High temperature method for the production of ribavirin

A method for the production of a 1,2,4-triazole nucleoside comprising the step of reacting a ribose donor with a triazole compound in the presence of an enzyme preparation derived from Brevibacterium acetylicum is disclosed. The method is characterized in that the ribose donor is guanosine and the temperature during at least a part of the reaction is at or above 65.degree. C. The method is capable of high production rates and high concentrations of the final product.

BACKGROUND OF THE INVENTION 
The present invention is directed to a method for the production of 
ribavirin and related compounds. The systematic name for ribavirin is 
1-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide. Compounds of this type 
are known antiviral agents. Reference is made to U.S. Pat. No. 3,798,209 
of Joseph T. Witkowski et al issued Mar. 19, 1974. Throughout the present 
specification, reference will be made to ribavirin. It will be understood 
that related compounds having a ribose group attached to a triazole are 
also intended. 
There are several methods for the production of ribavirin. Chemical methods 
(those methods not using enzymes) are expensive. Expensive starting 
materials and process steps characterized by low yields are common. 
As a result, bioconversion methods have been extensively studied. In these 
methods, an enzyme or enzymes are used to attach the ribose to the 
triazole. In some cases, the enzyme is first isolated and then used as the 
catalyst. Reference is made to U.S. Pat. No. 3,976,545 of Witkowski et al 
issued Aug. 24, 1976. In this patent there is disclosed a method wherein 
the ribose donor is ribose-1-phosphate. The triazole acceptor 
1,2,4-triazole-3-carboxamide is reacted with the donor in the presence of 
nucleoside phosphorylase at a temperature between 0.degree. C. and 
50.degree. C. The source of the enzyme is broadly disclosed. 
Methods involving fermentation are also well known. In these methods the 
1,2,4-triazole-3-carboxamide is added to a culture medium containing 
proliferating microorganisms such as a microorganism from the genus 
Brevibacterium. In this case, the necessary ribose donor comes from the 
fermentation medium as the organisms grow. Since the organisms are 
growing, the temperature is relatively low. Typical temperatures are 
between 20.degree. C. and 40.degree. C. Reaction times are very long, 
typically on the order of days. 
In U.S. Pat. No. 4,458,016 to Yamanaka et al issued July 3, 1984 there is 
described a method that is very similar to the method of the U.S. Pat. No. 
3,976,545 discussed above except that the temperature is between 
55.degree. and 65.degree. C. Rather than isolated enzyme, whole cells 
containing the necessary activity can be used. Comparative results in the 
specification of this patent with the specific materials used indicate 
that the amount of ribavirin that is produced is very low at 70.degree. C. 
and negligible at 75.degree. C. and 80.degree. C. (see table 3 at column 
7) The microorganism that was used in this test was Klebsiella pneuminiae 
and the ribose donor was either ribose-1-phosphate or uridine. 
In U.S. Pat. No. 4,614,719 to Fujishima et al issued Sept. 30, 1986 there 
is disclosed a method that is similar to the method of U.S. Pat. No. 
4,458,016 discussed above. In U.S. Pat. No. 4,614,719 a Brevibacterium 
acetylicum microorganism is used under nonproliferative conditions. A wide 
variety of ribose donors can be used according to this disclosure and the 
temperature can be between 40.degree. C. and 80.degree. C. However, in the 
examples, inosine is predominantly used as the donor and the temperature 
is usually 60.degree. C. In example 2 a variety of donors are tested with 
the B. acetylicum at 60.degree. C. and in Example 4 a variety of 
temperatures up to 70.degree. C. are tested with that microorganism and 
inosine as the donor. The yield of ribavirin dramatically decreases 
between 65.degree. and 70.degree. C. 
The bioconversion method of U.S. Pat. No. 4,458,016 and U.S. Pats. No. 
4,614,719 offer many advantages over the previous chemical method and the 
methods where the enzyme had to be isolated before use. However, 
additional improvements were still needed. For example, these methods 
produce ribavirin at a rate that is slower than desired. In U.S. Pat. No. 
