WO1995016785A1

WO1995016785A1 - Use of thermophilic bacteria to manufacture triazole nucleosides

Info

Publication number: WO1995016785A1
Application number: PCT/CA1994/000261
Authority: WO
Inventors: Gordana Opacic; Ljubinka Gligic; Zeljka Radulovic; Valentia Zivkovic; Lola Matic; Robert Smith
Original assignee: Gordana Opacic; Ljubinka Gligic; Zeljka Radulovic; Valentia Zivkovic; Lola Matic; Robert Smith
Priority date: 1993-12-14
Filing date: 1994-05-12
Publication date: 1995-06-22
Also published as: AU6718294A; YU77593A; RS49561B; CN1111061A

Abstract

A method for producing pharmaceutically active compounds having a ribose group attached to a triazole by reacting a triazole with a ribose donor in the presence of a source of inorganic phosphate as a catalyst, and a thermophilic bacteria.

Description

TITLE OF INVENTION

USE OF THERMOPHILIC BACTERIA TO MAIXIUFACTURE TRIAZOLE NUCLEOSIDES FIELD OF INVENTION The present invention relates to the production (enzymatic synthesis) of pharmaceutical compounds having a ribose group attached to a triazole, for example triazole nucleosides and in some embodiments attached to a 1,2,4- triazole to form 1,2,4-triazole nucleosides. One such compound is the medicine, ribavirin, chemically known as l-β-D-ribofuranosyl-l,2,4-triazole-3- carboxamide.

BACKGROUND OF THE INVENTION

Pharmaceutical compounds having a ribose group attached to a triazole, for example, triazole nucleosides can be obtained by various methods of manufacture. The best known of such compounds is ribavirin, i.e. 1-β-D- ribofuranosyl-l,2,4-triazole-3-carboxamide. In some instances, complex chemical reactions are set up involving a number of chemicals and include the blocking of active groups of components or reactants before the reaction, the activation of ribose and the subsequent deblocking (by amidation) of intermediates. See Canadian Letters Patent 997,756 corresponding U.S. Patent 3,798,209, which relates to the manufacture of ribavirin. Disadvantages of these procedures include product impurity because of the many intermediate residues and technological operations and the high cost of production.

The manufacture of such compounds including ribavirin has also been carried out through fermentation and enzymatic synthesis. See for example U.S. Patents 3,976,545 and 4,458,016. Fermentation methods include aerobic cultivation of bacterial genera like: Brevibacterium, Cory nob acterium, Anthrobacterium, Bacillus, etc. within the period of two to eight days, in a culture medium which beside nutritive medium contains l,2,4-triazole-3- carboxamide and a ribose donor. Isolation of ribavirin, the final product for example, accumulated in the bacterial culture is then undertaken.

See also for example Furuya, Akira et al. Japan Kokai 75. 123. 882 ICI.C12D,C07D, 29 Sep. 1975, U.S. Patent 4,614,719, and an article entitled J.P.No. 29720/1975 (/Agric.Biol.M.50/1), 121-6, 1986.

The disadvantages however with the prior art methods referred to, include the long process time period which permits contamination with other microorganisms and, numerous undesired enzymatic reactions resulting in impurification with substantial reaction by-products which must be removed. These impurities reduce ribavirin yield and impede purification.

In this latter regard, the methods have included the presence of an enzyme source derived from bacteria which contributes substrate specific enzymes, (for example, phosphatases, pyrimidine and purine nucleoside phosphorylases), used to react the ribose group and triazole for manufacturing the triazole nucleoside (for example, ribavirin). In practice, however, these processes do not produce commercially desirable amounts of ribavirin. In some, the enzymes of the different heterotrophic mesophilic microorganisms made are active at temperatures insufficient to provide high yields. In some instances, the process is long, increasing contamination possibilities by other microorganisms. Thus, the process becomes complex resulting in a complex procedure for substrate specific enzyme purification.

It is therefore an object of this invention to provide improved processes for reacting a ribose group with a triazole to yield a pharmaceutically active compound comprising a triazole nucleoside, for example ribavirin.

It is a further object of the invention to provide such processes which produce the pharmaceutically active compounds in high yields with less impurities thereby providing highly efficient processes.

It is still a further object of the invention to provide such processes wherein the components are more readily available for better contact and interaction with one another in the reaction mixture. It is still a further object of this invention to employ processes which may employ phosphate and nucleoside phosphorylase enzyme for producing the active, for example, ribavirin, in high yield.

