JPS59179094A - Production of triazol-deoxyribonucleoside - Google Patents

Abstract

PURPOSE:The reaction between 1,2,4-triazol-8-carboxamide and 2-deoxyribose donor is carried out in the presence of a nucleoside phospholase to produce the titled substance having a strong antiviral activity against a wide range of DNA and RNA viruses. CONSTITUTION:The reaction of 1,2,4-triazol-8-carboxamide with 2-deoxyribose is conducted in the presence of uncleoside phospholase at 4-10, preferably 6-8pH and 28-80, preferably 40-65 deg.C to produce triazole-deoxyribonucleoside of the formula. The compound shows high antiviral activity against a wide rang of DNA and RNA viruses.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing triazole oxides/lihonucleons using enzymes.

In the present invention, triazo-luteoquine ribonucleonot means a compound represented by the structural formula [1]
-y O+n-β-D-erythro-pentofuranone)
-1,2,4-triazole-3-carbo4-samide. This compound is an analog of Ribavirin, a J compound that exhibits strong antiviral activity over a wide range of DNA and RNA viruses.
This compound itself is expected to be an antiviral agent.

Prior Art Conventionally, as a method for producing triazoledeoquine ribonucleondo, 3-methoxoluponyl-1,2,4
- triazole or 3-noano1.2,4. -1-
A method is known in which, after condensing lyazole and theogynolyhonucleondo derivative, a metquine carbonyl, L, or noa group is converted into a carhoki→nomito group (J
CarbohydraLcs-Nucleosid
esNucleotides, 2(11,1-36(
1975)).

Such synthetic methods require complicated reaction operations and yields are poor.

In addition, methods for producing triazole nucleotides such as ribavirin enzymatically or using small η-compounds are known (!1'41' No. 5029720,
JP-A-50-123882-zu and JP-A-57-14
No. 6598 H<4). However, no enzymatic method or method using microorganisms has been adopted for the production of triazole deoquinolihonucleons.

(It) Summary of the Invention The present inventors began by producing triazole deoxoribonucleons by the action of an enzyme using a 1,2,4-1-lyazole derivative and a 2-deoxine lyphosate as substrates. This was successful, and the present invention was completed based on this.

The present invention provides 1,2,4-triazole-3-calhoki a
t MiF (hereinafter sometimes abbreviated as 1-triazolyl compound) and 2-teoxolihose compound are reacted in the presence of a nucleondopos d-lylase source to form a compound represented by the above structural formula [1]. The purpose of the present invention is to provide a method for producing a triazole deoquinoliponucleond, which has a 4'-1f feature of obtaining a triazole deoquinoliponucleond.

In the present invention, [nucleonodophosphorylase source-1] refers to the nucleonodophosphorylase source that acts on 1.2.4-1-lyazole-3-carfoxamide and 2-thioquinolibose (J(') 3 body defined later), and A substance that contains a single enzyme or a group of enzymes that tilt nucleotides, and contains at least nucleonode phosphorylase.

In addition, here [nucleonodophosphorylase-1]
2′-deoquinolive nucleotide is phosphorolyzed to give 2
- An enzyme responsible for the action of synthesizing triazole deoxoliphonucleosides from 2-thioquinolibose-1-phosphate and triazole compounds. means.

The nucleonodophosphorylase source may also include enzymes other than nucleonodophosphorylase.

These enzymes are enzymes that actively participate in the reaction of the present invention, for example, by acting on the raw material compound to generate a substrate for nucleonodophosphorylase, or In reaction step A1, the enzyme may be an enzyme that does not inhibit the reaction of the present invention. An example of an enzyme that actively participates in the reaction is phosphatase when a thioquinoribonucleotide is used as a 2-thioquine ribose donor.

The above-mentioned nucleonodophosphorylase sources may be of any origin and form during the reaction (soluble enzyme, immobilized enzyme, culture, live bacterial cells, processed bacterial cells, etc.), as long as they meet the purpose of the present invention.
No questions asked.

In other words, enzyme-containing substances derived from microorganisms (fungi) such as bacteria, yeasts, actinomycetes, filamentous fungi, and basidiomycetes, animal organs or tissues, or plants can be used, and in particular enzyme-containing substances derived from microorganisms can be used. Enzyme-containing materials are preferred. The most preferred Scleocytopospolyracea whale (L/Hiba/7 Tel)
Microorganisms belonging to the genus A (13revibacLerium) are exemplified.

