, fructose, maltose, sutucarose, trehalose Negative: arabinose, xylose, galactose, lactose, sorbitol, inosit, glycerin (12) Growth PH range: PH6 .0 to 9.0 (13) Optimum growth temperature: 25 to 37°C.
The search was conducted using the classification criteria of Bacteriology, 7th edition (1957). As a result, strain AT-6-7 was identified as a strain belonging to the genus Brevibacterium because it is a short bacillus, almost like a coccus, is Gram-positive, does not form filaments, and produces acid from carbohydrates. , and named Brevibacterium acetylicum AT-6-7.

The identification and attribution of the above strains is from Virgies.
This is based on the 7th edition of the Manual of Determinative Bacteriology, and if the identification and attribution of these strains is made using a different classification standard due to changes in the classification standards, they may be assigned to other species or other strains. Although the microorganisms named as above in the present invention can be used as sources of nucleoside phosphorylases for the purpose of the present invention, and have the above-mentioned mycological properties or equivalent It includes microorganisms that basically have mycological properties and can be uniquely identified.

Regarding this strain, the 1981 Ministry of International Trade and Industry Notification No.
In accordance with No. 178, we filed an application for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the deposit was made on January 13, 1981, with the deposit number being FERM P-6305. has been granted. Other exemplary strains include Brevibacterium imperiale (B.
Imperiale) ATCC8365 can be exemplified.

In addition, the above-mentioned strains may undergo induced mutations by general mutagenesis methods such as physical treatments such as irradiation with ultraviolet rays, The mutants thus derived may also be used in the present invention, as long as they do not lose enzymatic activity as a source of nucleoside phosphorylase.

Furthermore, if the gene for the enzyme as a source of nucleoside phosphorylase obtained from the above-mentioned suitable strain is incorporated into a microorganism other than the genus Brevibacterium and such a trait is expressed, such microorganism It should be considered equivalent to the genus Brevibacterium.

When culturing these microorganisms in order to prepare the microorganisms used in the present invention, the culture medium and culture method used are not particularly limited as long as these microorganisms grow.

The medium used contains appropriate amounts of carbon sources and nitrogen sources that can be assimilated by these microorganisms, and if necessary, inorganic salts, trace growth-promoting substances, antifoaming agents, etc. are added. Specifically, carbon sources include glucose, fructose, maltose, ribose, sutucarose, starch, starch hydrolyzate,
Sugars such as molasses and blackstrap molasses or their derivatives such as fatty acid esters, natural carbohydrates such as wheat, rice, etc., alcohols such as mannitol, methanol, and ethanol, gluconic acid, pyruvic acid, acetic acid,
Fatty acids such as citric acid, normal paraffin,
Carbohydrates such as kerosene, amino acids such as glycine, glutamic acid, glutamine, alanine, asparagine, etc. are selected from among common carbon sources, one or two or more of which are appropriately selected in consideration of the assimilation ability of the microorganisms used. do it. Nitrogen sources include meat extract, peptone, yeast extract, dried yeast, soybean hydrolyzate, soybean flour, milk casein,
Organic nitrogen compounds such as casamino acids, various amino acids, corn staple liquor, cotton seed meal or its hydrolysates, fruit meal or its hydrolysates, hydrolysates of other animals, plants, and microorganisms, ammonia, ammonium nitrate,
Select one or more of ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium acetate, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea, taking into consideration the assimilation ability of the microorganisms used. and use it. Furthermore, one or more of phosphates, hydrochlorides, nitrates, carbonates, nitrates, acetates, etc. of magnesium, manganese, iron, zinc, copper, sodium, calcium, potassium, etc. are added as appropriate as inorganic salts. If necessary, add vegetable oil, antifoaming agents such as surfactants, vitamin _B1 ,
B ₂ , nicotinic acid, pantothenic acid, biotin, p
-Minor growth promoting substances such as aminobenzoic acid may be added. Furthermore, when using microorganisms that exhibit nutritional requirements, it is necessary to add to the medium a substance that satisfies the growth of the microorganisms.

Cultivation is carried out in a liquid medium containing the above-mentioned medium components by selecting a culture method suitable for the microorganism used from among conventional culture methods such as shaking culture, aerated agitation culture, static culture, and continuous culture.

Culture conditions may be appropriately selected depending on the type of microorganism and medium used, but the pH at the start of culture is usually adjusted to about 6 to 8, and culture is carried out at a temperature of about 25 to 35°C. The culture period may be a period sufficient for the growth of the microorganism used, and is usually 1 to 3 days.

