Biosynthesis of anti-HCV compounds using thermophilic microorganisms

Article abstract of DOI:10.1016/j.bmcl.2012.08.045

This work describes the application of thermophilic microorganisms for obtaining 6-halogenated purine nucleosides. Biosynthesis of 6-chloropurine- 2′-deoxyriboside and 6-chloropurine riboside was achieved by Geobacillus stearothermophilus CECT 43 with a conversion of 90% and 68%, respectively. Furthermore, the selected microorganism was satisfactorily stabilized by immobilization in an agarose matrix. This biocatalyst can be reused at least 70 times without significant loss of activity, obtaining 379 mg/L of 6-chloropurine-2′-deoxyriboside. The obtained compounds can be used as antiviral agents.

Full text of DOI:10.1016/j.bmcl.2012.08.045

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6059–6062
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biosynthesis of anti-HCV compounds using thermophilic microorganisms
a
a
b
a
a,
⇑
Cintia W. Rivero , Eliana C. De Benedetti , Jorge E. Sambeth , Mario E. Lozano , Jorge A. Trelles
a
Laboratorio de Investigaciones en Biotecnología Sustentable (LIBioS), Universidad Nacional de Quilmes. Roque Saenz Peña 352, Bernal (B1868BXD), Argentina
Centro de Investigación y Desarrollo en Ciencias Aplicadas Dr. Jorge J. Ronco. Universidad Nacional de La Plata.47 N° 257, La Plata (B1904CMC), Argentina
b
a r t i c l e i n f o
a b s t r a c t
Article history:
This work describes the application of thermophilic microorganisms for obtaining 6-halogenated purine
nucleosides. Biosynthesis of 6-chloropurine-2 -deoxyriboside and 6-chloropurine riboside was achieved
Received 21 June 2012
Revised 10 August 2012
Accepted 13 August 2012
Available online 21 August 2012
0
by Geobacillus stearothermophilus CECT 43 with a conversion of 90% and 68%, respectively. Furthermore,
the selected microorganism was satisfactorily stabilized by immobilization in an agarose matrix. This
biocatalyst can be reused at least 70 times without significant loss of activity, obtaining 379 mg/L of 6-
0
chloropurine-2 -deoxyriboside. The obtained compounds can be used as antiviral agents.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Geobacillus stearothermophilus
Nucleoside analogues
Green chemistry
Entrapment immobilization
It is known that 6-halogenated purine nucleosides and their
derivatives play an important role in antiviral therapies. The activ-
ronmentally friendly methodology. These compounds are used as
antiviral compounds, prodrugs or antileukemic agents.
0
ity of 6-chloropurine-2 -deoxyriboside and 6-chloropurine riboside
The ability of Geobacillus stearothermophilus to hydrolyze uri-
dine (Urd) and thymidine (dThd) was evaluated in order to identify
the optimal strain for 6-halogenated purine nucleoside biosynthe-
sis (Fig. 1A). G. stearothermophilus CECT 43 showed the highest
activity for both nucleosides tested. Hydrolysis was quantitatively
evaluated by HPLC analysis, which was performed on Nucleodur
1
2
against hepatitis C virus (HCV) and SARS coronavirus (SARS Co-V)
has already been demonstrated. Moreover, the antitumoral activity
3
of 6-chloropurine riboside has been assayed on leukemic cells.
Nucleoside analogues are principally synthesized by chemical
methods. Such methods require organic solvents, multiple reaction
steps and the removal of protective groups, yielding undesirable
100-5 C18 column (5
l
m, 125 mm ꢀ 5 mm) using a UV detector
4
racemic mixtures, decreasing conversion and affecting product
(254 nm). The column was operated using isocratic mobile phase
water/methanol (90:10, v/v) at a flow rate of 1.2 ml/min.