Strains with particularly strong enzyme activity targeted by the present invention include:
Specifically, it was isolated from the sand of Koshien Stadium in Nishinomiya City, 42nd Prefecture. The AT to 6-7 strains can be mentioned. The mycological properties of this strain are described below.

A-morphology (1) Cell morphology and size: short rod-like, 08-1, 0
X 1.0-1.2 Itm (2) Spore formation: None 3) Durham staining: Positive B Growth status in various media (1) Juice agar plate culture (28°C, 48 hours) ■ Shape of colony Circular ■Uplift on the surface of the village:
Flat, smooth (3mo, o th
) ■Size: 2-4 mm ■Color: yellow to pinkish yellow (2) Juice agar slant culture (28°C, 148 hours) ■Good growth ■2 symptoms of growth (E C11i n u l a t)
e) (3) Broth liquid culture (28°C, 948 hours) A ring was formed on the growth surface, and a slight sediment (Se
diment).

(4) Meat juice seratin puncture culture (20°C, 6 to 1 hour): Liquefied into 3 traitiforms.

(5) Litmus milk medium (28°C, 4 to 1 year)
: Slight coagulation and peptonization is also observed.

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

(2) Generation of hydrogen sulfide (28°C, 5 days): Not generated.

(3) Starch hydrolysis: Degradable.

(4) Catalase: positive (5) Generation of indole: Not generated.

(6) Production of ammonia from peptone and arginine; negative (7) Methyllet test: negative t81 V-P test: positive (9) Attitude towards oxygen: aerobic (11) Production of acid from sugars; positive: curcose , mannose, fructose, maltose, sucrose.

)・Levalose negative: arabinose, kinlose, galactose, lactose, tsuruhito.

Innonot, Chrycerin (12) Growth pH range: pH 6.0 to 90 (I3) Optimum growth temperature: 25 to 37°C or above
Bergey's Manual
of l）eterminative Bacter
The test was carried out according to the classification criteria of 7th edition (1957).

As a result, the AT-6-7 strain is a short bacillus that is almost similar to cocci, is Durham positive, forms filaments, and produces acid from carbohydrates, so Brevibacterium (B.
A bacterial strain belonging to the genus Brevibacterium was identified, and Brevibacterium acetylicum (Breviba
cterium acetylicum) A T-6
It was named -7.

The identification and attribution of the above bacterial strains is based on Virginie's Manual of Non-Destructive Nocterology, 7th edition, and due to changes in the classification standards, these strains may be classified according to different classification criteria. If identification is made, the microorganisms named above may belong to other species or genera. It includes microorganisms that can be identified and that basically have the above-mentioned mycological properties or equivalent Dutch properties, and can be uniquely identified.

Regarding this strain, Ministry of International Trade and Industry Notification No. 178 of 1982
A deposit application was made to the Institute of Microbial Technology, Agency of Industrial Science and Technology in accordance with RR, and it was received on January 130, 1981, and the deposit number was 6305.
FERM P-6305) is given as an example. Other exemplary strains include Brevibacterium imperiale (B, imper.
iale) ATCC8865 can be given as an example.

In addition, the strains listed in iii) can be mutated by physical treatment such as irradiation with ultraviolet rays, Mutant strains induced by natural mutations are also 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 nucleoside phosphorylase source 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 is considered to be 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, JllI salt, trace growth promoters, antifoaming agents, etc. are added. Specifically, as a carbon source (:l, glucose, fructose, maltose, lipose, sucrose, starch, starch hydrolyzate, saccharide, sugars such as blackstrap molasses or their derivatives such as fatty acid esters), wheat , natural carbohydrates from tsuzumi and rice, alcohols such as mannitol, methanol, and ethanol, fatty acids such as curconic acid, pyruvic acid, acetic acid, and sodium citrate, normal paraffin, hydrocarbons from keronnato, glycino, curtamic acid, Amino acids such as glutamine, alanine, and asparagine,
- One or more carbon sources may be selected and used as appropriate, taking into consideration the assimilation ability of the microorganisms used. Nitrogen sources include meat extract, peptone, yeast 1d extract, dried yeast, soybean hydrolyzate, soybean flour, milk casein, casamino acids, various amino acids, corn staple liquor, conoton seed meal or its hydrolyzate, and fitz meal. or its hydrolyzate, other animals.