After culturing the microorganisms as described above, the obtained culture, viable bacterial bodies collected from the culture by conventional methods such as centrifugation, sedimentation, and flocculation, or appropriate treatment of viable bacterial bodies. The bacterial cell-treated product obtained by subjecting can be used as a nucleoside phosphorylase source in the present invention. Here, the term "culture" refers to a state in which the culture medium and cultured bacterial cells are unseparated after cultivation.
In addition, the treated bacterial cells include dried bacterial cells, cell membrane/wall-denatured bacterial cells, crushed bacterial cells, immobilized bacterial cells, bacterial cell extracts, and bacteria that have enzyme activity as a source of nucleoside phosphorylase for the purpose of the present invention. It refers to a protein fraction of a body extract or a purified product thereof, or an immobilized product of a protein fraction or a purified product thereof. In addition, in the present invention, these may be collectively referred to as "microbial cells, etc.".

The method for obtaining the treated bacterial cell product is illustrated below. That is, physical treatments such as freeze-thaw treatment, freeze-drying treatment, ventilation drying treatment, acetone drying treatment, heating treatment under acidic or alkaline conditions, grinding treatment, ultrasonic treatment, osmotic pressure difference treatment, etc. For example, enzyme treatment such as lysozyme, cell wall lytic enzyme, toluene,
By subjecting the bacterial cell extract to chemical or biochemical treatments such as contact treatment with solvents such as xylene or butyl alcohol (butanol) or surfactants alone or in combination, for example, salting out treatment, By applying enzyme separation and purification methods such as isoelectric focusing treatment, organic solvent precipitation treatment, various chromatographic treatments, and dialysis treatment alone or in combination, live bacterial cells, bacterial cell extracts, or purified products thereof can be subjected to comprehensive treatment. A bacterial cell-treated product can be obtained by applying enzyme immobilization means such as crosslinking treatment, adsorption treatment to a carrier, etc.

Reaction substrate The reaction substrate in the enzyme reaction of the present invention is 1, 2,
4-triazole-3-carboxamide and 2
- is a deoxyribose donor.

1,2,4-triazole-3-carboxamide can be used in either a free form or a salt such as a sodium salt.

In the present invention, "2-deoxyribose donor" refers to a 2-deoxyribose donor capable of transferring a 2'-deoxyribose residue to the ^N1 position of 1,2,4-triazole-3-carboxamide by the action of a nucleoside phosphorylase source. It is a deoxyribose derivative.
Thus, the term "2-deoxyribose donor" not only refers to a compound that can serve as a substrate for a nucleoside phosphorylase and, by its immediate action, donates a 2-deoxyribose residue to a triazole compound, but also as a source of a nucleoside phosphorylase. It also means a compound that can be converted into a direct substrate of the above-mentioned nucleoside phosphorylase by the action of an enzyme other than those mentioned above contained in the above-mentioned.

Specifically, 2′-deoxyribonucleoside,
Examples include 2'-deoxyribonucleotide, 2-deoxyribose-1-phosphate, or salts thereof.

As a 2′-deoxyribonucleoside, 2′-
Deoxyadenosine, 2′-deoxyinosine,
Examples of the 2'-deoxyribonucleotide include 2'-deoxyguanosine, 2'-deoxycytidine, thymidine, and 2'-deoxyuridine.
2'-deoxyadenosine-
5′-monophosphoric acid, 2′-deoxyadenosine-5′-
Diphosphoric acid, 2'-deoxyadenosine-5'-triphosphoric acid, 2'-deoxyinosine-5'-monophosphoric acid,
2'-deoxyinosine-5'-diphosphoric acid, 2'-deoxyinosine-5'-triphosphate, 2'-deoxyguanosine-5'-monophosphate, 2'-deoxyguanosine-5'-diphosphoric acid acid, 2'-deoxyguanosine-
5'-triphosphoric acid, 2'-deoxycytidine-5'-monophosphoric acid, 2'-deoxycytidine-5'-diphosphoric acid, 2'-deoxycytidine-5'-triphosphoric acid, thymidine-5' -monophosphoric acid, thymidine-5'-diphosphoric acid, thymidine-5'-triphosphoric acid, 2-deoxyuridine-5'-monophosphoric acid, 2'-deoxyuridine-5'-diphosphoric acid, 2' Free forms such as -deoxyuridine-5'-triphosphoric acid or alkaline salts such as sodium salts may be mentioned.

Reaction Substrate Solution The substrate solution used in the enzyme reaction of the present invention is basically an aqueous liquid in which the above-mentioned reaction substrate is dissolved or suspended in an aqueous medium.