When hydrolysis of other sugar donors was evaluated (Fig. 1B),
purification. However, biocatalysis emerged as an alternative to
these drawbacks owing to its high catalytic efficiency, inherent
selectivity and simple downstream processing. Biosynthesis of
0
G. stearothermophilus CECT 43 was able to hydrolyze 93% of 2 -
6
-modified purine nucleosides by transglycosylation using
deoxyuridine (dUrd) after 2 h of reaction. On the other hand, no
5
microorganisms was previously reported. These reactions can be
performed stereo-selectively by nucleoside phosphorylase (NP)
enzymes.
significant activity was detected when uracil 1-b-D-arabinofurano-
0
0
side (araUra) or 2 ,3 -dideoxyuridine (ddUrd) were evaluated at
short reaction times (2 h). Therefore, subsequent assays were per-
formed using dUrd as nucleoside sugar donor.
6
Thermophilic microorganisms are a source of enzymes with ex-
7
9
treme stability. The application of these microorganisms as bio-
It is known that G. stearothermophilus has NPs. These enzymes
catalysts is attractive for industrial bioprocesses.
catalyze reversible phosphorolytic cleavage of N-glycosidic bonds
of nucleosides to form a free base and its respective activated pen-
tose moiety, which is then coupled to the desired modified base to
yield a nucleoside analogue. These enzymes are involved in a
nucleoside salvage pathway and their expression during different
growth phases may vary. For this reason, 6-chloropurine-2 -
deoxyriboside (6ChPurdRib) biosynthesis was evaluated at differ-
ent stages of microorganism growth (Fig. 2). All reactions were
quantitatively analyzed by HPLC as described above and product
characterization was performed by MS-HPLC using a LCQ-DECA-
XP4 Thermo Finnigan Spectrometer with the Electron Spray Ioniza-
tion method (ESI) and one ion trap detector. The experimental
Microorganism stabilization by immobilization techniques is a
good method to carry out biotransformations because it allows
high operational stability, easy upstream separation and bioproc-
ess scale-up feasibility. Cell entrapment techniques are the most
widely used for whole cell immobilization.8
6
0
The present work describes an efficient one-pot biosynthesis of
6
-halogenated purine nucleosides using a smooth, cheap and envi-
⇑
Corresponding author. Tel.: +54 1143657100x5645; fax: +54 1143657132.
E-mail address: jtrelles@unq.edu.ar (J.A. Trelles).
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.045

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C. W. Rivero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6059–6062
10
Figure 1. Selection of G. stearothermophilus strain for 6-halogenated purine nucleosides biosynthesis. Hydrolysis reactions were carried out three times using 1 ꢀ 10 CFU
(
colony forming units), 2 mM nucleoside in 0.5 ml of potassium phosphate buffer (30 mM, pH 7) at 55 °C and 200 rpm. The reaction medium without microorganisms was
⁄
used as control. (A) Hydrolysis of dThd (white) and Urd (gray) using different G. stearothermophilus strains at 1 h of reaction. Significant differences when dThd ( P <0.001) and
Urd ( P <0.001) were used. (B) Hydrolysis of different sugar donors: dThd (triangle), Urd (square), dUrd (circle), araUra (inverted triangle) and ddUrd (rhombus) using G.
stearothermophilus CECT 43. Thermophilic microorganisms were kindly supplied by the ‘Colección Española de Cultivos Tipo (CECT)’, Universidad de Valencia (Spain).
⁄⁄
Figure 3. Biosynthesis of 6ChPurdRib at different temperatures. Reactions were
carried out three times in 0.5 ml of potassium phosphate buffer (30 mM, pH 7)
1
0
during 2 h using 1 ꢀ 10
CFU, 2 mM 6ChPur and 6 mM dUrd at 200 rpm.
Figure 2. Biosynthesis of 6ChPurdRib at different growth stages. G. stearothermo-
philus was grown at 55 °C in media contained 10 g/L meat peptone, 5 g/L yeast
Conversion was calculated as: (mmol product/mmol limiting reagent)
Significant differences respect to 30 and 45 °C: P <0.001.
⁄
100.