Organic nitrogen compounds such as hydrolysates of plants and microorganisms, ammonia, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate,
One or more of ammonium salts such as ammonium acetate, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea are selected and used in consideration of the assimilation ability of the microorganisms used. In addition, trace amounts of makui num, mankan, Vl, zinc, copper, sodium,
One or more of carnoum, potassium phosphate, hydrochloride, sulfate, carbonate, nitrate, acetate, etc. are added as appropriate, and vegetable oil, surfactant, etc. are added as necessary. antifoaming agent, vitamin B1. B2, nicotinic acid, pantothenic acid, biotin, p-amine benzoic acid, and trace amounts of growth promoting substances may be added. Furthermore, when using microorganisms that exhibit nutritional requirements, it is of course necessary to add substances to the culture medium that will satisfy 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 the usual 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 usually the pH at the start of culture is adjusted to about 6 to 8, and culture is carried out at 614 degrees at about 25 to 35 degrees Celsius. The culture period may be sufficient for the growth of the microorganism used (usually between 1 and 30 hours).

After culturing microorganisms as in 2, the obtained culture
In the present invention, the nucleondoposporirer is a viable bacterial cell collected from a culture by a conventional method such as centrifugation, sedimentation, or flocculation, or a treated bacterial cell product obtained by subjecting the viable bacterial cell to an appropriate treatment. It can be used as a source. Here, the culture refers to a culture medium and cultured bacterial cells in an unseparated state.

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 bacterial cells as a source of nucleondophosphorylase, which is the object of 1 of the present invention. The following is an example of a method for obtaining a protein-purchased microbial cell-treated product of a microbial cell extract having nucleation activity.

zlJ, q) For example, 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 for living bacterial cells. Physical treatment means such as treatment, or chemical treatment such as enzymatic treatment with lysozyme, cell wall lytic enzymes, contact treatment with a medium or surfactant such as toluene, kiln, butyl alcohol (butanol). By applying target or biochemical treatments alone or in combination, By applying enzyme separation and purification methods alone or in combination, and by applying enzyme immobilization methods such as entrapment treatment, crosslinking treatment, and adsorption treatment to carriers to live bacterial cells, bacterial cell extracts, or purified products thereof. It is possible to obtain processed bacterial cells.

Reaction Substrate The reaction JA substance in the enzyme reaction of the present invention is 1,2°4-triazole-3-carboxami) and 2-thioquine ribose donor.

1.2.4-) Lyano-3-carboxamide can be used in its free form or as a salt such as the sodium salt.

In the present invention, [2-thioquine ribose donor JJ body 1] is 1.2
．． 2 at the N1 position of 4-1 lyazo-3-carboxamide
It is a 2-thioquine ribose derivative capable of transferring a '-thioki/lipose residue. That is, the term "2-thioquine ribose donor" refers to a compound that can serve as the group TI of nucleondophosphorylase and provide a 2-thioquinolipose residue to a triazole compound by its direct action. It also refers to compounds that can be converted into direct substrates of the above-mentioned nucleonthosporylases by the action of enzymes other than those mentioned above contained in the source of Bos-osporase.

Specific examples thereof include 27-thioquinoliponucleondo, 2'-thioquinoliboscleotide, 2-thioquinolibose-1-phosphate, or salts thereof.

Examples of the 2'-thioquinoliponucleoside include 2'-deoxoanonone, 2'-thioquinioninon, 2'-deoxoguanoone, 2'-thioquinotinone, thymidine,
Examples of 2'-thioquinoliphonucleotides include monophosphates at the 3' and/or 5' positions of the 2'-deoquinolihonucleotides; Whether all phosphoric acid esters are included, a typical example is 2'-thioki/
Azo7nono-5'-monophosphoric acid, 2'-thioquinadenonone-5'-nophosphoric acid, 2'-deoxoanonone 5'
-h phosphoric acid, 2'-thioquininonone-5'-monophosphoric acid, 2'-thioquine/ynonone-5'-nophosphoric acid, 2'
-thio-1noinononone-5'-triphosphoric acid, 2'-thioquihaanoone-5'-monophosphoric acid, 2'-thioquinoguanoone-5'-diphosphoric acid, 2'-deoxoguanonone −
5'-triphosphoric acid, 2'-thioquinonotine-5'-monophosphoric acid, 2'-thioquinonotine-5'-nophosphoric acid,
2'-thioquinosotidine-5'-triphosphoric acid, thymidine-5'-monophosphoric acid, thyminone-5'-nophosphoric acid, thymidine-5'-triphosphoric acid, 2'-thioquinostidine-5'-triphosphoric acid
Examples include free forms such as '-monophosphoric acid, 2'-thioquinuridine-5'-7-phosphoric acid, and 2'-thioquinourinone-5'-1-phosphoric acid, and alkali salts such as sodium salt.

