Ribavirin: Biotechnological synthesis and effect on the reproduction of Vaccinia virus

Article abstract of DOI:10.1023/B:RUBI.0000049772.18675.34

The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied on the culture of Vero cells. The combination of ribavirin and vidarabin was shown to provide the antiviral effect at lesser concentrations than with these compounds taken separately.

Full text of DOI:10.1023/B:RUBI.0000049772.18675.34

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 6, 2004, pp. 553–560. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 6, 2004, pp. 613–620.
Original Russian Text Copyright © 2004 by Konstantinova, Leont’eva, Galegov, Ryzhova, Chuvikovskii, Antonov, Esipov, Taran, Verevkina, Feofanov, Miroshnikov.
Ribavirin: Biotechnological Synthesis and Effect
on the Reproduction of Vaccinia Virus
,
1
I. D. Konstantinova* , N. A. Leont’eva**, G. A. Galegov**, O. I. Ryzhova*,
D. V. Chuvikovskii*, K. V. Antonov*, R. S. Esipov*, S. A. Taran***, K. N. Verevkina***,
S. A. Feofanov***, and A. I. Miroshnikov*
*
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
*
*Ivanovskii Institute of Virology, Russian Academy of Medical Sciences, ul. Gamalei 16, Moscow, 123098 Russia
*
**Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Pushchino Branch), Russian Academy of Sciences,
pr. Nauki 6, Pushchino, Moscow oblast, 142292 Russia
Received July 17, 2003; in final form, November 17, 2003
Abstract—The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of
immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of rib-
avirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was
studied on the culture of Vero cells. The combination of ribavirin and vidarabin was shown to provide the anti-
viral effect at lesser concentrations than with these compounds taken separately.
Key words: 6-azauridine, immobilized enzymes, purine nucleoside phosphorylase, ribavirin, uridine phospho-
rylase, Vaccinia virus, vidarabin
1
INTRODUCTION
Modified nucleosides whose structures are similar
medical practice. An outstanding role in the herpes
therapy play acyclic guanine-containing nucleosides,
e.g., acyclovir (acycloguanosin). Ribavirin (1-β-D-
ribofuranosyl-1,2,4-triazol-3-carboxamide) that exhib-
its a wide spectrum of antiviral activities toward a great
number of RNA and DNA viruses is used in systemic
therapy of a number of distributed viral infections of
humans, such as viral hepatitis C and the respiratory-
syncytial and adenoviral infections [1, 2].
to those of intermediate products of DNA and RNA
biosynthesis constitute a large group of antiviral prepa-
rations. The mechanism of antiviral action of the
purine and pyrimidine analogues consists in their inclu-
sion into the structure of growing viral DNA instead of
natural nucleosides in the composition of the corre-
sponding nucleotides. In a number of cases, this results
in the inhibition of DNA and RNA polymerases, stop-
2
The human smallpox virus does not circulate in
page of the growth of the chain of viral nucleic acid, nature for more than 20 years and is not dangerous from
and, consequently, the loss of its virulence.
the epidemic and medicinal points of view. Neverthe-
less, the representatives of pox viruses are being inter-
esting not only for researchers. Currently, the probabil-
ity of using some single orthopox viruses highly patho-
genic for humans as the weapon of aggression
A pronounced inhibiting effect toward the DNA-
containing viruses, including the viruses of herpes sim-
plex and pox and adenoviruses, exhibits a group of
modified nucleosides: 5-bromo-2'-deoxyuridine, 1-β-
D-arabinofuranosyladenine, and AraA; they are used in
(
bioterrorism) seems not to be purely utopian. The
effective therapeutics for the orthopox virus infections
are some known nucleosides: ribavirin, AraA, cidofovir
1
Corresponding author; phone: +7 (095) 330-7247; e-mail:
kid@kou.ibch.ru
[
(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cyto-
2
sine, vistide], nucleotides, isatin β-semicarbazones,
Abbreviations: AraA, vidarabin (9-β-D-arabinofuranosylade-
nine); AzUr, 6-azauridine; CE , 50% cytotoxic infectious dose and recombinant α-interferon [3–5]. The combined
5
0
for the noninfected Vero cells; CEF, chicken embrio fibroblast;
action of antipox-viral compounds on an adequate
model of the Vaccinia virus is only slightly studied. We
believe that the application of combinations of real anti-
CPE, viral cytopathogenic effect; IE and IE , doses of prepara-
5
0
90
tions inhibiting viral cytopathogenic effect by 50 and 90%; PFU,
plaque-forming unit; Puo-phosphorylase, a purine nucleoside
phosphorylase obtained using the genetic engineering overpro- pox-viral drugs (e.g., a combined use of modified
ducing Escherichia coli strain BL21(DE3)/pERPUPHO1; TCA,
nucleosides and nucleotides and α-interferon) could be
1
,2,4-triazol-3-carboxamide; TCID , 50% tissue cytotoxic infec-
50
promising in the medicinal control of the human poxvi-
ral infection. Our study is devoted to studying the
action of ribavirin and its combinations with AraA and
tious dose; and Urd-phosphorylase, uridine phosphorylase from
the genetic engineering overproducing E. coli strain
BL21(DE3)/pERUPHO1.