⁄
1
0
extract, 5 g/L NaCl and 4 g/L glucose at pH 7, and 1 ꢀ 10 CFU were collected at
several times. Biosynthesis was carried out during 2 h of reaction in 0.5 ml of
potassium phosphate buffer (30 mM, pH 7) at 55 °C and 200 rpm using 2 mM
6
ChPur and 6 mM dUrd as substrates. All reactions were performed three times and
conversion was calculated as: (mmol product/mmol limiting reagent)
Significant differences respect to the other growth times: P <0.001.
⁄
100.
tional facility requirements, the selected temperature for subse-
quent trials was 30 °C.
⁄
Different reaction variables were tested in order to optimize
other reaction parameters for obtaining 6-halogenated purine
nucleosides. The importance of the presence of phosphate in the
reaction for nucleoside phosphorolysis by NP has been previously
conditions were: voltage source 5.0 kV, capillary voltage 14 V, gas
1
1
flow rate (35 arbitrary units), temperature 200 °C. For 6ChPurdRib
reported. Preliminary tests were performed to optimize different
phosphate concentrations (20, 30 and 40 mM), pH values (5, 6, 7
and 8) and stirring speed (100, 200 and 300 rpm). No significant
differences were observed among several parameters (see Supple-
mentary data). Therefore, we decided to use 30 mM potassium
phosphate buffer at pH 7 and 200 rpm as standard conditions.
It has been widely reported that transglycosylation reactions
+
(
M : 272.6) biosynthesis, an isocratic water/methanol (0.1% acetic
acid) (85:15 v/v) mobile phase was used at a flow rate of 200
min.
lL/
The best conversion was achieved with cultures at early station-
ary phase (8 h). Therefore, for the following assays, we grew G. ste-
arothermophilus strain CECT43 to this phase.
1
2
Previously, we had shown that temperature influences enzyme
are reversible and it was shown that an excess of substrates im-
proves transglycosylation. We studied 6ChPurdRib biosynthesis in
the presence of an excess of 6-halogenated base, an excess of
nucleoside donor (dUrd) or equal molar quantities (Fig. 4). Lower
yields of 6ChPurdRib productivity were obtained when equimolar
quantities of substrates were used. Productivity was 0.63 mM/h
when 2:6 mM ratio (6ChPur/dUrd) was evaluated. However, when
an excess of 6-halogenated base was tested (6:2 mM ratio),
6ChPurdRib productivity was increased significantly (0.9 mM/h).
1
0
activity, thus its effect was evaluated on the conversion reaction.
Therefore, 6ChPurdRib biosynthesis was performed at 30, 45 and
5 °C (Fig. 3). The best conversion values were obtained at temper-
5
atures lower than the optimum temperature for growth of the
microorganism. Based on the obtained results, we have observed
hydrolase activity on the chlorine atom when the reaction was car-
ried out at high temperatures, decreasing 6ChPurdRib conversion.
As similar yields were obtained at 30 and 45 °C and due to opera-

C. W. Rivero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6059–6062
6061
Table 1
Biosynthesis of 6-halogenated purine nucleosides using G. stearothermophilus CECT 43
as biocatalyst
Sugar
donor
6-Halogenated
purine base
Reaction
time (h)
Conversionb
(%)
Productivity
(mM/h)
a
dUrd
Urd
dUrd
Urd
dUrd
Urd
6ChPur
6ChPur
6BrPur
6BrPur
6IPur
2
8
4
8
24
8
90
68
60
50
85
65
0.90
0.17
0.30
0.13
0.07
0.16
6IPur
10
Reactions were performed at least three times with 1 ꢀ 10 CFU at 30 °C in
potassium phosphate buffer (30 mM, pH 7) and 200 rpm using 6 mM base/2 mM
nucleoside ratio. 6ChPur: 6-chloropurine, 6IPur: 6-iodopurine and 6BrPur: 6-
bromopurine.
a
The best times of reaction are shown.
b
mmol product
Conversion (%) =
ꢁ 100.
mmol limiting reagent
Figure 4. 6ChPurdRib productivity using different initial molar ratios of substrates.