Further and other objects of the invention will be realized by those skilled in the art from the following summary of the invention and detailed description of embodiments thereof. SUMMARY OF THE INVENTION

According to one aspect of the invention, this invention provides a method of making pharmaceutically active compounds having a ribose group attached to a triazole, for example, triazole nucleosides e.g. ribavirin by reacting triazole (for example l,2,4-triazole-3-carboxamide) with a ribose donor group (for example ribose-1-phosphate, pyrimidine nucleosides such as uridine, and cytidine, purine nucleosides such as guanosine, inosine and adenosine, pyrimidine nucleotides such as 5 -uridine monophosphate (5 -UMP) and purine nucleotides such as 5 - adenosine monophosphate (5 -AMP) in the presence of a thermophilic bacteria (for example and preferably Thermothrix thiopara sp. (also termed Thermothrix thioparus)) a source of enzymes which catalyzes the reaction. A thermophilic bacteria may be defined as a bacteria which is viable at higher temperatures for example up to about 90 - 95 °C and includes Thermothrix thiopara sp.., which is viable at 40 - 80°C.

Thermothrix thiopara sp. occurs naturally in New Mexico hot springs (a geothermal spring) at a temperature of 74°C, a pH of

7.0 and an HS~ concentration of lmg/litre. The organism is gram-negative, non-motile 0.5-1.0 X 3-20 μmetre and forms cell chains up to 1 cm in length. The resulting filaments do not possess a sheath. Sulfur is deposited extracellularly. The organism was and can be isolated using an autotrophic medium with HS^" as the energy source and NO3" as the terminal electron acceptor. Anaerobically either NO2^" or NO3^" is required. NO2^" is formed from NO3^" and no observable gas is evolved. Oxygen can also be used as the terminal electron acceptor, but growth is poor because of the decreased solubility of O₂ at temperatures required for growth. Alternate energy sources used aerobically and anaerobically include hexose, HS^" , SO3", and S₂θ₃ ⁼. The temperature optimum is 70-73°C and growth occurs from 62 to 77°C. ("Thermothrix thioparus gen. et sp. nov. a facultatively anaerobic facultative chemolithotroph living at neutral pH and high Temperature" written by Douglas E. Caldwell, Sarah J. Caldwell, and J. Paul Laycock, Can. J. Microbiol. 22: 1509-1517)

The samples containing Thermothrix thiopara sp. can be treated to be initially cultivated, after which the enriched cultures can be purified.

As the natural habitat of thermophilic microorganisms (like Thermothrix thiopara sp.) is geothermal springs, (where no other types of bacteria, which are not thermophilic, occur naturally) and whose temperatures range from 40 to 80°C, we have chosen the thermal waters of three spas in Serbia according to the data on the composition and temperatures of the water from the spas having the highest temperatures as the source of the thermophilic bacteria used in our invention:

- Josanicka banja (62 - 78°C)

- Vranjski banja (78 - 80°C) - Sijarinska banja (41 - 63°C)

Habitat Characteristics:

(a) Josanicka banja is located at the foot of the Kopaonik mountain. It has four geothermal springs of medicinal, mineral water. Water temperatures are between 62 and 78°C, and the pH value of the water ranges from 7.5 to 7.8. (b) Vranjska banja is located at the foot of Veliki Pester. Its mineral water springs are the warmest in Yugoslavia, and among the warmest in

Europe. The water is sulfuric, alkaline and hyperthermic, while the pH value ranges from 7.8 to 8.0. In places where the samples were taken, the water temperature was within the 78 to 80°C range, although in certain localities it ranges form 63 to 92°C. (c) Sijarinska banja is located on the farthest reaches of the Golak mountain, 52 kilometers from Leskovac. It has 16 mineral water springs which are alkaline, ferrous, with temperatures ranging from 41 - 63°C. Material Sampling:

Slimy deposits of bacteria are present on rocks permanently flushed by warm water. Material sampling was performed under sterile conditions. Bacterial deposits were taken together with the water using pipettes and transferred into an empty conical flask. A bacteriological loop was used to remove the deposits and inoculate them onto a nutritive medium prepared in tubes (liquid media) and onto slanting agars (solid media). When removing the material using the loop, the deposits stretch into strands 10 to 15 cm long. The following media were used: nutritive bouillon, peptone 1.5%, meat extract 0.3% NaCl 0.5%, KH₂P0₄ 0.03%, and nutritive agars with the same composition as the nutritive bouillon but with 1.6% agar added.