The aqueous liquid contains at least a triazole compound and one or more of the above-mentioned 2-deoxyribose donors, and in addition to these reaction substrates, a phosphate ion donor, an organic solvent, a surfactant, and a metal salt. , coenzymes, acids, bases, sugars, and other substances that promote enzyme reactions, substances that inhibit interfering enzyme activity, substances that improve the solubility of reaction substrates, and substances that improve contact between enzymes and reaction substrates. You can leave it there. Furthermore, the medium may contain the above-mentioned medium components that can be assimilated by the microorganisms used.

As the aqueous medium, water or various buffers suitable for enzyme reactions (phosphate buffer, imidazole-hydrochloric acid buffer, veronal-hydrochloric acid buffer, Tris-hydrochloric acid buffer, etc.) can be used.

As the phosphate ion donor system, any substance that can be dissociated into phosphate ions in an aqueous medium may be used, such as free phosphoric acid itself, inorganic phosphates, alkali metals such as sodium and potassium, calcium, Salts with alkaline earth metals such as magnesium and ammonium are preferably used. Phosphate ion donating systems include systems that can liberate phosphate ions in substrate solutions for enzyme reactions, such as combinations of various phosphate ester derivatives and phosphatases, combinations of nucleotides and nucleotidase, nucleobases, and ribose-1.
-Phosphoric acid or a combination of 2-deoxyribose-1-phosphate and phosphorylase can be used.

The above-mentioned phosphoric acid donor system may be added from outside the system during the enzyme reaction, or may be contained as a component of the microorganism used. That is, as long as it is in a form that can be used for the enzymatic reaction, the above-mentioned substances alone or a combination of two or more of them, or microorganisms containing the above-mentioned substances or their processed products can be used in the enzymatic reaction of the present invention. In this case, these substances may be added separately to the reaction solution, or these substances contained in the nucleoside phosphorylase source may be used as they are.

Examples of organic solvents include methanol, ethanol, propanol, butanol, acetone,
Examples include methyl ethyl ketone, ethyl acetate, toluene, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide, and 2-ethoxyethanol.

Reaction Method The reaction of the present invention is achieved by contacting the enzyme source, ie, the nucleoside phosphorylase source, with a reaction substrate in an aqueous medium.

The contact method may be selected as appropriate depending on the form of the enzyme source, but it is usually a batch method in which the enzyme source is suspended or dissolved in a reaction substrate solution and preferably stirred or shaken while heating, or a contact method is used. Mix with appropriate carriers, auxiliaries, and adsorbents as necessary,
Alternatively, a column method may be applied in which the reaction substrate solution is passed through the reaction substrate by supporting the reaction material in a column. In the case of the batch method, after the reaction, bacterial cells are collected by a conventional method such as filtration (including pressure filtration, vacuum filtration, etc.), centrifugation, sedimentation separation, flocculation separation, etc., and the reaction substrate is collected. It can be used repeatedly by contacting it with a solution. In the case of a column method using an immobilized nucleoside phosphorylase source, separation operations for bacterial cells and the like are not required, but it can be similarly used repeatedly or continuously for enzymatic reactions. Furthermore, as described above, a method in which a reaction substrate is added to the medium during culturing of the microorganism used and the enzyme source and the reaction substrate are reacted can also be adopted in the present invention.

Concentrations or Added Amounts of Reaction Substrates and Enzyme Sources During the reaction, the substrate concentration of the reaction solution is not particularly limited, and a substrate concentration below the saturation concentration of each substrate in the aqueous medium used at the reaction temperature is usually adopted. The substrate concentration can also be increased by adding the above-mentioned organic solvents to the reaction substrate solution. Alternatively, each substrate can be present in a suspended state at a saturation concentration or higher in the reaction solution, and each substrate can be dissolved as the reaction progresses. Alternatively, each substrate can be added sequentially during the reaction and maintained at an appropriate concentration. When each substrate is added and dissolved, the substrate concentration is usually about 5 to 200 mM, preferably about 10 to 100 mM for the triazole compound or its salt, and usually about 1 to 300 mM, preferably about 1 to 300 mM for the 2-deoxyribose donor. is about 1-150mM.

The amount of the enzyme source to be used can be easily determined by those skilled in the art through preliminary experiments and the like, taking into consideration the type of microorganism, its mode of use, reaction efficiency, economic efficiency, and the like.

Reaction Conditions The reaction conditions of the present invention are such that the reaction substrate reacts with the action of bacterial cells, etc., and triazole deoxyribonucleosides are efficiently produced, regardless of whether the microorganism used is under non-growth conditions or under growth conditions. Not particularly limited. However, the reaction under non-growth conditions of the microorganism used is particularly efficient.