4,619,714 examples, the typical reaction time is 20 or 24 hours and the 
amount of ribavirin produced is relatively low. (Calculated to be at most 
about 10 g/L based on the data given.) Thus, while the % yield (actually 
the % conversion as described below) might be acceptable in these methods, 
the productivity of these methods is less than desired. Further, since the 
ribavirin is produced in only dilute solution, the recovery is more 
expensive than desired. 
SUMMARY OF THE INVENTION 
In accordance with the present invention there is provided an improved 
method for the production of a 1,2,4-triazole nucleoside comprising the 
step of reacting a ribose donor with a triazole compound in the presence 
of an enzyme preparation derived from Brevibacterium acetylicum. The 
improvement according to the present invention is that the ribose donor is 
guanosine or a guanosine derivative and the temperature during at least a 
part of the reaction is at or above 65.degree. C. The method according to 
this invention is capable of achieving higher concentrations of the 
desired product in the reaction mixture. 
In accordance with a further improvement, the initial concentration of the 
ribose donor and the triazole is greater than 100 mM. The highest 
concentration in the examples of U.S. Pat. No. 4,614,719 is about 50 mM. 
We have found that the present method is capable of utilizing the higher 
concentration because of the donor selected and the higher temperatures 
used. 
At extremely high concentrations of the reactants, we have found that there 
is a tendency for the reaction mixture to gel. In a preferred embodiment 
of the invention, this problem is avoided by adding the enzyme preparation 
before gelling occurs. 
In the typical method, the enzyme preparation is separated from the 
reaction mixture and discarded. We have found that significant amounts of 
the desired product are associated with the enzyme preparation. In a 
preferred embodiment of this invention, the enzyme preparation is 
separated from the reaction mixture and is washed so as to recover 
additional product. 
In accordance with a further improvement of the method, there is provided 
the additional step of adding the ribose donor and the triazole during the 
course of the reaction. This method is capable of producing ribavirin and 
related compounds in very high concentrations. 
DETAILED DESCRIPTION OF THE INVENTION 
Through the use of a specific ribose donor we found that higer than usual 
temperatures could be used. The result is an increase in the producitivity 
of the reaction with little or no decrease in the conversion. This result 
was surprising since the prior art suggested that the conversion decreased 
dramatically at higher temperatures. This was confirmed by comparative 
experiments described below. Using different donors, for example inosine, 
it was found that at a temperture of 70.degree. C., the rate was not as 
high as desired nor was the conversion of the starting materials to the 
desired product as high as desired. 
We have also found that not only is the initial rate higher, but the 
production of ribavirin continues at a high rate for an extended period 
thereby producing a high concentration of the desired product in the final 
reaction mixture. While not wishing to be bound by any particular theory, 
it may be that this occurs because the by-product of guanosine is less 
soluble than the by-product of other donors. Therefore, there could be 
less of a chance that the by-product inhibits the action of the enzyme. 
Therefore, the high rate of ribavirin production continues at a high level 
and high concentrations are attained. 
The final concentration of the ribavirin in the reaction mixture is an 
important component of the overall cost of carrying out the method. High 
concentrations allow for better economics since the recovery from 
concentrated solutions is less expensive than from dilute solutions. Using 
the method of the present invention, very high ribavirin concentrations, 
on the order of 100 g/L in preferred embodiments, can easily be achieved. 
In accordance with the present invention, the temperature should be at or 
above 65.degree. C. during at least part of the method. Any temperature 
above this limit can be used but as a practical matter, the conversion 
does decrease as the temperature increases, even though the rate of 
production remains high. Thus, a temperature of about 70.degree. C. is 
preferred. 
The ribose donor is guanosine. It can be purchased commercially and is 
found in the hydrolysate of RNA, for example yeast RNA. Derivatives of 
guanosine can also be used such as guanylic acid. 
The microorganism that is employed as the source of the catalytic activity 
is a Brevibacterium acetylicum. Any strain of this species can be used. 
The strain identified as ATCC 39311 available from the American Type 
Culture Collection and which is described in U.S. Pat. No. 4,614,719 
referenced above is preferred. 
The microorganism can be prepared by conventional fermentation processes 
such as the process described in Preparation 1 just prior to the present 
examples. A sample of the microorganism is inoculated into a fermentor 
with suitable nutrients and caused to grow to a stationary phase. The 
resulting fermentation broth can be used directly as the catalytic 
material. The cells can also be removed from the broth by filtration or 
centrifugation and used as a cell paste. The cells can be treated so as to 
increase their permeability. Treatments such as freeze thawing, and other 
treatments described in U.S. Pat. No. 4,614,719 are useful for this 
purpose. 
According to the present invention any enzyme preparation that is derived 
from B. acetylicum is useful. This includes the preparations described 
above and also any preparations from microorganisms that express the gene 
or genes isolated from B. acetylicum that is responsible for the 
nucleoside phosphorylase activity of this microorganism. 
The concentration of the reactants in the reaction mixture can vary widely. 
The concentration of the guanosine donor can be between about 10 mM and 
500 mM; the triazole between about 10 mM and 500 mM; and the cells present 
in an amount of between about 2 g/L and 100 g/L based on the cell dry 
weight. Since the reaction uses one mole of donor for each mole of 
triazole, it is preferred that these reagents be in the reaction mixture 
in this ratio. 
The starting pH can also vary widely and can range between about 6.0 and 
9.2. While it is not critical to control the pH during the reaction, pH 
control is desirable. The optimum pH for ribavirin production is about 
7.2. 
In preferred methods according to the invention, the concentration of the 
starting materials is higher than that usually found in similar methods. 
For example, guanosine is preferably present in an amount of between 100 
mM and 200 mM while the triazole is preferably present in an amount of 
between 100 mM and 200 mM. 
We have found that reaction mixtures using the donor guanosine tend to gel 
at high concentrations of guanosine at relatively high temperatures. 
Accordingly, it is preferred according to the present invention to add the 
catalyst before gelling occurs. Thus, the catalyst can be first mixed with 
one of the reagents and the other of the reagents added to that mixture. 
As the other reagent is added, the reaction begins immediately thereby 
preventing gel formation. Alternatively, the reactants can be mixed at low 
temperature and the catalyst added as the temperature is increased but 
before gelling occurs. These procedures are not suggested in the 
references cited above. In U.S. Pat. No. 4,614,719, for example, the 
reagent mixture is first formed and the catalyst is added to that mixture. 
In a particularly preferred embodiment, ribose donor and triazole are added 
during the course of the reaction. These reagents can be added 
continuously or in batches over time for example, every eight hours. The 
rate of addition is preferrably about 40 mM/hour although higher and lower 
rates can be used. As noted previously, concentrations of ribavirin near 
100 g/L can be achieved over the course of the reaction. Depending on the 
desired ending concentration, the time of reaction can vary widely, for 
example between about 6 to 30 hours. 
In addition to the ribose donor and the triazole, the reaction mixture 
preferably contains phosphate ion as this may be required by the enzymes. 
A useful source of phosphate ions is potassium monophosphate and the 
concentration is typically between about 25 mM and 100 mM. Lower levels of 
phosphate are useful if the pH is controlled during the reaction. 
In conventional methods of the present type, the cells that are used as the 
catalyst are removed from the reaction mixture and discarded. We have 
found that these cells contain a significant amount of the desired 
product. Thus, in a preferred method, the enzyme preparation is recovered, 
such as by centrifugation and washed. Additional product is then recovered 
from the wash liquid. The wash liquid is preferably water. 
In the examples below, the % conversion is referred to. The % conversion is 
the amount of ribavirin, on a molar basis, divided by the initial amount 
of starting materials, based on the molar amount of the limiting reactant. 
This is believed to be referred to as yield in the prior art art 
references. More precisely, yield refers to the amount of product produced 
divided by the amount of starting material that reacts. To calculate 
yield, the final amount of starting material must be known. In the present 
examples, no effort was made to measure the remaining amount of starting 
material at the end of the reaction. If the starting materials go only to 
the desired product, then % conversion and % yield are the same. 
The following preparation and examples are submitted for a further 
understanding of the invention. 
PREATION 1 
Preparation of: Brevibacterium acetylicum 
Ten liters of an aqueous cultivation medium at pH 7.2 was prepared, 
sterilized, and combined in a fermentor. The composition of the medium is 
shown in Table I. An inoculum was prepared by culturing Brevibacterium 
acetylicum ATCC 39311 in a Fernbach flask containing 500 mL of medium for 
20 hours at 30.degree. C. The Fernbach medium was identical to that used 
in the fermentor, except it lacked magnesium sulfate. After transfer of 
the inoculum, the fermentor was cultured at 30.degree. C. for 20 hours. 
The pH was controlled at 7.2 with potassium hydroxide. Additional glucose, 
amounting to 20 g/L of broth, was added at 12 hours into the fermentation. 
At the end of the fermentation, centrifugation of the broth yielded 50 
grams of wet cells per liter of broth. The cells were washed by 
re-suspending them in a 10mM Phosphate buffer. They were then collected by 
centrifugation and stored as a frozen paste. 
TABLE I 
______________________________________ 
Fermentation Medium 
Concentration 
Component (g/L) 
______________________________________ 
1. Nutrient broth 20 
2. K.sub.2 HPO.sub.4 
14 
3. KH.sub.2 PO.sub.4 
5.5 
4. Sodium citrate 0.025 
5. MnCl.sub.2 .multidot.4H.sub.2 O 
0.015 
6. ZnCl.sub.2 0.01 
7. FeCl.sub.3 .multidot.6H.sub.2 O 
0.01 
8. MgCl.sub.2 .multidot.6H.sub.2 O 
0.25 
9. CuCl.sub.2 .multidot.2H.sub.2 O 
0.001 
10. CaCl.sub.2 .multidot.2H.sub.2 O 
0.00375 
11. CoCl.sub.2 .multidot.2H.sub.2 O 
0.001 
12. NaMoO.sub.4 .multidot.2H.sub.2 O 
0.0005 
13. Polyglycol P-2000* 
2 
14. Glucose 50 
15. MgSO.sub.4 .multidot.7H.sub.2 O 
0.75 
16. Thiamine.multidot.HCl 
0.0002 
17. p-Aminobenzoic acid 
0.0002 
18. Pyridoxine.multidot.HCl 
0.0002 
19. Nicotinic acid 0.0002 
20 Riboflavin 0.0002 
21. Calcium d-pantothenate 
0.0002 
22. Folic acid 0.000002 
______________________________________ 
*available from Dow Chemical Midland Mich. USA

EXAMPLE 1 
Performance of the Bioconversion as a Function of Temperature and pH using 
Guanosine as the Ribose Donor 
Bioconversion media were prepared by combining 20 millimoles of guanosine, 
20 millimoles of 1,2,4-triazole-3-carboxamide, and 20 millimoles of 
KH.sub.2 PO.sub.4 with 200 mL water in 500 mL flasks. The pH of each flask 
was adjusted to the desired value with potassium hydroxide or sulfuric 
acid. 
The flasks were placed in temperature-controlled baths, and the contents of 
the flasks were allowed to equilibrate at the desired temperature. Forty 
grams of B. acetylicum cell paste (thawed at room temperature) were then 
added to each flask to initiate the bioconversion. The contents of the 
flasks were stirred via a magnetic stirrer. 
Samples were removed periodically from the bioconversion mixtures for 
analysis of the ribavirin concentration. The concentration in the 
cell-free solution was determined. The initial reaction rate calculated 
over the first hour of the bioconversion, the cell-free ribavirin 
concentration and conversion after seven hours of reaction, and the final, 
cell-free ribavirin concentration and conversion (measured after 20 to 27 
hours) are shown in Table II as a function of the reaction temperature and 
initial reaction pH. The conversion is expressed on a molar basis with 
respect to the initial concentration of guanosine in the cell-free 
solution. 