1
068-1620/04/3006-0553 © 2004 MAIK “Nauka /Interperiodica”

5
54
KONSTANTINOVA et al.
AzUr on the reproduction of Vaccinia virus in the Vero glycosylation by means of immobilized nucleoside
cell culture.
A steady growth and occurrence of human viral dis-
phosphorylases obtained by genetic engineering.
eases and the practically proven efficiency of the
medicinal use of medicines on the basis of modified
nucleosides provide for a high interest to the develop-
ment of modern biotechnological methods of manufac-
turing of antiviral preparations of a new generation for
their wide use in practical medicine.
RESULTS AND DISCUSSION
A Biotechnological Method
of Obtaining Modified Nucleosides
The modified nucleosides ribavirin,AraA, andAzUr
are used in the modern medicinal practice for the com-
In this connection, we describe in this work an bined therapy of viral infections and the treatment of
improved highly efficient method of the preparation of particularly dangerous diseases of humans, such as
ribavirin and some other modified analogues of nucle- bronchiolitis in children of the first life year, various
osides based on the reaction of microbiological trans- forms of herpetic illnesses, and chronic hepatitis C.
H
H
O
O
N
NH2
N
N
HN
N
N
N
N
N
N
O
O
O
HO
N
HO
HO
O
HO
HO
OH
Ribavirin
HO
HO
AzUr
OH
AraA
The chemical synthesis of modified nucleosides is a
In this work, we improved and optimized the tech-
complex and multistage process [6–9], the key stage of nological scheme of preparation of the preparations of
which is the glycosylation of a modified nucleic base ribavirin, AraA, and AzUr using the previously devel-
with a protected derivative of ribose (arabinose). The oped genetic engineering nucleoside phosphorylases:
performing of this reaction with selective chemical Puo-phosphorylase from the overproducing Escheri-
methods leads to the formation of a racemic mixture of chia coli strain BL21(DE3)/pERPUPHO1 (EC 2.4.2.1)
α and β anomers with 90–95% predominance of the [16, 17] and uridine phosphorylase (Urd phosphory-
natural β anomer. The separation of minor impurities of lase) from the overproducing E. coli strain
the unnatural α anomer is a rather laborious process, BL21(DE3)/pERUPHO1 (EC 2.4.2.3) [18]. They were
which is also complicated by the necessity of removal immobilized on an aminopropylated macroporous
of protective groups. Therefore, the number of stages in glass AP-CPG-170 (Sigma, United States). The use of
the chemical methods of synthesis of modified nucleo- these immobilized enzymatic preparations significantly
sides is varied on the average from five to nine, and the simplifies the technology of preparation of the modified
total yields of the target products are consequently nucleosides and allows a multiply repeated use of the
reduced.
An alternative method of the synthesis of natural
catalytic activity of the enzymatic preparations (up to
1
5 manufacturing cycles).
and modified nucleosides is based on the reaction of
microbiological transglycosylation, which is achieved
by the use of nucleoside phosphorylases (Scheme 1)
The enzymes were immobilized on the aminopropy-
lated macroporous glass AP-CPG-170 by glutaric dial-
dehyde by the method [19]. A covalent bond between
the aldehyde groups and the amino groups of the sor-
bent and proteins is formed in the presence of sodium
borohydride [20].
[
10–13]. A transfer of a carbohydrate residue from a
natural purine or pyrimidine nucleoside to a natural or
a modified heterocyclic base occurs during the reaction.