Methods for carrying out the reaction under conditions in which microorganisms do not grow include a method in which the enzyme reaction temperature is set within a temperature range in which the microorganisms used do not grow (however, a temperature range in which the enzymes involved in the reaction of the present invention are not inactivated); A method in which microorganisms are rendered incapable of growing by physically, chemically, or biochemically treating the microbial cells as described above, and then subjected to a reaction.Inhibiting the growth of the microorganisms used, such as toluene, during the reaction. The method of adding a substance to the reaction substrate solution may be used alone or in combination, but the method of manipulating the reaction temperature is particularly effective and simple.

The reaction of the present invention proceeds in a temperature range of 28 to 80°C, but in consideration of practicality, a range of 37 to 70°C is preferable, and a temperature of 40 to 60°C is particularly optimal.

The liquid properties of the reaction substrate solution should be generally maintained within the range of PH4 to 10, preferably PH6 to 8.
When the pH fluctuates, it may be corrected to a preferred pH range using an acid such as hydrochloric acid, sulfuric acid, or phosphoric acid, or an alkali such as sodium hydroxide, potassium hydroxide, aqueous ammonia, or ammonia gas.

The reaction time can be determined while checking the conversion rate of the reaction substrate to the target product, but in the case of a batch method, the reaction time is usually about 2 to 45 hours, preferably 24 to 36 hours. The reaction may be carried out by setting appropriate conditions according to the method.

Separation and Purification After the reaction, if necessary, bacterial cells and the like are separated and removed by a conventional method such as filtration, centrifugation, sedimentation, or flocculation, and subjected to a triazole deoxyribonucleoside separation and purification step.

The separation and purification of triazole deoxyribonucleosides may be carried out by known methods or by applying them, such as ion exchange chromatography,
Various chromatography methods such as adsorption chromatography, partition chromatography, and gel filtration methods, methods that utilize distribution between two liquid phases such as countercurrent distribution and countercurrent extraction, and solubility such as concentration, cooling, and addition of organic solvents. General separation and purification methods, such as methods that utilize the difference between the two, may be used alone or in appropriate combinations.

Analysis In the Examples of the present invention, triazole deoxyribonucleosides and 1,2,4-triazole-3-carboxamides were analyzed by high performance liquid chromatography. When analyzed using the equipment and conditions shown below, triazole deoxyribonucleosides were found to have 1,
2,4-triazole-3-carboxamide is eluted around a retention time of 4.64 minutes, and the amount of each can be calculated from the calibration curve.

Equipment: Shimadzu high-performance liquid chromatograph LC-3A model (manufactured by Shimadzu Corporation) Column: Micro Bondapak (μBONDAPAK) C ₁₈ , 4.6 mm x 250 mm (manufactured by Nippon Waters Limited) Eluent: Contains 2% acetonitrile 20mM Tris-hydrochloric acid buffer (PH7.5) Flow rate: 1ml/min Measurement wavelength: 225nm Column operating temperature: room temperature The present invention will be explained in more detail with examples below, but this is one embodiment of implementation. This does not limit the scope of the present invention.

Example Brevibacterium acetylicum AT-6-7 (FEI Bacterium Co., Ltd.) was added to 1.5% yeast extract medium 5.
6305) was inoculated and cultured at 28°C for 24 hours. The cells were separated from the culture solution by centrifugation.

Substrate solution containing 4.488 g of 1,2,4-triazole-3-carboxamide, 2'-deoxyuridine and 2.722 g of monopotassium phosphate (PH7.0)
60 g of the above wet bacterial cells were added to 1 and reacted at 45°C for 24 hours. When this reaction solution was analyzed, the production rate of triazole deoxyribonucleoside was 92.45.
It was %. Note that the production rate here refers to 1,
It is the molar percentage of triazole deoxyribonucleoside produced relative to 2,4-triazole-3-carboxamide.

After separating the bacterial cells from the reaction solution, the pH of this solution was adjusted to 11.5, and the formed precipitate was removed by centrifugation. Add this solution to anion exchange resin (chlorine type) 400
ml column. The effluent and the washing solution were combined and adjusted to pH 5.3, and the mixture was adsorbed onto a 400 ml column of activated carbon. After washing this with water, 20 containing 0.25% ammonia
Elution was performed with 3200 ml (8 CV) of % ethanol solution. Concentrate this solution to 500ml, adjust the pH to 10.0,
The solution was passed through 50 ml of anion exchange resin (boric acid type). The passing liquid and washing liquid are combined and adsorbed onto the activated carbon column again.
After elution, concentrate to 100 ml, freeze-dry, and obtain 6.840
g of triazole deoxyribonucleoside was obtained.

Claims (1)

[Claims] 1 1,2,4-triazole-3-carboxamide and a 2-deoxyribose donor are reacted in the presence of a nucleoside phosphorylase source to form a compound having the structural formula [] A method for producing a triazole deoxyribonucleoside, which comprises obtaining a triazole deoxyribonucleoside represented by: