CN1111061A - Use of thrmophilic bacteria to manufacture triazole nucleosides - Google Patents

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

Thermophilic bacterium is used to make the purposes of triazole nucleosides

The present invention relates to have the medicinal compound of the ribosyl that is connected with triazole with enzymatic synthesis method production, triazole nucleosides for example, in some instances with 1,2, the 4-triazole connects, and forms 1,2, the 4-triazole nucleosides.A kind of compound is the medicine ribavirin, its chemistry 1-β by name-D-ribofuranosyl-1,2,4-triazole-3-methane amide.

Medicinal compound with the ribosyl that links to each other with triazole, for example triazole nucleosides can make with different methods.Known best compound is a ribavirin, i.e. 1-β-D-ribofuranosyl-1,2,4-triazole-3-methane amide.In some cases, cause the chemical reaction of the complexity that relates to many chemical reagent, the active group of component or reactant is protected before being included in reaction,, intermediate is gone to protect then (by amidation) ribose activation.The Canadian Patent 997756 that this can consult corresponding to United States Patent (USP) 3798209 has related to the manufacture method of ribavirin in the literary composition.The shortcoming of these methods is that impurity in products is many, because the impurity that has many intermediate residues and processing method to bring, in addition, production cost is also high.

Making also available fermentation of compound and the enzymatic synthesis method that this class comprises ribavirin carries out.See for example United States Patent (USP) 3,976,545 and 4,458,016.Fermentation process comprises the aerobic cultivation of bacterium, as brevibacterium sp, and corynebacterium, Anthrobacterium, the aerobic cultivation of bacteriums such as bacillus, fermentation time 2-8 days, in the substratum except that nutrient media, also contain 1,2,4-triazole-3-methane amide and ribose are given body.Then accumulative final product such as ribavirin in the inoculum are separated.

Also can consult for example Furuya, people such as Akira disclose 75123882 ICIC12D in Japan special permission, C07D, on September 29th, 1975, United States Patent (USP) 4614719 be entitled as J.P No.29725/1975 (/Agric.Biol.M.50/1), 121-6,1986 article.

Yet the shortcoming of the method for above mentioned prior art comprises that fermentation time is long, can be by other microbial contamination, and the assorted of a large amount of side reaction products that many undesirable enzymatic reaction meetings cause removing dyes.These impurity have reduced ribavirin output and have hindered purge process.

This back a bit on, these methods comprise the enzyme source of existence from bacterium, for substrate provides specific enzymes (for example phosphoesterase, pyrimidine and purine nucleoside phosphorylase), are used for making the reaction of ribosyl and triazole to make Ribavirina (for example ribavirin).Yet in fact these methods can not produce the ribavirin of commercial aequum.In some cases, the enzyme of having a liking for warm microorganisms by different heterotrophisms is activated being not enough to produce under the temperature of high yield.In some cases, this process is very long, has increased by the possibility of other microbial contamination.Like this, it is complicated that this process becomes, and caused the method complexity when the substrate specificity enzyme purification.

Therefore, the purpose of this invention is to provide the modification method that makes ribosyl and triazole reaction, to obtain for example such Ribavirina medicinal activity compound of ribavirin.

Another object of the present invention provides the method that produces the few medicinal activity compound of high yield and impurity, thereby is very efficient method.

Another object of the present invention provides such method and makes the easier contact of all compositions, interacts better each other in reaction mixture.

Another object of the present invention is active substance such as the ribavirin that used method phosphoric acid esterase and nucleoside phosphorylase produce high yield.

Other purpose of the present invention for those skilled in the art by can being very clearly in the detailed description of following summary of the invention and embodiment thereof.

One aspect of the present invention has provided the method that a kind of preparation has the medicinal activity compound of the ribosyl that links to each other with triazole, for example prepare Ribavirina, as ribavirin, be as the enzyme source of catalyzed reaction in the presence of thermophilic bacterium (as being preferably the desulphurization thermophile bacteria), with triazole (as 1,2,4-triazole-3-methane amide) gives body (ribose-1-phosphoric acid for example with ribosyl, pyrimidine nucleoside, as uridine and cytidine, purine nucleoside is as guanosine, inosine and adenosine, pyrimidine nucleotide as 5 '-uridylic acid (UMP) (5 '-UMP) and purine nucleotides as 5 '-adenylic acid (5 '-AMP) reaction.

Thermophilic bacterium may be defined as at comparatively high temps for example up to about 90-95 ℃ of bacterium that still can survive, and also comprises the desulphurization thermophile bacteria, and it is still survival under 40-80 ℃.

Natural New Mexico's hot spring (the earth constitutes spring), 74 ℃ of temperature, pH7.0, the HS of being present in of desulphurization thermophile bacteria ^-Concentration 1mg/ liter.This microorganism is a Gram-negative bacteria, non-mobility bacterium, and 0.5-1.0 * 3-20 μ m can form the cell chains that length reaches 1cm.The filament that forms does not have pod membrane.Sulphur deposition is in the extracellular.This microorganism can separate HS in the substratum with the autotrophy substratum ^-Be the energy, NO ₃ ^-Be whole last electron acceptor.Anaerobic condition needs NO ₂ ^-Or NO ₃ ^-By NO ₃ ^-Form NO ₂ ^-, no gas evolution.Oxygen also can be used as last electron acceptor eventually, but because of O under the required temperature of growth ₂Solubleness reduced, thereby this bacteria growing is relatively poor.Aerobic and anaerobism is cultivated the used alternately energy and is included hexose, HS ^-, SO ₃ ⁼, and S ₂O ₃ ⁼Optimum temps is 70-73 ℃, growth temperature is 62-77 ℃ (" thermophilic Pseudomonas of desulphurization and the facultative chemolithotrophy bacterium of an amphimicrobian novel species; in neutral pH and high temperature growth down ", draw E.Caldwell from Douglas, Sarah J.Caldwell, with J.Paul Laycoct, Can.J.Microbiol.22:1509-1517).

After the sample that contains the thermophilic bacterial classification of desulphurization is just cultivated, again to enrichment culture liquid purifying in addition.

Because being the earth, the natural habitat of thermophilic microorganism (as the thermophilic bacterial classification of desulphurization) constitutes spring (bacterium that does not have not thermophilic naturally occurring other type herein), temperature range 40-80 ℃, we are according to the composition and the temperature data of mineral water, the source of the thermophilic bacterium that the hot water of having selected three mineral springs in Serbia from the mineral spring with top temperature is used as the present invention:

-Josanicka?banja(62-78℃)

-Vranjski?banja(78-80℃)

-Sijarinska banja (41-63 ℃) habitat feature: (a) Josanicka banja is positioned at Kopaonik at the foot of the hill, has the earth of the medicinal mineral water in 4 places to constitute spring.Water temperature 62-78 ℃, the pH scope 7.5-8.5 of water.(b) Vranjska banja is positioned at Veliki Pester at the foot of the hill, is the most warm mineral water spring in Yugoslavia, also is that Europe is the most warm.Contain sulphur and alkali in the water, water is ultrahigh-temperature, and pH scope 7.8-8.0.Sampling water temperature range 78-80 ℃ of part, but the water temperature range 63-92 in some location ℃.(c) Sijarinska banja is positioned at the distal-most end on Golak mountain, apart from 52 kilometers of Leskovac, 16 mineral water springs is arranged, and contains alkali and iron, temperature range 41-63 ℃.Sampling:

The bacterial deposition thing of stick-slip is present in often by on the rock of warm water washing.Under aseptic condition, take a sample, the bacterial deposition thing is taken into together with water, transfer in the Erlenmeyer flask, take out settling, be inoculated on the nutrient media (liquid nutrient medium) and agar slant (solid medium) of test tube with the bacterium ring with transfer pipet.When pipetting with the bacterium ring, settling can pull into the long silk of 10-15cm.Use following substratum: the meat soup nutritive medium (contain peptone 1.5%, meat extract 0.3%, NaCl 0.5%, KH ₂P0 ₄0.03%) with nutrient agar medium (it forms identical with meat soup nutritive medium composition, just contains agar 1.6%).

The biomaterial sampling is put in the Erlenmeyer flask, also is inoculated in the nutrient media, transports the laboratory back.Material enrichment and purifying:

Originally the biomaterial of sampling is seeded in the liquid nutrient solution cultivates, in 45 and 70 ℃ of overnight incubation.The nutrient solution that " spends the night " is with 20 times of same medium, again in 45 ° and 70 ℃ of cultivations.And then dilution, in 45 ℃ and 70 ℃ of overnight incubation.Make initial biomaterial breeding with this round trip method.Separate pure bacterial cultures with " piece ware method ", the single bacterium colony that obtains is different as shape, size, color and denseness on morphology, and the growth pattern on culture dish also is different.On culture dish, cultivate the back and dissimilar bacterium colonies occurs.Every kind of bacterium colony streak inoculation is to nutrient agar medium, in 45 ℃ and 70 ℃ of cultivations.The bacterium colony of turning out inoculates on agar slant.Make the biomaterial enrichment and the purifying that pick up from Josanicka, Vranjska and Sijarinskabanja in this way.Isolate different bacterium colony on 4 kinds of morphology from Josanicka banja, be called J ₁, J ₂, J ₃(desulphurization thermophile bacteria) and J ₄Isolated two kinds of bacterium colonies are called V in the water of Vranjska banja ₁And V ₂Only isolate a kind of bacterial cultures from Sijarinska banja, therefore behind the sample purifying, isolate 7 kinds of thermophile bacteria (bacterium colony) that morphology is different altogether, in fact the bacterium colony that obtains from Vranjska banja is the same (V with the bacterium colony that obtains from Josanickabanja ₁=J ₂, V ₂=J ₃).The separation of the pure growth of isolated thermophile bacteria and preservation: (a) isolated bacterial cultures is kept on culture dish and the agar slant, 4 ℃ of preservations, and transferred species was 1 time in per 1 month.(b) all isolated culture freeze-drying, standby in 4 ℃ of storages.

Another aspect of the present invention has provided the Ribavirina of medical active such as the method for ribavirin of producing, and this process is to take place in the presence of by thermophile bacteria (as the desulphurization thermophile bacteria) excretory nucleoside phosphorylase.

Another aspect of the present invention is a Ribavirina of producing medical active with thermophile bacteria, ribavirin for example, the preferred desulphurization thermophile bacteria of this class bacterium.

Therefore, another aspect of the present invention is because the existence of thermophile bacteria (preferred desulphurization thermophile bacteria) has unexpectedly improved the value of making this class active compound method.

Know that all inorganic phosphate Yanyuan (preferably phosphoric acid potassium dihydrogen) has participated in the biochemical route of ribose phosphorylation as catalyzer as phosphate source.

Enzymatic synthetic reaction scheme of the present invention is as follows:

R can be: 1) NH ₂: ribavirin

2)OH

3) alkoxyl group (when not making ribavirin, the product that generates being transformed into ribavirin) by any method well known by persons skilled in the art

() ^*This step is chosen wantonly, and this depends on that it is Nucleotide or nucleosides that ribose is supplied with body, for example step (e) step (e)

Step (f) is begun by ribose-1-phosphoric acid, and step is as follows:

(because ribose-1-phosphoric acid costliness and less stable, be uneconomic, not as other material such as uridine worthwhile) as the raw material of synthesis example such as ribavirin.

Because thermophilic bacterium allows to improve temperature of reaction, and make with improved enzymatic reaction method, thereby synthetic expense has reduced, for example increased speed of reaction, increased the solubleness of raw material, the cytolysis in the reaction makes enzyme contact better with substrate, has reduced the danger of competitive microbial contamination, reduced the possibility of competitive reaction has taken place simultaneously, and improved total reaction yield.

A concrete instance is that synthetic method of the present invention can be used to make ribavirin.For this reason, make 1,2,4-triazole-3-methane amide (TCA) and a kind of ribose are supplied with body (for example preferably uridine), concentration should be a little more than TCA molar weight (for example 1.2: 1), at inorganic phosphate Yanyuan such as potassium primary phosphate as enzymatic synthesizing triazazole nucleoside in the presence of the catalyzer.Be used for the non-reproductive ability cell of the preferably desulphurization thermophile bacteria of enzyme source of this reaction mixture, these cells dissolve subsequently.Non-reproductive ability cell is meant does not grow and nonpropagating cell.The dissolving subsequently of cell can make enzyme or enzyme material directly contact to carry out for example enzymatic building-up reactions of ribavirin with substrate, rather than it is synthetic by some other metabolic process of viable cell, cell is alive under latter instance, the synthetic meeting of ribavirin is carried out in cell, promptly carries out under different conditions.PH is the scope of 5-9 preferably, and preferred temperature is 50-80 ℃.Reaction was generally finished in 1-48 hour.

The suitable supply body of ribose can be ribose-1-phosphoric acid, pyrimidine and purine nucleoside and pyrimidine and purine nucleotides.Pyrimidine nucleotide comprises 5 '-uridylic acid (UMP) (5 '-UMP); Purine nucleotides comprises 5 '-adenylic acid (5 '-AMP), pyrimidine nucleoside comprises uridine (preferably) and cytidine; Purine nucleoside comprises guanosine, inosine and adenosine.

Be that certain density wet bacterial biomass is incorporated in the reaction mixture in the example.Wet bacterial biomass in reaction mixture is 50-60mg/ml.The preparation of wet bacterial biomass in an example is a concentration matter of making the desulphurization thermophile bacteria by method commonly used appreciated by those skilled in the art.

When desulphurization thermophile bacteria cells whose development that is used for example and preparation, the nutrient media of cultivating usefulness has following composition: peptone I, meat extract, sodium-chlor, potassium primary phosphate, Sulfothiorine pentahydrate and distilled water.The preparation of master culture is that inoculum is inoculated in the nutrient media, cultivates 18 hours in the 100rpm continuously stirring on vibrator in the presence of air at 50 ℃ then.After cultivating then, cell and nutrient media are separated with clock in 2700rpm centrifugal 30, abandoning supernatant is collected the sediment that is made of the wet cell biomass.

In this preparation, carry out following operation:

The composition of nutrient media and preparation medium are formed:

Peptone I 6g

Meat extract 1.3g

NaCl 2.0g

KH ₂PO ₄ 0.12g

Na ₂S ₂O ₃·5H ₂O 0.099g

Distilled water is added to 440ml

The peptone of the above-mentioned amount of weighing, meat extract, NaCl and KH ₂PO ₄The capacity of being added to is in the glassware of 200ml respectively, dissolves each composition respectively with 100ml distilled water, slowly mixes, and is clear and bright fully up to solution.Merge the dissolved composition, place the 1000ml glassware, adding distil water is to 440ml (when medium was sterilized in autoclave, moisture approximately evaporated 10%).

Regulate medium pH to 7.0-7.2 with the 2M NaOH aqueous solution, be poured into the medium that places in the 1000ml Erlenmeyer flask.

Medium sterilization is sterilization 15 minutes in the 1.5 atmospheric autoclaves in 120 ℃, pressure.

Under aseptic condition, measure the 100ml medium and place in 4 1000ml Erlenmeyer flasks, add the 0.1M sodium thiosulfate solution of 1ml prepared fresh behind the cool to room temperature.Na ₂S ₂O ₃The preparation of liquid

Weighing 0.248g Na ₂S ₂O ₃5H ₂O is dissolved in the 10ml distilled water, under aseptic condition in biofilter =0.45 μ is filled into the aseptic Glass Containers of lucifuge (aluminium foil).The preparation of inoculum (seed culture fluid)

From the 100ml Erlenmeyer flask, measure the medium that 5ml prepares previously, transfer on the agar slant.Scrape that the bacterium of getting on the agar slant suspends again and suspension transferred to blank pipe that (in the 16cm * 10mm), jolting (using the vortex vibrator) 1-2 minute obtains uniform suspension, and microscopically be can't see mycelia and bacterium sediment with aseptic bacterium ring.

The bacterial suspension that makes like this is inoculated in the Erlenmeyer flask that contains the 95ml medium.

Clog postvaccinal medium bottle with asepsis plug, asepsis plug is made with cotton and gauze, and jolting was cultivated 6 hours in 50 ℃ and air, and 100rpm stirs and ventilation (K _{1 Λ}=0.64mM O ₂/ minute).After cultivating then, check that inoculum (seed) pH that obtains should be 7.4-7.8.If the pH of inoculum is not right, then can not be used for preparing master culture.The preparation of master culture

Each 5ml of inoculum that makes above is inoculated in 3 Erlenmeyer flasks that contain the 95ml medium.With aseptic interlayer plug (duplicating paper cotton) obturation, (50 ℃ of temperature, 100rpm stirs, ventilation K to cultivate 18 hours by above-mentioned inoculum preparation condition in air on vibrator _{1 Λ}=0.64mM O ₂/ minute).

After cultivating end, master culture is merged in the 1000ml Erlenmeyer flask, detect pH (should be 8.3-8.6).If pH is not right, it is synthetic that this master culture can not be used for enzymatic.Measure the concentration (C of the wet biomass of bacterium in the master culture ₁) to determine the required volume (V of master culture ₁) the mensuration optical density(OD)

The 0.5ml master culture is joined in the 4.5ml medium, make homogenizing with the violent jolting of vortex vibrator.Compare the optical density(OD) of measuring the 610nm place with the medium that does not add bacterium.

Calculate the wet biomass concentration (C of bacterium of master culture with following formula ₁):

C ₁=3.89 * OD _610mm+ 10.76 for example: if OD _610mm=4.8, C then ₁=29.6mg/ml (the wet biomass concentration of bacterium in the main sample liquid)

If OD _610mm=5.5, C then ₁=32mg/ml (the wet biomass concentration of bacterium in the main sample liquid).For carrying out the volume (V that enzymatic reaction determines that master culture is required ₁)

Need to determine with following formula by the centrifugal reaction mixture volume (V that makes ₂) main nutrient solution volume (V ₁): $V_1=\frac{C_2\×V_2}{C_1}$ C in the formula ₁For using standard curve determination OD ₆₁₀And the wet biomass concentration C of bacterium in the master culture of determining ₂Optimum concn (58mg/ml) V for the wet biomass of bacterium in the reaction mixture ₂Volume for reaction mixture.For example: if C ₁=29.6mg/ml, V ₂=50ml, then when making the wet biomass concentration of bacterium final in the reaction mixture be 58mg/ml, the required volume of centrifugal master nutrient solution is 100.0ml.

If C ₁=32mg/ml, V ₂=50ml, then V ₁=90.5ml.Isolated cell method in the autonomous nutrient solution: with V ₁Ml master culture (90.5-100ml) centrifugal (2700rpm) 30 minutes, sedimentation matter, the supernatant liquor that inclines is suspended in the sediment (about 2.9g) that obtains in the aseptic double distilled water of 5ml again, transfers in the sterile test tube, contains 2.0 * 10 in this suspension ⁷-10 ⁹Individual viable cell/ml is used to carry out enzymatic reaction.Enzymatic reaction with separate

The wet cell piece of thermophile bacteria is suspended in the distilled water again, this bacterial suspension is incorporated in the solution, make all composition ultimate densities be:

Constituent concentration

TCA 100mM

Uridine 120mM

KH ₂PO ₄ 50mM

Thermophile bacteria 58mg/ml

(not having nutrition in the bacterial biomass in an example) makes enzymatic reaction mixture.

Enzymatic reaction mixture is incubated 24 hours (because of the hypertonicity of nonnutritive thing and reaction medium, making the viable cell dissolving in the insulating process) in 60 ℃.

After the enzyme reaction fully,, separate and purifying with the by product of reaction and residual reaction reagent with active medicinal matter such as ribavirin.This extraction, purifying and lock out operation for example are by step-by-step precipitation method, carry out based on the different solubility of product, by product and raw material in the reaction medium, also can use other extraction, purifying and isolating method known to those skilled in the art.

Available column chromatography, for example with suitable ion exchange column, for example in the time will removing uridine, uridylic, TCA, uridylic acid (UMP) and inorganic phosphate, their available reinforcing yin essence ion exchange resin (Dowe for example ^, Cl ^-Type) removes.As the cationic impurity (K that will remove trace ⁺, NH ₄ ⁺, heavy metal etc.) time, also need strong cation-exchanging resin (as Dowe ^50W, H ⁺Type).Pharmaceutical grade other through the water wash-out with from concentrating, can being recovered after refrigerative elutriant (preferably the adding ethanol again) crystallization, yield is 80-90%.

When making ribavirin, it is as shown in table 1 to the yield effect of ribavirin that ribose is supplied with body:

Table 1

Ribose is supplied with body ribavirin yield (%)

Guanosine 21.0

Cytidine 32.0

Uridine 80.0

Inosine 4.0

Adenosine 39.9

5-UMP 40.0

Table 2 has been listed and has been made ribose with uridine and supply with body and 1,2, and the temperature of reaction mixture insulation is to the influence of ribavirin output during 4-triazole-3-formamide:

Table 2:

Temperature of reaction (℃) ribavirin yield (%)

50 56.7

60 84.6

65 55.3

70 29.7

Uridine is that ribose is supplied with body, as the potassium primary phosphate (KH of catalyzer ₂PO ₄) concentration table 3 is listed in the influence of ribavirin output.

Table 3:

KH ₂PO ₄Concentration (mM) ribavirin yield (%)

25 80.36

50 84.94

75 82.17

100 73.77 (mM is a mmole)

The preparation of ribavirin is undertaken by following reaction process in the presence of the desulphurization thermophile bacteria:

(enzyme in the desulphurization thermophile bacteria is preferred for by uridine and 1,2,4-triazole-3-methane amide synthesizing triazazole nucleoside.Uridine and inorganic phosphate are transformed into ribose-1-phosphoric acid and uridylic under the effect of pyrimidine-nucleoside phosphorylase.The degraded that uridine takes place under the effect of nucleosidase simultaneously generates uridylic and ribose.At last, purine nucleoside phosphorylase catalysis ribose-1-phosphoric acid and 1,2,4-triazole-3-formamide generates inorganic phosphate and ribavirin).

Obviously can make under the temperature that is reflected at raising as the enzyme source of enzymatic reaction with thermophile bacteria and to carry out, thereby improve the active of enzyme and improved the solubleness of TCA.Because short and the both economical method for preparing bacterial biomass (operating) with untreated bacterial cell, because cytolysis discharges enzyme, these enzyme-to-substrates are contacted preferably and increase the efficient of enzymatic reaction, owing to shortened the sex change and the sepn process of cell protein, owing to select suitable chromatography column to shorten layer utmost point purge process, total preparation time shortened.Except operation under comparatively high temps, also owing to selected suitable ribose to supply with the mol ratio and the catalyzer of body, reagent, owing to prepare and selected the wet biomass of bacterium of sufficient quantity, the pH of reaction and the condition of having determined separation and purifying have been selected, this enzymatic reaction has higher yield (conversion percentage), and higher yields is also arranged in the separation of end product.Synthetic other microbiological contamination that prevented of bacterial biomass preparation and enzymatic under the temperature that improves.Owing to bacteriolysis takes place in reaction process, thereby do not have the inactivation problem of cell after enzymatic reaction fully.

Embodiment with following enzymatic synthesizing triazazole nucleoside is illustrated the present invention now.Embodiment 1

The desulphurization thermophile bacteria joined be rich in energy Na ₂S ₂O ₇Aseptic nutrient media in, ventilate following 50 ℃ of incubations 18 hours of the latter's pH7.0, the substratum of inoculating.After incubation finishes, reach required biomass, go out bacterium in the 2700rpm centrifugation, the cell that settles down is suspended in the distilled water again, mixing.

The undressed intact cell bacterial biomass of desulphurization thermophile bacteria joined contain 120mM uridine, 100mMTCA and 50mM KH ₂PO ₄PH be in 6.8 the reaction mixture, making the ultimate density of wet biomass in the reaction mixture is 58mg/ml.Reaction mixture is constantly mixed, in 60 ℃ of incubations 24 hours.In the incubation process, viable cell dissolving in reaction mixture gradually (because hypertonicity of supply of nonnutritive thing and reaction medium).Incubation is finished, and reaction mixture is centrifugal, removes sediment.Supernatant liquor made enzyme deactivation and sex change, albumen precipitation in 20 minutes in 90 ℃ of heat treated.Concentrate under the supernatant liquor vacuum that obtains, making uridylic concentration is 20-40mg/ml (measuring with HPLC), and the uridine of the uridylic of most of residual quantity, TCA and part amount is removed at this moment.Behind the regulator solution pH to 9, isolate a large amount of inorganic phosphates, the supernatant liquor that adjusting obtains is to pH11.5, with strong anionic resin (Dowex is housed ^1 * 8Cl ^-Type) ion exchange column carries out purifying.Ribavirin water wash-out on post goes out, and the uridine of residual quantity, uridylic, TCA, uridylic acid (UMP) and inorganic phosphate are still stayed on the post.Water elution liquid is through Dowe ^50W H ⁺Type ion exchange resin, and the impurity of the cationic form of trace is stayed on the post.Elutriant through concentrate and cooling after, the crystallization drug is with other ribavirin of level, yield 80-90%.Embodiment 2

Aforesaid thermophilic bacterium J1 is inoculated in the sterile media of pH7.0, cultivates 24 hours (" stagnate and cultivate ") in 60 ℃ of thermostat containers.The elementary bacterial classification that obtains inoculates in the fresh nutrient media, and cultivates 24 hours down in similarity condition.Second class inoculum inoculates in the fresh medium, cultivates 18 hours down in similarity condition, makes master culture.Make the bacterium sedimentation in 2700rpm is centrifugal, the sediment resuspending that obtains in distilled water, mixing.With J ₁The wet biomass of bacterium joins in the 10ml reaction mixture, and the latter is contained 32nM TCA, 60mM uridine and 50mM KH ₂PO ₄, pH7.0, the ultimate density that makes bacterium in the reaction mixture is 76mg/ml.After the reaction mixture homogenizing, in 45 ℃ of incubations 24 hours, the ribavirin output that obtains was 14.9mM (16.6%).Embodiment 3

To be inoculated in by the desulphurization thermophile bacteria that agar slant obtains and be rich in 30ppm Na ₂S ₂O ₃In-3 the aseptic nutrient media, with elementary bacterial classification inoculation.Stir after aerobic is cultivated 24 hours down in 50 ℃ and 100rpm, elementary bacterial classification is inoculated in the fresh nutrient media, under similarity condition, second class inoculum was cultivated 24 hours.Second class inoculum is inoculated in the fresh nutrient media inoculation master culture and incubation 18 hours under similarity condition.Centrifugal in 2700rpm then, the bacterium sediment resuspending that obtains is in distilled water, and vibration makes evenly.The wet desulphurization thermophile bacteria biomass of above-mentioned preparation is joined in the 10ml reaction mixture, wherein contain 100mM TCA, 120mM ribose supply body (listing in the table) and 25mM KH ₂PO ₄, pH68.After mixture homogenization, 60 ℃ of incubations 24 hours, the ribavirin productive rate that obtains is listed in table 1.

Art methods and the inventive method prepare yield more as shown in table 4 of ribavirin: I. the applicant's invention temperature 50-80 ℃ of time (hour) 1-48 ribose: 1.2: 1 yields (%) of TCA ratio 85II. United States Patent (USP) 3976545

Enzyme source is little cattle spleen

0-50℃

0.08

2∶5

54.1III. United States Patent (USP) 4458016

Enzyme source is a klebsiella pneumoniae

55-65℃

0.17-20

4.9∶1 ^**

24.2 ^** supply with molar concentration rate * * ribose-1-phosphoric acid of body and TCA for ribose, the ribavirin yield is 24.2%, ribose: TCA ratio is 4.9: 1.Supply with body if make ribose by United States Patent (USP) 4458016 usefulness uridines, then the ribavirin yield is 11.5%, ribose: TCA=4.6: 1.

Owing to can carry out many changes to the present invention in the case without departing from the scope of the present invention, so all material that this paper comprises all is unconfined intention for the present invention is described.

Claims (43)

1. One kind thermophilic bacterium with as the inorganic phosphate Yanyuan of catalyzer in the presence of make triazole and ribose supply with precursor reactant production to have the method for the medicinal activity compound of the ribosyl that links to each other with triazole.
2. 2. the process of claim 1 wherein that bacterium is the desulphurization thermophile bacteria.
3. 3. the method for claim 2, wherein medicinal activity compound is a ribavirin.
4. 4. the method for claim 2, wherein ribose is supplied with body and is selected from ribose-1-phosphoric acid, pyrimidine nucleoside, purine nucleoside, pyrimidine nucleotide and purine nucleotides.
5. 5. the method for claim 2, wherein triazole is 1,2,4-triazole-3-methane amide.
6. 6. the method for claim 2, wherein ribose is supplied with body and is selected from ribose-1-phosphoric acid; Pyrimidine nucleoside, uridine and cytidine; Purine nucleoside, guanosine, inosine and adenosine; Pyrimidine nucleotide, 5 '-uridylic acid (UMP) (5 '-UMP) and purine nucleotides, 5 '-adenylic acid (5 '-AMP).
7. 7. the method for claim 4, wherein ribose is supplied with body and is selected from ribose-1-phosphoric acid; Pyrimidine nucleoside, uridine and cytidine; Purine nucleoside, guanosine, inosine and adenosine; Pyrimidine nucleotide, 5 '-uridylic acid (UMP) (5 '-UMP) and purine nucleotides, 5 '-adenylic acid (5 '-AMP).
8. 8. the method for claim 5, wherein ribose is supplied with body and is selected from ribose-1-phosphoric acid; Pyrimidine nucleoside, uridine and cytidine; Purine nucleoside, guanosine, inosine and adenosine; Pyrimidine nucleotide, 5 '-uridylic acid (UMP) (5 '-UMP) and purine nucleotides, 5 '-adenylic acid (5 '-AMP).
9. 9. the method for claim 5, wherein ribose is supplied with molar weight that body exists greater than 1,2, the molar weight of 4-triazole-3-methane amide.
10. 10. the method for claim 6,7 or 8, wherein ribose is supplied with molar weight that body the exists molar weight greater than triazole.
11. 11. claim 6,7,8 or 10 method, wherein ribose supply body is a uridine.
12. 12. the method for claim 2,5 or 9, wherein ribose supply body is a uridine, and the molar weight of its existence is greater than the molar weight of triazole.
13. 13. the method for claim 5, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
14. 14. the method for right claim 6, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
15. 15. the method for claim 7, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
16. 16. the method for claim 8, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
17. 17. the method for claim 9, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
18. 18. the method for claim 10, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
19. 19. the method for claim 11, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
20. 20. the method for claim 12, wherein the inorganic phosphate as catalyzer is a potassium primary phosphate.
21. 21. above-mentioned any one method can obtain ribavirin.
22. 22. claim 1,2,4,6,7,10,11,14 or 15 method also comprises the step for preparing ribavirin.
23. 23. produce the method for medical active Ribavirina, wherein reacting by the nucleoside phosphorylase preparation in thermophilic bacterium source with in the presence of as the inorganic phosphate Yanyuan of catalyzer.
24. 24. the method for preparing nucleosides of claim 23, wherein bacterium is the desulphurization thermophile bacteria.
25. 25. the method for claim 24, wherein reaction is to carry out in the presence of ribose supply body and triazole.
26. 26. the method for claim 25, wherein reaction is to carry out in the presence of ribose supply body and triazole.
27. 27. the method for claim 25, wherein medicinal activity compound is a ribavirin.
28. 28. the method for claim 25, wherein triazole is 1,2,4-triazole-3-methane amide.
29. 29. the method for claim 28, wherein ribose is supplied with body and is selected from ribose-1-phosphoric acid; Pyrimidine nucleoside, uridine and cytidine; Purine nucleoside, guanosine, inosine and adenosine; Pyrimidine nucleotide, 5 '-uridylic acid (UMP) (5 '-UMP) and purine nucleotides, 5 '-adenylic acid (5 '-AMP).
30. 30. claim 25,26,27 or 28 method, wherein ribose is supplied with the molar weight of the molar weight of body existence greater than triazole.
31. 31. the method for claim 25 or 27, wherein ribose is supplied with the molar weight of the molar weight of body existence greater than triazole, and it is uridine that ribose is supplied with body.
32. 32. the method for claim 26,27 or 29, wherein ribose is supplied with the molar weight of the molar weight of body existence greater than triazole, and it is uridine that ribose is supplied with body.
33. 33. the method for claim 30,31 or 32, wherein ribose is supplied with the molar weight of the molar weight of body existence greater than triazole, and it is uridine that ribose is supplied with body.
34. 34. claim 23,24,25,27 or 31 method also comprises the step for preparing ribavirin.
35. 35. claim 23,24,25,26,27,28,29,30,31,32 or 33 method also comprises the step for preparing ribavirin.
36. 36. method with desulphurization thermophile bacteria enzymatic synthesizing triazazole nucleoside, comprise with the potassium primary phosphate being catalyzer and in the presence of heat stable pyrimidine nucleoside and purine nucleoside phosphorylase, make ribose supply with body and 1,2,4-triazole-3-formamide, Starch phosphorylase is from complete desulphurization thermophile bacteria, this bacterium does not breed in reaction mixture, and the pH scope is 5-9, and temperature range is 50-80 ℃, time is 1-48 hour, and wherein the concentration of bacterium in reaction mixture is 50-60mg/ml.
37. 37. the method for claim 36 is wherein separated and the purifying ribavirin is a column chromatography with the ion exchange resin of selected type, according to the difference of each components dissolved degree in the reaction mixture by product and residual reactant is separated.
38. 38. the method for claim 36, wherein ribose is supplied with body and is selected from ribose-1-phosphoric acid, pyrimidine nucleoside, and purine nucleoside, pyrimidine nucleotide and purine nucleotides, its molar weight is greater than 1,2, the molar weight of 4-triazole-3-methane amide.
39. 39. the method for claim 36, wherein to supply with body be uridine to ribose, and its molar weight is greater than 1,2, the molar weight of 4-triazole-3-methane amide.
40. 40. the method for claim 36, the concentration of the potassium primary phosphate that uses as catalyzer wherein, with 1,2 of every 100mM, 4-triazole-3-methane amide meter is 25-100mM.
41. 41. the method for claim 40 or 42, wherein the separation of ribavirin and purifying are to remove residual dissolved cell and albumen through thermal treatment, with the supernatant liquor pH regulator to 11.5 of vacuum concentration in advance, remove the reactant and the by product of residual quantity with step-by-step precipitation method, through chromatography purification, wash the ribavirin of full dose with the post of regenerated in advance of the ion exchange resin that contains selected type with water.
42. 42. the method for claim 44, wherein chromatography column loads Dowex respectively ^1 * 8, Cl ^-Type and Dowex ^50W, H ⁺Reinforcing yin essence ion and strong cation-exchanging resin.

    

    Wherein R is selected from NH ₂(generation ribavirin), OH or alkoxyl group

    

    Wherein R is selected from NH ₂(generation ribavirin), OH or alkoxyl group

    
43. 49. prepare the method for ribavirin in the presence of the desulphurization thermophile bacteria, this method comprises the following steps:

    P herein _iIt is inorganic phosphate