The biological material thus sampled in a conical flask, as well as the one inoculated in nutritive media was transported to the laboratory. Material Enrichment and Purification:

The initial cultivation of the sampled biological material was carried out by seeding into liquid nutritive media, which were incubated overnight at 45 and 70°C. The developed "overnight" cultures were diluted in the same medium 20 fold and again incubated at 45 and 70°C. After incubation, the cultures were again diluted and incubated overnight at 45 and 70°C. In this way, using a double passage, the initial biological material was multiplied. Using the "patchy" plate method we separated mixed from pure bacterial cultures, and obtained separate individual colonies which differ morphologically by the shape, size, colour, consistency and type of growth on the petri plate. After incubation various types of colonies appeared on the petri plate. Each type of colony was streaked onto a petri plate with nutritive agar and incubated at 45 to 70°C. After incubation the colonies were reseeded onto slanting agars. In this way the biological material from the samples taken at Josanicka, Vranjska, and Sijarinska banja were enriched and purified. Four morphologically different types of colonies denoted as J--., J₂ , J (Thermothrix thiopara sp.) and J₄ were isolated from Josanicka banja. Two types of colonies denoted as V₁ and V₂ were isolated from the water of Vranjska banja. From Sijarinska banja only one bacterial culture was isolated, so that after sample purification, we isolated a total of seven morphologically different types of thermal bacterial (colonies), with the colonies from Vranjska banja being virtually identical to those from Josanicka banja V_τ = J₂ and V₂ = J₃ )•

Isolation and Maintenance of Isolated Pure Cultures of Thermophilic Bacteria:

(a) The isolated bacterial cultures are kept on petri plates and slanting agars at 4°C and maintained by passing at monthly intervals.

(b) All isolated cultures were lyophilized and were stored in that form at +4°C ready for use.

According to another aspect of the invention, this invention provides a process for producing a pharmaceutically active triazole nucleoside for example ribavirin wherein the process takes place in the presence of a nucleoside phosphorylase enzyme derived from the thermophilic bacteria (for example Thermothrix thiopara sp. bacteria).

According to another aspect of the invention, the use of the thermophilic bacteria to produce pharmaceutically active triazole nucleosides for example ribavirin is provided. Preferably such bacteria is Thermothrix thiopara sp. Thus, according to another aspect of the invention, the process of manufacture of such active compounds is enhanced unexpectedly by the presence of thermophilic bacteria (preferably Thermothrix thiopara sp.).

As is known, a catalyst being a source of inorganic phosphate preferably potassium dihydrogen phosphate acts as a source of phosphate which participates in the biochemical pathway of ribose phosphorylation.

The following reaction schemes of enzymatic synthesis according to aspects of the invention are therefore set up: ase)

Step (b)

R may be: 1) NH₂: RIBAVIRIN

2) OH

3) ALCOXY

[Where ribavirin is not prepared, it may be prepared from the product produced by conversion in any manner understood by a person skilled in the art]

Step (c)

O O— P— e)

OH

H

Step (d)

uracil

Thermothrix thiopara sp. (phosphatase) ase)

5'-Uridine Monophosphate

H

[ ] * This step is optional depending on the ribose donor whether a nucleotide or nucleoside for example Step (e)

Step (e) uracil

Thermothrix thiopara sp.

HO- (nucleoside phosphorylase)

HO OH alkaline dihydrogen

phosphate

Uridine

H Step (f)

Starting with ribose- 1-phospate the following process presents itself

H

(Because ribose-1-phosphate is an expensive and somewhat unstable material, it is not as preferred as a starting material in a commercially viable and economically sound synthesis of for example ribavirin, as other materials such as uridine.)

Because of the thermophilic bacteria allowing the elevated reaction temperature, and the processes of manufacture using the improved enzymatic reaction methods, improved economics of synthesis are achieved, for example, increased reaction rates, increased solubility of the starting material, lysis of the cells during the reaction providing better contact of the enzyme and substrate, decreased potential for contamination from competing organisms, decreased potential for concurrent competitive reactions, and improved overall reaction yields.

In one embodiment, the process or method of synthesis taught by the invention may be used to manufacture ribavirin. In this regard, the enzymatic synthesis of ribavirin reacts l,2,4-triazole-3-carboximide (TCA) with one of a number of ribose donors (for example and most preferably uridine), preferably in a concentration slightly greater than the molar amount of TCA (for example 1.2:1) in the presence of a catalyst being a source of inorganic phosphate for example, potassium dihydrogen phosphate. The source of the enzyme used in the reaction mixture is preferably the thermophilic bacteria Thermothrix thiopara sp. in a form of non-proliferating cells which are subsequently lysed. The term non proliferating cells is meant to denote cells which do not grow and do not multiply. The subsequent lysis of the cells enables the reaction to proceed towards enzymatic synthesis of for example ribavirin through a direct contact of the enzyme or enzymatic material as the case may be, with the substrate and not through some other metabolic processes of the living cell, in which other case when the cell is alive, the ribavirin synthesis would be taking place intracellularly, i.e. under different conditions. Preferably the pH is in a range of from 5 to 9. Preferably the temperature is 50 - 80°C. The process usually takes from 1 - 48 hours to complete.

Suitable donors of ribose may be ribose-1-phosphate, pyrimidine and purine nucleosides and pyrimidine and purine nucleotides. Pyrimidine nucleotides include 5 uridine monophosphate (5 -UMP); purine nucleotides include 5 -adenosine monophosphate (5 -AMP), pyrimidine nucleosides include uridine (which is preferred) and cytidine; purine nucleosides include guanosine, inosine and adenosine.

In one embodiment, a concentration of a wet bacteria biomass is incorporated in the reaction mixture. The wet bacteria biomass in the reaction mixture is 50 - 60 mg/ml. The wet bacteria biomass is, in one embodiment made up of a concentrated mass of Thermothrix thiopara sp. cells which has been prepared by common methods familiar to those skilled in the art.

In developing and preparing the cells of the thermophilic bacteria Thermothrix thiopara sp. for use in an embodiment, the thermophilic bacteria was cultured in nutritive medium having the following constituents: peptone

I, meat extract, sodium chloride, potassium dihydrogen phosphate, sodium thiosulfate pentahydrate, and distilled water. The main culture was prepared by inoculation of a seed culture in the nutritive medium followed by incubation for 18 hours at 50°C in the presence of air with continuous agitation on a shaker at 100 rpm. Upon completion of the incubation period, the cells were separated from the nutritive medium by centrifugation at 2700 rpm for 30 minutes. The supernatant was discarded, and the sediment comprising the wet biomass of cells was collected. In this preparation, the following was carried out:

COMPOSITION AND PREPARATION OF NUTRITIVE MEDIA Medium composition:

Peptone I 6 g

Meat extract 1.3 g NaCl p.a. 2.0 g

KH₂P0₄ p.a. 0.12 g

Na₂S₂0₃ x 5π₂0 p.a. 0.099 g Distilled water up to 440 ml

Measured the mentioned quantities of peptone, meat extract, NaCl and KH₂P0₄ into separate laboratory glasses a 200 ml. Dissolved each of the mentioned medium components in 100 ml of distilled water, gradually mixed until the solution became completely transparent. Combined the dissolved components in a 1000 ml glass and added distilled water up to 440 ml (cca 10% of the water evaporated during the sterilization of the medium in an autoclave).

Adjusted medium pH to 7.0 - 7.2 using 2M water solution of NaOH and poured the medium in a conical flask a 1000 ml.

Sterilized the medium, thus prepared, in an autoclave for 15 minutes at 120° and a pressure of 1.5 atmospheres.

Under aseptic conditions measured 100 ml each into four conical flasks of 1000 ml and after cooling at room temperature added 1 ml of freshly prepared 0.1M aqueous solution of Na₂S₂0 . Preparation of NaoSgC^

Measured 0.248g of Na₂S₂0₃ x 5 H₂0 and dissolved in 10 ml of distilled water and filtered, under aseptic conditions, through bacteriological filter 0 = 0.45 μ, into a sterile glass vessel protected from the light (Al-foil). INOCULUM PREPARATION (SEED CULTURE)

From a conical flask a 100 ml pipetted 5 ml of previously prepared medium and transferred to slanting agar. Resuspended bacteria from slanting agar by scraping the material using sterile bacteriological loop and transferred the suspension into an empty tube (16 cm x 10 mm). Shake (using Vortex) 1 - 2 minutes to obtain a homogenous suspension without microscopically visible filaments and bacterial sediment.

Inoculated the bacterial suspension thus prepared into a conical flask containing 95 ml of the medium.

Stoppered the seeded medium using a sterile stopper made of cotton and gauze and incubated on a shaker in air during 6 hours at 50°C, stirred at 100 rpm and aerated (K_{1 Λ} = 0.64 mM 0₂/l min). After cultivation was completed checked the pH of the obtained inoculum (SEED), which should be between 7.4 - 78. If the inoculum pH did not comply, did not use for preparing the main culture. PREPARATION OF THE MAIN CULTURE

Inoculated 5 ml of the previously prepared seed culture into each of three conical flasks containing 95 ml of medium. Closed using sterile "sandwich" stoppers (double layer of paper cotton) and incubated during 18 hours on a shaker in air under the same conditions as for the inoculum (temperature 50°C, stirred 100 rpm, aerated K_1Λ = 0.64 mM 0₂/l min).

After incubation was finished the main cultures were joined into one conical flask of 1000 ml and pH checked (this should be between 8.3 and 8.6). If the pH is not adequate, the main culture could not be used for enzymatic synthesis.

DETERMINATION OF CONCENTRATION OF WET BIOMASS OF BACTERIA IN THE MAIN CULTURE ( ) IN ORDER TO DETERMINE THE NEEDED VOLUME OF THE MAIN CULTURE (VI) Measuring Optical Density

Added 0.5 ml of the main culture into 4.5 ml of the medium and vigorously homogenized using Vortex. Determined optical density at 610nm (OD₆₁₀) as compared to unseeded medium. Calculated the concentration of wet bacterial biomass in the main culture ( ) using the following formula: = 3.89 x OD₆₁₀ n + 10.76 EXAMPLE: If OD₆₁₀ nm = 4.8, C-, = 29.6 mg/ml (concentration of wet bacterial biomass in the main sample) If OD₆₁₀ nxn ⁼ 5-5/ = 32 mg/ml (concentration of wet bacterial biomass in the main sample)

Determination of the needed volume of the main culture (V-i) for the enzymatic reaction Determined the volume of the main culture (V-i ) which needed to be centrifuged for the defined volumne of reaction mixture V₂ using the following equation.

C₂ x V₂

Vi =

Ci where:

C_j concentration of wet bacterial biomass in the main culture determined by measuring OD₆₁₀ using the standard curve C₂ optimal concentration of wet bacterial biomass in the reaction mixture (58 mg/ml) V₂ volume of the reaction mixture

EXAMPLE: If = 29.6 mg/ml, and V₂ = 50 ml of the require volume of the main culture (V_α) for centrifuging is 100.0 ml, in order to have the end concentration of wet bacterial biomass on the reaction mixture (C₂) of 58 mg/ml. If = 32 mg/ml, and V₂ = 50 ml, V-, = 90.5 ml. ISOLATION OF CELLS FROM THE MAIN CULTURE PROCEDURE: Centrifuged V-, ml of the main culture (90.5 - 100 ml) during 30 min. at 2700 rpm. After sedimentation, decant the supernatant, and resuspended the obtained sediment (cca 2.9 g) in 5 ml of sterile redistilled water, and transferred into sterile tube. The suspension contained 2.0 x 10⁷ - 10⁹ live cells /ml, and was used for the enzymatic reaction.

ENZYMATIC REACTION AND ISOLATION

The enzymatic reaction was prepared by resuspending the wet cell mass of thermophilic bacteria in distilled water, and introducing the bacterial suspension into a solution such that the final composition was as follows: Component Concentration

TCA 100 mM

Uridine 120 mM

KH₂P0₄ 50 mM thermophilic bacteria 58 mg/ml (No nutrient is provided for the biomass in one embodiment)

The enzymatic reaction mixture was incubated at 60°C for 24 hours. (During the course of incubation, because of the unavailability of nutrient for the biomass and because of the hypertonicity of the reaction medium, the live cells are lysed.) After completion of the enzymatic reaction, the pharmaceutical grade active, for example, ribavirin, was isolated and purified by separation of the by¬ products and residual amounts of the reactants from the active, for example, ribavirin. This isolation, purification and separation may be carried out for example by gradual precipitation on the basis of the differential solubilities of the products, by-products and starting materials in the reaction media, although other isolation, purification and separation procedures known to persons skilled in the art may be employed.

Thus column chromatography may be employed, for example, with suitable ion exchange columns, for example, where uridine, uracil, TCA, uridine monophosphate and inorganic phosphates are to be removed, they may be removed on a strong anionic exchange resin, for example Dowex®, Cl^" form. A strong cationic exchange resin, for example Dowex® 50W, H⁺form, maybe required where traces of impurities in cationic form (K+, NH₄ ⁺, heavy metals, etc.) are to be removed. A pharmaceutical grade of ribavirin may then be recovered by elution with water and after being crystallized from concentrated and cooled eluate with preferably subsequent addition of ethanol. Yields are in the order of 80 - 90%.

In the manufacture of ribavirin, the effect of the ribose donor on the manufacture of ribavirin is given in Table 1.

TABLE 1

Ribose Donor Ribavirin Yield (%) Guanosine 21.0

Cytidine 32.0

Uridine 80.0

Inosine 4.0

Adenosine 39.9 5-UMP 40.0

Effect of the reaction mixture incubation temperature on ribavirin production from uridine as ribose donor and l,2,4,-triazole-3-carboxamide, is given in the Table 2.

TABLE 2 Reaction Temperature CO Ribavirn Yield (%)

50 56.7

60 84.6

65 55.3

70 29.7 Effect of potassiumdihydrogen phosphate (KH2PO ) concentration used as a catalyst on ribavirin production is given in the Table 3 when the ribose donor is uridine.

TABLE 3

KHnPO, Concentration (mM. Ribavirin Yield (%) 25 80.36

50 84.94

75 82.17

100 73.77

(mM is millimole) Thus, the manufacture of ribavirin follows the following reaction scheme in the presence of Thermothrix thiopara sp.:

uracil + ribose carboxamide

Pi = inorganic phosphate (for example KH₂PO₄)

(Enzymes from Thermothrix thiopara sp. are preferably used in the synthesis of ribavirin from uridine and l,2,4-triazole-3-carboxamide. Uridine and inorganic phosphate are converted to ribose-1-phosphate and uracil by pyrimidine nucleoside phosphorylase. Concurrent degradation of uridine to uracil and ribose is catalyzed by nucleosidase. Ultimately, purine nucleoside phosphorylase catalyzes the reaction of ribose-1-phosphate and l,2,4-triazole-3- carboxamide, to form inorganic phosphate and ribavirin.) It is thus clear that the use of thermophilic bacteria as the source of enzymes for enzymatic synthesis enables the performing of the reaction at an elevated temperature, which enhances the activity of the enzyme and improves TCA solubility. The total duration is reduced by a shorter and more economic procedure of biomass preparation (work with untreated bacterial cells), by increased efficiency of the enzymatic reaction, through better contact of the substrate and enzymes released by cell lysis, by a shortened process of denaturation and separation of cell proteins, by shortening the process of chromatographic purification through the choice of adequate columns. Higher yields in the enzymatic reaction (conversion percentage) and in the isolation of the final product are provided, apart from working at higher temperatures, also by the choice of an adequate ribose donor, the molar ratio of the reagents and catalysts, by the preparation, and choice of adequate quantity of wet biomass, by the choice of reaction pH, and by defining the conditions of isolation and purification. Biomass preparation and enzymatic synthesis at elevated temperatures prevents contamination with other microorganisms. Since the bacterial lysis takes place during the reaction there is no problem with inactivation of the cells after the completion of the enzymatic reaction.

The invention will now be illustrated with reference to the following examples of the enzymatic synthesis of ribavirin. Example 1

Thermothrix thiopara sp. bacteria were added to the sterile nutritive medium enriched by the energy source Na₂S₂0₃ which pH value is 7.0, and the resulting seeded culture medium was incubated for 18 hours at 50°C with aeration. Upon completion of the cultivation which achieved the required biomass, the bacteria were separated by centrifuging at 2700 rpm, and the sedimented cells resuspended in distilled water and thoroughly mixed. Into the reaction mixture of pH 6.8, containing 120 mM uridine, 100 mM

TCA, and 50 mM KH P0₄, was introduced the bacterial biomass of untreated intact cells of Thermothrix thiopara sp., so that the final concentration of wet biomass was 58 mg/ml of the reaction mixture. The reaction mixture was mixed continuously and incubated for 24 hours at 60°C. During the course of incubation, the live cells gradually lysed in the reaction mixture (because of the unavailability of any nutrient and because of the hypertonicity of the reaction medium). When incubation was completed, the reaction mixture was centrifuged, and the sediment removed. The supernatant was subjected to thermal treatment by heating at 90°C for 20 minutes to inactivate enzymes and to denature and precipitate proteins. The supernatant so obtained was concentrated under vacuum up to the uracil concentration of 20 - 40 mg/ml as measured by HPLC, at which most of residual amounts of uracil, and TCA, and partial amount of uridine were removed. After adjusting the pH of the solution to 9, the largest amount of inorganic phosphates was separated. The obtained supernatant was adjusted to pH 11.5 and applied to an ion exchange column packed with strong anionic resin type Dowex® 1 x 8 Cl^" form. Ribavirin was eluted from the column by water whereas residual amount of uridine, uracil, TCA, uridine monophosphate and inorganic phosphates remain on the column. Water eluate was passed through ion exchange resin of the type Dowex® 50W, H⁺ form, whereas traces of impurities in cationic form remain on the column. Pharmaceutical grade ribavirin was crystallized from concentrated and cooled eluate, in the yield of 80 - 90%. Example 2

The thermophilic bacteria J₂ described previously, was inoculated in a sterile medium, pH 7.0, and cultivated for 24 hours at 60°C in a thermostat ("stagnant culture"). The obtained primary seed was reseeded into fresh nutritive medium and cultivated under the same conditions for 24 hours. The main culture was prepared by reseeding the secondary seed onto fresh medium and by cultivation during 18 hours, under the same conditions. Bacteria were subsequently sedimented by centrifuging at 2700 rpm, and the obtained sediment was resuspended in redistilled water and homogenized. The wet biomass of J_j bacteria was introduced into the reaction mixture, volume 10 ml, containing 32 mM TCA, 60 mM uridine and 50 mM KH₂P0 , pH 7.0, so that the final concentration in the reaction mixture was 76 mg/ml. The reaction mixture was homogenized and incubated for 24 hours at 45°C. The obtained yield of ribavirin was 14.9 mM (16.6%). Example 3