Reaction Substrate Solution The substrate mit& 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-thioquine phosphate donors, and in addition to these reaction substrates, it also provides a phosphate ion donor!
J-based substances, organic solvents, surfactants, metal salts, coenzymes, acids, bases, sugars, etc. Substances that promote enzyme reactions, substances that inhibit interfering enzyme activity, substances that improve the solubility of reaction substrates, enzymes It may contain a substance that promotes contact between the reaction substrate and the reaction substrate. It may also contain the above-mentioned culture medium components that are assimilated by the microorganisms used.

As the aqueous medium, water or various buffers suitable for enzymatic reactions (such as phosphate slow-forming, imidasol-hydrochloric acid buffer, heronal-hydrochloric acid buffer, 1.lis-hydrochloric acid buffer, etc.) can be used.

As the phosphate ion donor system, any substance that can be dissociated into phosphate ions in the water leaf medium may be used (for example, free phosphoric acid itself, inorganic phosphates, such as sodium, potassium, etc.). Salts of alkali metals, alkaline earth metals such as calcium and magnesium, and ammonium are preferably used.Also, as a phosphate ion donor system, phosphate ions are liberated in the substrate solution of enzyme reaction. For example, combinations of various phosphate ester derivatives and bosphatase, combinations of nucleotides and nucleotidases, combinations of nucleobases and ribose-1-phosphate or 2-thioquinolihose-1-phosphate, and phosphorylase. You can use it.

The phosphoric acid donating system mentioned above may be added from outside the system during the enzymatic 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 enzymatic reactions, a system of the substances listed in ``1'' alone or in combination with 2 rCrry-1=, or a microbial cell or its The treated bacterial cells may be separately added to the reaction solution during the enzyme reaction of the present invention, or these substances contained in the nucleondosporylase source may be used as they are.

Examples of organic solvents include methanol, ethanol,
propatool, butanol, acetone', methyl ethyl ketone, ethyl acetate, toluene, tetrahydrofuran,
Examples include dioxane, dimethylsulfokitamine, and 2-engineered toquinoethanol.

Reaction method The reaction of the present invention is achieved by bringing the above-mentioned enzyme source, ie, nucleondophosphorylase source, into contact with a reaction substrate in an aqueous medium.

The contact force 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 turbid or dissolved in a reaction substrate solution, preferably stirred or shaken without heating; Alternatively, a column method may be used in which the enzyme source is mixed with appropriate carriers, auxiliary agents, and adsorbents as necessary, or is supported on these, packed in a column, and the reaction substrate m ii & is passed through. In addition, in the case of the No-Nochi method, after the reaction, bacterial cells etc. are filtered (including pressure filtration and vacuum filtration),
Bacteria can be collected by conventional methods such as centrifugation, sedimentation, and flocculation, and can be used repeatedly by contacting with a reaction substrate solution. Immobilization Nu. In the case of a column method using a creondophosphorylase source, there is no need for separation operations for bacterial cells, etc., or they can be used repeatedly or continuously in the enzyme reaction. Furthermore, as described above, the method of adding a reaction substrate to the medium and allowing the enzyme source and the reaction substrate to react when culturing the microorganism used is also employed in the present invention.

Concentration or Addition of Reaction Substrate and Enzyme Source "During the IX reaction, the substrate concentration of the reaction solution is not particularly limited, and a substrate concentration below the saturation concentration of each substrate with respect to the aqueous medium used at the reaction temperature is generally adopted. Alternatively, the substrate concentration can be increased by adding the above-mentioned organic solvent to the reaction substrate solution.Also, each substrate can be present in a turbid state at a saturation concentration or higher in the reaction solution, and the reaction progresses. Alternatively, each substrate can be added sequentially during the reaction and maintained at an appropriate degree.

When adding and dissolving each substrate, the substrate concentration is usually 5-20 for triazole compounds or their salts.
About 0mM, preferably 10-100m
For the 2-thioquine ribose donor, it is about 1 to 800 mM, preferably about 1 to 15
It is about 0 mM.

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

Reaction conditions The conditions for the reaction of the present invention are such that the reaction substrate is produced by bacterial cells, etc.
The conditions under which the reaction is carried out and the reasol nucleont is produced with high efficiency are not particularly limited, regardless of whether the microorganism used is under non-growth conditions or under growth conditions. However, the reaction is particularly efficient under conditions where the microorganism used does not grow.