All the known works devoted to the preparation of
modified nucleosides by chemoenzymatic methods
The synthesis of ribavirin was further carried out by
used bacterial strains of microorganisms, obtained by a Scheme 2. The presence of inorganic phosphate is nec-
microbiological selection, as a source of nucleoside essary in the reaction of enzymatic transglycosylation
phosphorylases. It is the intact cells of these microor- for the efficient formation of the intermediate α-D-ribo-
ganisms of the enzymes themselves isolated from the syl-1-phosphate [11]. The optimal conditions for the
bacteria that served as catalysts of the biochemical reaction are: 60°ë and pH 7.0. The characteristics of the
transformations [10–15].
transglycosylation reaction in the synthesis of modified
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

RIBAVIRIN: BIOTECHNOLOGICAL SYNTHESIS
HO
555
HO
B
B'
O
HO
O
O
E
E, B'
Y
O
Y
X
Y
B
O P O–
OH
X
OH
OH
OH
X
where X = OH, Y = H, B = Gua, B' = 1,2,4-triazol-3-carboxamide;
X = H, Y = OH, B = Ura, B' = Ade;
X = OH, Y = H, B = Ade, B' = 6-azauracil;
E – Puo/Urd phosphorylases.
Scheme 1.
nucleosides are more comprehensively described in virus, logBFU/ml, was determined; see figure) demon-
Table 1.
strated that the combination of the two modified nucle-
osides is effective and a synergic antiviral effect was
observed even after a four times decrease in the ribavi-
rin concentration and two times decrease in AraA con-
centration.
We demonstrated a possibility of obtaining the mod-
ified nucleosides AraA and AzUr using a similar
scheme and a binary system of immobilized Puo- and
Urd-phosphorylases.
Thus, an efficient biotechnological method of prep-
aration of a number of modified nucleosides using
nucleoside phosphorylases obtained by genetic engi-
neering and immobilized on an aminopropylated
macroporous glass. The simplicity and versatility of
this approach allow scaling up the process and prepare
development batches of modified nucleosides for bio-
logical testing.
The results allow one to predict a basic possibility of although their combinations inhibit CPE at lower con-
the development of efficient contemporary technologi- centrations. This is also true in respect of the Vaccinia
cal sets for the synthesis of a number of modified nucle- virus resistant to 5-iodo-2'-deoxyuridine. The combina-
osides with the use of immobilized nucleoside phos-
phorylases obtained by genetic engineering.
The development of the viral infection was deter-
mined from the value of virus-induced CPE (Table 2).
The antiviral activity of the studied compounds (indi-
vidually or in combination) was evaluated according to
their ability to inhibit the development of the virus-
induced CPE by 50% (IE ) and almost completely
5
0
(
IE ). Table 2 shows that the three modified nucleo-
90
sides protect cells and prevent the development of CPE,
tions of ribavirin and AraA turned out to be the most
effective, whereas the efficiencies of AzUr and AraA
combinations were less pronounced. On the basis of the
mechanism of action of AraA and its 5'-phosphate, one
could conclude that the reproduction of Vaccinia virus
could be destroyed directly at the stage of synthesis of
The Study of Antiviral Activity
of Modified Nucleosides
The results of studying the effects of the synthesized viral DNA [21]. At the same time, literature data sug-
ribavirin, AraA, and their combinations on the repro- gest that ribavirin (in the form of its 5'-triphosphate)
duction of Vaccinia virus (the infection titer of the decreases the translation efficiency of mRNA of the
Table 1. The characteristics of transglycosylation reaction in the synthesis of modified nucleosides
Substrates for enzymatic reaction
60°C, pH 7.0)
Reaction
Content of the target
product*, %
Immobilized enzyme
Puo-phosphorylase
Yield**, %
68
(
time, days
Guo (60 mM)
TCA (40 mM)
KH PO (50 mM)
1.5
0.2
1
Ribavirin, 92
2
4
6
-Azauracil (3 mM)
Puo/Urd-phosphory-
lases
6-Azauridine, 19.3
Vidarabin, 45.7
45
56
Adenosin (10 mM)
KH PO (0.4 mM)
2
4
1
-β-D-Arabinofuranosyluracil (5 mM)
Puo/Urd-phosphory-
lases
Adenine (6 mM)
KH PO (5 mM)
2
4
*
HPLC-monitoring of incubation mixture.