10
Reactions were performed during 1 h (white) and 2 h (gray) with 1 ꢀ 10 CFU at
3
0 °C in potassium phosphate buffer (30 mM, pH 7) and 200 rpm using 6ChPur and
0
activity (less than 50% of initial activity) before 12 reuses (see Sup-
plementary data).
We selected this biocatalyst to perform a preliminary test for
bioprocess scale-up. These trials were conducted in a 10 ml batch
reactor in the conditions previously optimized and the results were
similar to those obtained at microscale.
dUrd at different ratios (base/2 -deoxyriboside). All reactions were performed three
times and productivity was calculated relative to the limiting reagent concentra-
tion. Significant differences at 1 h ( P <0.001) or 2 h ( P <0.001) of reaction respect
⁄
⁄⁄
to other ratios.
This difference could be due to an excess of 6ChPur that shifts the
equilibrium favoring 6ChPurdRib formation, since the modified
base competes more effectively against natural purines within
microorganisms.
Currently, environmental factor (E-factor) values to produce
these compounds by pharmaceutical companies vary from 25 to
1
5
1
00. However, the use of safe microorganisms, the characteristics
of the matrix and the simple recovery of excess substrates favor E-
factor decrease more than 10-fold.
Once the reaction parameters were optimized, we evaluated the
0
biosynthesis of different 6-halogenated purine ribo- and 2 -deoxy-
In this report, a new green bioprocess for 6-halogenated purine
nucleosides production has been described by direct transglycosy-
lation using G. stearothermophilus CETC 43 immobilized in an agar-
ose matrix. This biocatalyst meets the requirements of high
activity, stability and short reaction times needed for low cost pro-
duction in a future preparative application using an environmen-
tally friendly methodology.
ribosides using 6-chloro, 6-bromo and 6-iodopurine as starter pur-
ine bases and dUrd or Urd as sugar donors. Quantitative analysis
was carried out by HPLC and product identification was performed
by MS-HPLC under the above mentioned conditions. 6ChPurRib
+
+
+
(
M : 287.5), 6IPurdRib (M : 327.1), 6IPurRib (M : 343.0), 6BrPur-
+
+
dRib (M : 316.2) and 6BrPurRib (M : 332.2). We were able to obtain
acceptable productivity for subsequent bioprocess scale-up. How-
ever, a higher conversion was obtained when dUrd was used as su-
0
Acknowledgments
gar donor instead Urd, because 2 -deoxyriboside hydrolysis was
greater. G. stearothermophilus strain CECT43 was able to synthesize
0
This research was supported by Agencia Nacional de Promoción
Científica y Tecnológica and Universidad Nacional de Quilmes. J A
T, J E S and M E L are research members at CONICET; C W R and
E C D are CONICET fellows, Argentina.
6
-halogenated purine ribosides and 2 -deoxyribosides with a con-
version greater than 50% in all cases (Table 1). The presence of
two PNPs (PNPI and PNPII) in G. stearothermophilus could account
9
for the acceptance of different purine bases. In conclusion, we have
been able to prepare a broad spectrum of different 6-halogenated
purine nucleoside analogues reaching significant productivities.
Entrapment techniques are widely used for microorganism sta-
bilization and allow reuse of biocatalyst. Different agarose (2%, 3%,
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.
045.
4
% and 5%) and polyacrylamide (15%, 20% and 25%) concentrations
were assayed for G. stearothermophilus immobilization as previously
described by Rivero and col.13 The minimum matrix percentage for
preventing microorganism release into the reaction medium was
assessed, being 4% and 25% the optimum percentages for agarose
and polyacrylamide immobilization, respectively. This thermophilic
microorganism was successfully immobilized in agarose and polya-
crylamide, obtaining 70% (at 6 h) and 61% (at 24 h) of 6ChPurdRib
conversion, respectively. It was observed that the immobilized bio-
catalysts required longer time than free microorganisms to reach
successful conversion values and it is well known that this differ-
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