Methods for conducting the reaction under conditions where microorganisms do not proliferate include a method in which the enzyme reaction temperature is set within a temperature range in which the microorganisms used cannot proliferate (in a temperature range in which the enzyme involved in the reaction of the present invention is not inactivated); A method for preparing the microorganisms to be used and making them incapable of growth by physically, chemically or biochemically treating them as described above, and then subjecting them to a reaction. The most effective and simple method is to add a substance that inhibits the reaction to the reaction substrate solution, either singly or in combination, or in particular, to manipulate the reaction temperature.

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 range of 40 to 65°C is particularly optimal.

The liquid properties of the reaction substrate solution are usually maintained within the range of pH 4 to 10, preferably pH 6 to 8. If the pH changes during the reaction, acid such as hydrochloric acid, sulfuric acid, phosphoric acid or hydroxylation should be used. The reaction time should be determined without checking the conversion rate of the reaction substrate to the target product. In the normal method, it is sufficient to react for about 2 to 45 hours, preferably for about 24 to 36 hours, and in the column method, the reaction can be carried out by setting appropriate conditions according to the method. Yes.

After the separation and purification reaction, bacterial cells may be filtered, centrifuged, or applied, as necessary, such as ion exchange chromatography, adsorption chromatography, partition chromatography, gel chromatography, etc. Filtration methods, various chromatography methods, countercurrent distribution, countercurrent extraction, methods that utilize partitioning between two liquid phases, and concentration.

General separation and purification methods such as cooling, addition of an organic solvent, and methods utilizing differences in solubility may be used alone or in appropriate combinations.

Analysis In the Examples of the present invention, triazole teoquinoliponucleont and 1.2.4-1-lyazole-3-carboxamide were analyzed by high performance liquid chromatography. When analyzed using the equipment and conditions shown below, the triazolethioquine ribonucleond has a retention time of 900%.
Near the fraction, 1°2.4. -triazole-3-calhoki→normide was eluted around the retention time of 464 minutes, and the retention time was 11 minutes.
) Calculate each quantity from the line.

Equipment: Shimabara High Performance Liquid Chromatograph LC-3A type (manufactured by Shimabara Seisakusho) Column Micro Hontapanoku <tlBONDAPA
K) Cts, 46 mm x 250'' (manufactured by Nippon Waters Limited) Eluent: 2 On 1M Tris-HCl buffer containing 2% acetonitrile (I) H7,
5) Flow rate: 1 m 11 min Measurement wavelength + 2251 m Column operating temperature Below room temperature The present invention will be explained more specifically with examples, but this does not represent the scope of the present invention. It is not a restriction.

Example 1 250 ml of a pre-culture solution of Brevibacterium acetylicum AT-6-7 (Kijoken Bacteria 6305 Yumi) was inoculated into 54% yeast extract medium 54, and cultured at 280G for 24 hours. Bacterial cells were isolated from the culture solution by centrifugation.

1.2.4-Triazole-3-carboxamide 4.4
Substrate solution in which 88!7.2'-thioquinourine and potassium phosphate 2.722'/ were dissolved (pH 7,0)
1. e Add 60q of wet bacterial cells and heat at 45°C.
The mixture was reacted for 24 hours. When this reaction solution was analyzed, the production rate of triazoleteoquinolifonucleond was 924.
It was 5%. Note that the production rate here refers to 1.2.
4. -) riasole-3-carboxamide) is the molar percentage of the triazolethioquinolibonucleosites produced.

After separating the bacterial cells from the reaction solution, the pH of this solution was adjusted to 11.5, and the generated precipitate was removed by centrifugation. This solution was passed through a 400 ml column of anion exchange resin (chlorine type). The passing liquid and the washing liquid were combined, adjusted to pH 5.8, and absorbed into a 400 πl column of activated carbon. After washing this with water, add 8.200 wl of 20% ethanol solution containing 0.025% anthenia, concentrate the solution eluted with gcvj to 500 ml, adjust the pH to O, O, and add anion exchange resin (boric acid type). The passing liquid and the washing liquid were combined, adsorbed again on the activated carbon column I, eluted, concentrated to 10.0 tel, freeze-dried, and 6.8
40 g of triazolethioquine ribonucleond was obtained.

Patent applicant (677) Yamasa Soy Sauce Co., Ltd. 1 Amisho 59
-179094 (8)

Claims (1)

[Claims] 1.2.4-triazole-3-carboxamide and 2-
A triazole deoxyribonucleoside is obtained by reacting a deokine ribose donor with a nucleondophosphorylase donor in the presence of a nucleondophosphorylase source to obtain a triazole deoxoribonucleoside represented by formula (1). Manufacturing method.