*
* After purification (see the Experimental section).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

5
56
KONSTANTINOVA et al.
combination of ribavirin and AraA provides an intensi-
O
N
fication of the antiviral effect at lower concentrations
than those of individual compounds. Ribavirin and
AraA effectively inhibit the reproduction of the Vac-
cinia virus variant resistant to 5-iodo-2'-deoxyuridine.
This effect is also retained at the use of combinations of
these modified nucleosides at lower concentrations. It
is real to use a combination of the two compounds with
different mechanisms of action on the reproduction of
Vaccinia virus also for the possible treatment of ortho-
pox-viral infection of humans.
N
HN
N
H N
2
HO CH2
O
H NOC C
N
2
+
N
C H
N
HO
OH
N
H
Guo
1,2,4-triazol-3-carboxamide
A high sensitivity of the reproduction of pox viruses
to α-interferon makes it topical to study its antiviral
activity in combination with ribavirin and AraA. This
might be an optimal scheme of treatment of the pox-
viral infection in humans. At the same time, a combina-
tion of α-interferon and ribavirin has been turned out to
be efficient at the treatment of the widely distributed
viral hepatitis C; this combination could also be prom-
ising for the therapy of the recent outbreak of the RNA-
viral infection of atypical pneumonia induced by a
crown virus.
O
C
E,
NH2
N
KH PO
O
2
4
HO CH2
N
O
N
+
HN
H N
N
H
N
2
OH OH
Ribavirin
Gua
E, immobilized Puo phosphorylase
EXPERIMENTAL
Scheme 2.
We used in this work domestic solvents and reagents
as well as some reagents from Sigma (United States)
and Chemapol (Czech Republic) and guanosine form
the Novopolotsk BVK plant (Belarus) without addi-
tional purification.
Vaccinia virus, because it precludes the formation of
cap-structure at the 5'-terminus of its RNA [22–24].
We also obtained a positive antiviral effect for the
ribavirin–AraA combination on the primary culture of
Substances were identified and their purity was
CEF. At the multiplicity of infection of 0.1 BFU per cell checked by TLC on the plates Kieselgel 60 F254 and
and the introduction of the compound directly after the
infection of cells, we obtained the following results (see
Table 3).
HPTLC Kieselgel 60 plates (Merck, Germany) in the
system 9 : 1 acetonitrile–water; spots were detected by
spraying with 70% H SO followed by heating.
2
4
It was shown that ribavirin biotechnologically
The homogeneity of substances, reaction course,
obtained in IBCh RAS efficiently inhibits the reproduc- and yields of products were monitored by HPLC on a
tion of Vaccinia virus in the Vero and CEF cell cultures Waters 740 chromatograph using an Altima (0.4 ×
at both a high and a low multiplicity of infection. The 25 cm) column filled with Nucleosil C18 5 µm sorbent.
Table 2. The development of the virus-specific CPE in the Vero cell cultures infected with the Vaccinia virus in the presence
of ribavirin, AraA, AzUr, and their combinations*
IE , µg/ml
IE , µg/ml
90
5
0
Series
Compound
Ribavirin
CE , µg/ml
50
VV
VV-R
VV
VV-R
I
3.0
2.0
3.0
2.5
3.0
5.0
–**
6.0
95
II
AraA
5.0
37
90
III
IV
V
AzUr
20
20
–**
Ribavirin/AraA
Ribavirin/AzUr
AraA/AzUr
0.22/0.22
3.0/3.5
3.75/1.0
0.22/0.5
3.0/3.5
3.75/1.5
0.5/0.5
7.5/7.0
7.5/2.0
0.5/0.5
7.5/7.0
7.5/3.0
≥>50/10
>25/25
>25/10
VI
*
Result 72 h after the cell infection. The multiplicity of infecting is 0.001 PFU/cell. VV, Vaccinia virus; VV-R, Vaccinia virus resistant
to 5-iodo-2'-deoxyuridine.