TABLE II 
______________________________________ 
Ribavirin Production as a Function of Temperature 
and pH with Guanosine as the Ribose Donor 
Initial Rate 
of Ribavirin 
Ribavirin 
Initial 
Temp. Production Conc. (g/L) 
% Conversion 
pH (.degree.C.) 
(g/L/Hr.) 7 Hr. Final 7 Hr. 
Final 
______________________________________ 
6.0 65 1.1 12.8 16.4 52 67 
70 1.0 6.8 7.8 28 68 
75 0.3 0.3 4.0 1 1 
6.8 65 2.8 14.0 16.8 57 69 
70 2.6 16.6 17.2 68 70 
75 2.8 4.0 4.0 16 16 
7.6 65 3.5 10.6 14.5 43 59 
70 4.6 15.0 16.8 61 69 
75 4.1 6.4 7.0 26 29 
8.4 65 3.7 14.2 17.0 58 70 
70 4.6 14.6 16.0 60 66 
75 3.3 6.0 6.0 25 25 
9.2 65 3.2 14.0 16.5 57 68 
70 3.8 11.4 14.2 47 58 
75 2.4 3.2 3.0 13 12 
______________________________________ 
EXAMPLE 2 
Comparison of Bioconversion Performance Using Various Nucleosides at 
70.degree. C. and pH 7.2 
Bioconversion mixtures were prepared as in Example 1, except that the 
ribose donor was varied. Bioconversions were tested using five different 
nucleosides. All bioconversions were operated at 70.degree. C. and an 
initial pH of 7.2. The results are given in Table III. The conversion is 
expressed on a molar basis with respect to the initial concentration of 
nucleoside in the cell-free solution. The final conversion was determined 
at 28.25 hours. 
TABLE III 
______________________________________ 
Ribavirin Production as a Function of Ribose Donor 
Initia1 Rate 
of Ribavirin 
Ribavirin 
Production 
Conc. (g/L) % Conversion 
Ribose Donor 
(g/L/Hr.) 6.5 Hr. Final 6.5 Hr. 
Final 
______________________________________ 
Adenosine* 
5.6 7.4 9.6 30 39 
Guanosine 4.3 16.2 18.1 66 74 
Inosine* 1.4 5.7 10.3 23 42 
Cytidine* 0 0 0 0 0 
Uridine* 2.3 2.3 2.3 9 9 
______________________________________ 
*Comparison 
EXAMPLE 3 
Gel Prevention and Cell Washing 
(A) Comparison 
Bioconversion media were prepared as in Example 1, except the amounts of 
guanosine and 1,2,4-triazole-3-carboxamide were 20, 40, or 100 millimoles. 
The two reactants were used in a 1:1 molar ratio. The initial pH of each 
flask was adjusted to pH 7.2 with potassium hydroxide. 
The flask with 100 millimoles of the two reactants gelled as it was heated 
to 70.degree. C.--before any cell paste was added. The flask with 40 
millimoles of the two reactants also gelled, but required a few minutes 
longer. Agitation via the stir bar was ineffective in reversing the 
gelling in both cases. 
(B) 
Bioconversion media were prepared as in Example 1, except the amounts of 
guanosine and 1,2,4-triazole-3-carboxamide were varied from 20 to 40 
millimoles. The two reactants were used in a 1:1 molar ratio. The initial 
pH of each flask was adjusted to pH 7.2 with potassium hydroxide. The 
bioconversions were initiated and operated as in Example 1, except that 
the temperature was 70.degree. C. The cells were added promptly as the 
temperature reached 70.degree. C. to avoid gelling of the bioconversion 
mixture. 
The final conversion was determined at 24 hours. Each bioconversion broth 
was centrifuged, and samples were taken from the supernatant for 
determination of the final, cell-free ribavirin concentration. 
The cell paste from each bioconversion (about 40 grams) was re-suspended in 
200 mL water. These solutions were stirred at 22.degree. C. for 1 hour to 
extract ribavirin from the cells. The solutions were then centrifuged, and 
samples were taken from the supernatant for determination of the extracted 
ribavirin. The extraction process was repeated a second time using fresh 
water and the cell paste from the first extraction. 