Thermothrix thiopara sp. bacteria from a slanting agar were inoculated into a sterile nutritive medium enriched with 30 ppm Na₂S 0₃ and the primary seed was seeded. After 24 hours of aerobic cultivation at 50°C and mixing at 100 rpm the primary seed was reseeded into fresh nutritive medium and the secondary seed was cultivated during 24 hours under the same conditions as the primary one. By reseeding the secondary seed into fresh nutritive medium the main culture was seeded and incubated during 18 hours under the same conditions. Bacteria were subsequently centrifuged at 2700 rpm, and the obtained sediment resuspended into redistilled water and homogenized on a shaker. Into a reaction mixture, volume 10 ml, containing 100 mM TCA, 120 mM ribose donors (listed in the table) and 25 mM KH₂P0₄ with a pH of 6.8, the above prepared wet Thermothrix thiopara sp. bacterial biomass was added. The mixture was homogenized and then incubated during 24 hours at 60°C. The obtained yields of ribavirin are presented in Table 1 (at page 15). A comparison with prior art processes of the identified patents gives the following yields of ribavirin: TABLE 4

I II III

Applicant's U.S. Patent Enzyme U.S. Patent Enzyme Invention 3,976,545 source is 4,458,016 source is

Temperature (°C) 50-80 0 - 50 calf spleen 55 - 65 Klebsiella pneumoniae

Time (hours) 1-48 0.08 .17-20

Ratio (ribose:TCA)* 1.2:1 2:5 4.9:1**

Yield (%) 85 54.1 24.2**

*ratio of molar concentrations of the ribose donor to that of TCA **using ribose-1-phosphate, ribavirin yield was 24.2% with ribose: TCA ratio of 4.9:1. When uridine is used in accordance with the teachings of U.S. Patent 4,458,016, ribavirin yield was 11.5% and the ribose: TCA ratio was 4.6:1.

As many changes can be made to the invention without departing from the scope of the invention, it is intended that all material contained herein by interpreted as illustrative of the invention and not in a limiting sense.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIV PROPERTY OR PRIVILEGE IS CLAIMED ARE AS FOLLOWS:

1. A method for producing pharmaceutically active compounds havin a ribose group attached to a triazole by reacting a triazole with a ribose donor i the presence of a source of inorganic phosphate as a catalyst, and a thermophili bacteria.

2. The process of claim 1 wherein the bacteria is Thermothrix thiopar sp.

3. The process of claim 2 wherein the pharmaceutically activ compound is ribavirin.

4. The process of claim 2 wherein the ribose donor is selected fro ribose-1-phosphate, a pyrimidine nucleoside, a purine nucleoside, a pyrimidin nucleotide and a purine nucleotide.

5. The process of claim 2 wherein the triazole is l,2,4-triazole-3 carboxamide.

6. The process of claim 2 wherein the ribose donor is selected fro ribose-1-phosphate; the pyrimidine nucleosides, uridine and cytidine; the purin nucleosides, guanosine, inosine and adenosine; the pyrimidine nucleotide, 5' uridine monophosphate (5'-UMP) and the purine nucleotide, 5'-adenosin monophosphate (5'-AMP).

7. The process of claim 4 wherein the ribose donor is selected fro ribose-1-phosphate; the pyrimidine nucleosides, uridine, and cytidine; the purin nucleosides, guanosine, inosine and adenosine; the pyrimidine nucleotide, 5' uridine monophosphate (5'-UMP) and the purine nucleotide, 5'-adenosin monophosphate (5'-AMP).

8. The process of claim 5 wherein the ribose donor is selected fro ribose-1-phosphate; the pyrimidine nucleosides, uridine, and cytidine; the purin nucleosides, guanosine, inosine and adenosine; the pyrimidine nucleotide, 5' uridine monophosphate (5'-UMP) and the purine nucleotide, 5'-adenosin monophosphate (5'-AMP).

9. The process of claim 5 wherein the ribose donor is present in a mol amount greater than the molar amount of the l,2,4-triazole-3-carboxamide.

10. The process of claim 6, 7 or 8 wherein the ribose donor is present i a molar amount greater than the molar amount of the triazole.

11. The process of claim 6, 7, 8 or 10 wherein the ribose donor is uridine

12. The process of claim 2, 5 or 9 wherein the ribose donor is uridine an is present in a molar amount greater than the molar amount of the triazole.