*
*Effect was not achieved.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

RIBAVIRIN: BIOTECHNOLOGICAL SYNTHESIS
557
Elution was carried out at a rate of 1 ml/min in the fol-
logPFU/ml
lowing solvent systems. System 1: water as solution A
and 1 : 1 acetonitrile–water as solution B, gradient of B
in A from 0 to 100% for 40 min, was used in the analy-
sis of ribavirin synthesis at the detection at 214 nm. Sys-
tem 2: water as solution A and 7 : 3 acetonitrile–water
as solution B, 7 min at 0% B and gradient of B in A
from 0 to 100% for 40 min, was used in the analysis of
AzUr synthesis at the detection at 254 nm. System 3:
water as solution A and 7 : 3 acetonitrile–water as solu-
tion B, gradient of B in A from 0 to 100% for 40 min,
was used in the analysis of AraA synthesis at the detec-
tion at 254 nm.
8
7
6
5
4
3
2
1
I
II
III
IV
0
6
12
18
24
36
Time after infection, h
Ion exchange chromatography was carried out using
Effect of ribavirin, AraA, and their combinations on the
reproduction of Vaccinia virus in the Vero cell culture.
Infection multiplicity 5 PFU/cell. The preparations were
added at the following concentrations (µg/ml): I, 0; II, rib-
avirin (20); III, AraA (5); IV, ribavirin (5) + AraA (2.5).
–
a column (3 × 9 cm) filled with Dowex 1x8 (OH ), 20–
5
(
0 mesh) and a column (2.2 × 12 cm) with Dowex 50x8
+
H , 400 mesh, Serva).
Column chromatography was performed on ë18
Octadecyl=Si 100polyol (0.03 mm) sorbent (Serva,
United States).
When the protein content in supernatant became
UV spectra were measured on a UV-160 Shimadzu ≤0.5 mg/ml, the sorption was stopped, the sorbent was
spectrophotometer (Japan), and IR spectra, on a Hitachi three times washed successively with 50 mM potas-
1
sium phosphate (pH 7.3, 60 ml) and 2 M KCl (20 ml)
and treated with a solution (20 ml) of sodium borohy-
dride (90 mg) in distilled water for 1 h at 4°ë. Then the
sorbent was three times successively washed with
2
70-30 spectrophotometer. H NMR spectra were reg-
istered on a Bruker DRX-500 instrument in D O. Mass
2
spectra (ESI) were obtained on a Finnigan 900 S spec-
trometer.
5
0 mM potassium phosphate (pH 7.3, 60 ml) and 2 M
KCl (20 ml), the quantity of nonsorbed protein was
determined in the washings, and the quantity of protein
immobilized on 1 g of sorbent was calculated as a dif-
ference.
Immobilized Enzymic Preparations
An aminopropylated macroporous glass AP-CPG-
1
70 (10 g, 125–225 µmol of primary amino groups per
g, 200–400 mesh) was successively washed with dis-
tilled water (40 ml), ethanol (80 ml), distilled water
The yield of wet sorbent was 30 g, and it contained
9
0 mg of Puo phosphorylase (3 mg of protein per 1 g of
(
8
40 ml), and 0.1 M potassium phosphate (pH 7.3,
0 ml). The prepared sorbent was wrung out from the
buffer on a glass filter, mixed with 5 vol % glutaric dial-
wet sorbent from the results of three experiments).
The enzymatic activity of the recombinant immobi-
dehyde solution in 0.1 M potassium phosphate (pH 7.3, lized Puo-phosphorylase was determined according to
2
50 ml), stirred for 10 h, and wrung out from the reac- its ability to cleave inosine to hypoxanthine. The quan-
tion mixture on a glass filter and washed with 0.1 M tity of enzyme capable of the conversion of 1 µmol
potassium phosphate (pH 7.3, 80 ml). The sorbent was inosine into hypoxanthine for 1 min at 37°ë was taken
then wrung out on a glass filter and weighted. The wet
sorbent was prepared immediately before the applica-
tion of enzymic preparations and used for a day.
Table 3. The effect of ribavirin, AraA, and their combinations
on the reproduction of Vaccinia virus in the CEF cell culture
A lyophilized powder of Puo-phosphorylase
obtained
from
an
overproducing
strain
Concentration, Infection titer of virus,
Compound
control)
BL21(DE3)/pERPUPHO1 of E. coli [16, 17] has a spe-
cific activity of 20 U/mg of protein; 1 U of the enzyme
converts 1 µmol of inosine into hypoxanthine in 50 mM
potassium phosphate, pH 7.3, for 1 min at 37°ë. The
Puo-phosphorylase preparation was dissolved in
µg/ml
logPFU/ml, after 48 h*
(
–
7.4
6.6
6.0
6.3
5.7
5.2
4.2
Ribavirin
15
2
5
3
6
5
0 mM KH PO buffer, pH 7.3, to get 30 ml of a solu-
2 4
AraÄ
tion with the protein concentration of 5 mg/ml (deter-
mined by the Lowry method [25]).