The initial reaction rate calculated over the first hour of the 
bioconversion, the cell-free ribavirin concentration and conversion after 
seven hours of reaction, and the final, cell-free ribavirin concentration 
and conversion, are shown in Table V as a function of the initial 
guanosine concentration in the cell-free solution. The increase in the 
final conversion obtained by extracting ribavirin from the cells is shown 
in Table VI. 
TABLE V 
______________________________________ 
Ribavirin Production as a Function of Initial 
Guanosine and 1,2,4-triazole-3-carboxamide 
Concentrations Between 100 and 200 mM 
Initial Rate 
Initial of Ribavirin 
Ribavirin 
Guanosine Production Conc. (g/L) 
% Conversion 
Concen. (mM) 
(g/L/Hr.) 7 Hr. Final 7 Hr. Final 
______________________________________ 
100 3.9 14.8 16.8 61 70 
125 4.5 18.8 20.9 62 70 
150 5.4 22.7 25.1 62 70 
175 5.4 25.7 28.9 60 68 
200 5.5 28.3 32.8 58 68 
______________________________________ 
TABLE VI 
______________________________________ 
Impact of Extraction of Ribavirin from 
Cells on the Final Conversion 
Initial Final Conversion (%) 
Guanosine No One Two 
Concen. (mM) 
Extraction Extraction 
Extractions 
______________________________________ 
100 70 78 79 
125 70 79 80 
150 70 79 80 
175 68 77 78 
200 68 77 79 
______________________________________ 
EXAMPLE 4 Accumulation of Ribavirin to a High Concentration Using a 
Fedbatch Bioconversion 
Bioconversion media were prepared as in Example 1, except the initial 
amounts of guanosine and 1,2,4-triazole-3-carboxamide were 40 millimoles. 
The initial pH of each flask was adjusted to pH 7.2 with potassium 
hydroxide. The bioconversions were initiated as in Example 1, except that 
the temperature was 70.degree. C. The cells were added promptly as the 
temperature reached 70.degree. C. to avoid gelling of the bioconversion 
mixture. Forty millimole additions of both guanosine and 
1,2,4-triazole-3-carboxamide were made at 6 and 12 hours into the 
bioconversion. Thus after the second addition, a total of 120 millimoles 
of the two reactants had been added. Potassium hydroxide was added at 6 
hours to adjust the pH from 6.7 to 7.2. 
The cell-free ribavirin concentration, the conversion based on guanosine, 
and the overall reaction rate at 6, 12, and 24 hours are shown in Table 
VII. 
TABLE VII 
______________________________________ 
Performance of the Fed-batch Bioconversion 
Time Ribavirin Average Production 
(Hrs) Conc. (g/L) Rate (g/L/Hr.) 
% Conversion 
______________________________________ 
0 0 0 0 
6 25.8 4.3 53 
12 56.7 4.7 58 
24 94.9 4.0 65 
______________________________________ 
EXAMPLE 5 Batch Conversion with 500 mM Guanosine and 
1,2,4-triazole-3-carboxamide 
A bioconversion medium was prepared by combining 10 millimoles of guanosine 
and 10 millimoles of 1,2,4-triazole-3-carboxamide with 10 millimoles of 
KH.sub.2 PO.sub.4 in 100 mL of water in a 500 mL flask at 70.degree. C. 
Forty grams of cell paste were then added, followed next by 90 millimoles 
of guanosine and 90 milimoles of 1,2,4-triazole-3-carboxamide and finally 
by 10 millimoles of KH.sub.2 PO.sub.4 in 100 mL water at room temperature. 
The reaction mixture was vigorously stirred during this preparation with a 
top-driven laboratory stirrer. 
The resulting reaction mixture was of a pasty consistency but did not gel. 
Production of ribavirin was similar to that of Example 4. 
The invention has been described in detail with particular reference to 
preferred embodiments thereof, but it will be understood that variations 
and modifications can be effected within the spirit and scope of the 
invention.