13. The process of claim 5 wherein the inorganic phosphate as catalyst i potassium dihydrogen phosphate.

14. The process of claim 6 wherein the inorganic phosphate as catalyst i potassium dihydrogen phosphate.

15. The process of claim 7 wherein the inorganic phosphate as catalyst i potassium dihydrogen phosphate.

16. The process of claim 8 wherein the inorganic phosphate as catalyst i potassium dihydrogen phosphate.

17. The process of claim 9 wherein the inorganic phosphate as catalyst i potassium dihydrogen phosphate.

18. The process of claim 10 wherein the inorganic phosphate as cataly is potassium dihydrogen phosphate.

19. The process of claim 11 wherein the inorganic phosphate as cataly is potassium dihydrogen phosphate.

20. The process of claim 12 wherein the inorganic phosphate as cataly is potassium dihydrogen phosphate. 21. The process of any previous claim of recovering ribavirin.

22. The process of claims 1, 2, 4, 6, 7, 10, 11, 14 or 15 further comprisin the step of producing ribavirin.

23. The process for producing a pharmaceutically active triazol nucleoside wherein the process takes place in the presence of a nucleosid phosphorylase preparation enzyme derived from a thermophilic bacteria and source of inorganic phosphate as catalyst.

24. The process for producing the nucleoside of claim 23 wherein th bacteria is Thermothrix thiopara sp.

25. The process of claim 24 wherein the process takes place in th presence of a ribose donor group and a triazole.

26. The process of claim 25 wherein the pharmaceutically activ compound is ribavirin.

27. The process of claim 25 wherein the ribose donor is selected fro ribose-1-phosphate, a pyrimidine nucleoside, purine nucleoside, a pyrimidin nucleotide and a purine nucleotide.

28. The process of claim 25 wherein the triazole is l,2,4-triazole-3 carboxamide.

29. The process of claim 28 wherein the ribose donor is selected fro ribose-1-phcsphate, the pyrimidine nucleosides, uridine, and cytidine; the purin nucleosides, guanosine, inosine and adenosine; the pyrimidine nucleotide, 5' uridine monophosphate (5'-UMP) and the purine nucleotide, 5'-adenosin monophosphate (5'-AMP).

30. The process of claim 25, 26, 27 or 28 wherein the ribose donor i present in a molar amount greater than the molar amount of the triazole.

31. The process of claim 25 or 27 wherein the ribose donor is present in molar amount greater than the molar amount of the triazole and the ribose dono is uridine.

32. The process of claim 26, 27 or 29 wherein the ribose donor is prese in a molar amount greater than the molar amount of the triazole and the ribos donor is uridine.

33. The process of claim 30, 31 or 32 wherein the inorganic phosphate a catalyst is potassium dihydrogen phosphate.

34. The process of claims 23, 24, 25, 27 or 31 further comprising the ste of producing ribavirin.

35. The process of claim 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, furthe comprising the step of recovering ribavirin.

36. A process for the enzymatic synthesis of ribavirin usin thermophilic bacteria of Thermothrix thiopara sp., comprising reacting a ribos donor and l,2,4,-triazole-3-carboxamide, potassium dihydrogen phosphate a catalyst and in the presence of thermostable pyrimidine nucleoside and purin nucleoside phosphorylases, from intact of the thermophilic bacteria Thermothri thiopara sp. which are non-proliferating in the reaction mixture, within a p range of 5-9, temperature interval 50° - 80°C and time period 1-48 hours, wherei the concentration of bacteria in the reaction mixture is 50-60 mg/ml.

37. The process of claim 36 wherein obtained ribavirin is isolated an purified by separation of by-products and residual reactant amounts on the basis different solubilities of the components in the reaction mixture and colum chromatogrpphy using selected types of ion-exchange resins.

38. The process according to claim 36, wherein the ribose donor i selected from ribose-1-phosphate, a pyrimidine nucleoside, a purine nucleoside, pyrimidine nucleotide and a purine nucleotide in a molar amount greater tha the molar amount of l,2,4-triazole-3-carboxamide.

39. The process of claim 36 wherein the ribose donor is uridine prese in a molar amount greater than the molar amount of l,2,4-triazole-3-carboxamid 40. The process according to claim 36 wherein potassium dihydroge phosphate used as a catalyst is in the concentration of 25-100 mM in relation t each 100 mM of l,2,4-triazole-3-carboxamide.

41. The process according to claim 40 or 42 wherein the ribavirin i isolated and purified by removal of residual lysed cells and proteins by therma processing, gradual precipitation of residual amounts of reactants and by-product from previously concentrated supernatant liquid in vacuum whereupon the p of the supernatant is adjusted to 11.5, purified by chromatography usin previously recovered columns which contain selected types of ion-exchang resins during elution of total amount of ribavirin with water.

42. The process according to claim 44 wherein columns are packed wit strong anion and strong cation ion-exchange resins of types Dowex® 1 x 8, Cl^" -form and Dowex® 50 W, H⁺ -form, respectively.

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