A solution of Puo phosphorylase was added to a wet
sorbent, and the mixture was kept on a shaker for 18–
Ribavirin + AraÄ
10 + 2
2.5 + 3
1
2
1 h at 4°ë, avoiding the formation of foam and moni-
toring the protein concentration in supernatant by tak-
* Multiplicity of infection is 0.1 PFU/cell. Compounds were added
immediately after the infection of cells.
ing 100-µl aliquots.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

5
58
KONSTANTINOVA et al.
as 1 U. The specific activity of the immobilized Puo- for 4.8 h at 60°ë. The process was monitored by HPLC;
phosphorylase under these conditions was no less than it was considered to be complete at the AzUr content in
1
5 U/mg of the immobilized protein.
the reaction mixture of 18–19%. The reaction mixture
was separated from the immobilized enzymic prepara-
tion by decantation, and the sorbent with the immobi-
lized enzymes was washed with 50 mM potassium
phosphate buffer (pH 7.0, 30 ml) and repeatedly used in
the process of AzUr preparation. The solution was
evaporated in a vacuum to a small volume and passed
through a layer of sorbent ë Octadecyl=Si 100polyol
The preparation of Puo-phosphorylase was stored at
4°C no longer than four weeks.
–
The immobilized Urd-phosphorylase was similarly
prepared from the enzyme isolated from the E. coli
overproducing strain BL21(DE3)/pERUPHO1 [18].
The specific activity of the immobilized Urd-phospho-
rylase was no less than 25 U/mg of the immobilized
protein.
18
(
0.03 mm). AzUr was eluted with water. The fractions
containing more than 95% of the target product were
1-b-D-Ribofuranosyl-1,2,4-triazol-3-carboxamide. collected and evaporated in a vacuum to dryness, dried
A mixture of guanosine (17.0 g, 0.06 mol) and 1,2,4-tri- in desiccator to a constant weight, and crystallized from
azol-3-carboxamide (4.48 g, 0.04 mol), and immobi- ethanol. The white crystalline powder was dried at
lized Puo-phosphorylase (30 g) was incubated in 50°ë for 5 h, to get AzUr,; yield 66 mg (45% from
5
0 mM KH PO buffer (pH 7.0, 1 l) for 36 h at 60°ë. 6-azauracil); the content of the main substance 99%
2
4
The process was monitored by HPLC; it was approved (HPLC in system 2 and a comparison with an indepen-
−1
–1
to be complete when the ribavirin content in the reac- dent standard); mp 159–161°ë; λ , nm (ε, å cm ):
max
tion mixture reached 86–92%. The immobilized enzy-
mic preparation was separated from the reaction mix-
ture by decantation, washed with 50 mM KH PO
2
62.3 (6 100).
AraA. A mixture of adenine (0.162 g (1.2 mmol),
-β-D-arabinofuranosyluracil [26] (0.24 g, 1.0 mmol),
2
4
1
(
pH 7.0, 60 ml), and repeatedly used for the preparation
and immobolilized Puo- (2 g) and Urd-phophorylases
of ribavirin no less than 15 times. The reaction solution
was cooled to +4°ë, and the precipitated guanine was
separated by filtration and washed with water (30 ml).
The filtrate was evaporated in a vacuum to a small vol-
(
0
3 g) in 5 mM potassium phosphate buffer (pH 7.0,
.2 l) was stirred for 24 h at 60°ë. The process was
monitored by HPLC; it was regarded as complete at the
AraA content in the reaction mixture of 45–46%. The
immobilized enzymic preparation was separated from
the reaction mixture by decantation, washed with
+
ume and passed through a layer of Dowex 50 (H ) to
remove the traces of heavy metals and inorganic cat-
ions; the crude ribavirin was washed out with water.
The eluate was evaporated in a vacuum to a small vol-
5
0 mM potassium phosphate buffer (pH 7.0, 30 ml) and
–
repeatedly used in the process ofAraA preparation. The
reaction solution was evaporated in a vacuum to a small
ume, and passed through a layer of Dowex 1 (OH ) to
purify from the admixtures of starting compounds
volume and passed through a layer of sorbent C Octa-
(guanosine and 1,2,4-triazol-3-carboxamide), inor-
18
decyl=Si 100polyol (0.03 mm). AraA was eluted with
water. The fractions containing more than 95% of the
target product were collected and evaporated in a vac-
uum to dryness, dried in desiccator to a constant
weight, and crystallized from methanol. The white
crystalline powder was dried at 50°C for 5 h, to get
AraA; yield 0.15 g (56% from 1-β-D-arabinofuranosy-
luracil); the content of the main substance 98% (HPLC
in system 3 and a comparison with an independent stan-
ganic phosphate, and traces of guanine. Ribavirin was
eluted with water, and the resulting aqueous solution
was evaporated in a vacuum to dryness (55°ë). The res-
idue was dried in a desiccator to a constant weight, and
the product was crystallized from ethanol with water
added (up to 10 vol %) to give a white crystalline pow-
der, which was dried at 100°ë for 5 h. The yield of the
product was 6.60 g (68%); the content of the main sub-
2
0
stance 99.9% (HPLC, system 1); [α] –36.2 (c 1,
D
–1
–1
dard); mp 257–258°ë; λmax, nm (ε, å cm ): 259
H O); mp 168–170°ë (lit. data: mp 166–168°C [6]); R
2
f
(13100).
–
1
–1
0
.48; λmax, nm (ε, å cm ): 205.6 (11 300–11 400);
MS, m/z: 245.5 [M + H] , 267.1 [M + Na] , 282.9 [M +
K] , 487.9 [2 M] , 510.0 [2 M + Na] , 526.0 [2 M + K] ;
H NMR (D é, δ, ppm, J, Hz): 8.80 (1 I, br. s, H5), 6.20
1 I, d, J 3.44, H1'), 4.71 (1 I, dd, J 3.44, J 5.27,
+
+
+
+
+
+
Antiviral Activity of Ribavirin
and Other Modified Nucleosides
1
2
(
1', 2' 2', 1' 2', 3'
The antiviral activity was studied using the biotech-
H2'), 4.55 (1 I, dd, J3', 2' 5.27, J3', 4' 5.27, H3'), 4.28 (1 I,
dt, J4', 3' 5.27, J4', 5‡' 3.21, J4', 5b' 5.27, H4'), 3.93 (1 I, dd,
H5a', J5a', 5b' 12.6, J5a', 4' 3.21, H5a'), and 3.82 (1 I, dd,
J5b', 5a' 12.6, J5b', 4' 5.27, H5b').
nological ribavirin preparation from IBCh RAS
obtained by the above-described procedure with the use
of immobilized enzymes. In some experiments, ribavi-
rin from ICN Pharmaceutical (United States) was used
AzUr. A mixture of adenosine (0.53 g, 1.9 mmol) as a standard for comparison. AzUr was obtained from
and 6-azauracil (0.068 g, 0.06 mmol), and immobilized the Institute of Organic Chemistry (Prague, Czech
Puo- (2 g) and Urd-phophorylases (3 g) in 0.4 mM Republic); AraA and 5-iodo-2'-deoxyuridine, from
potassium phosphate buffer (pH 7.0, 0.2 l) was stirred Sigma (United States).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

RIBAVIRIN: BIOTECHNOLOGICAL SYNTHESIS
559
Cells. A transferable culture of the Vero cells (the influenza
A
infection in the MDCK (strain
cells from kidney of African green marmosets) was H3N2/Aichi/68) cell culture were determined. The sup-
used. They were grown on a Eagle’s support medium port medium for the virus-infected cell culture included
MEM containing 10% fetal calf serum (PANEKO, Rus- trypsin. Both ribavirin preparations reduced the influ-
sia). The cells were cultured in 96-well plastic plates enza virus reproduction within the range of noncytotoxic
(
Linbro, Flow Lab., UK) in an atmosphere enriched concentrations 6–25 µg/ml by 2.15–3.1 logTCID /ml.
50
with CO2.
Virus. The strain 113 obtained from the Virus
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