Two-step efficient synthesis of 5-methyluridine via two thermostable nucleoside phosphorylase from Aeropyrum pernix

Article abstract of DOI:10.1016/j.bmcl.2011.12.142

5-Methyluridine has been synthesized in high yield using guanosine and thymine as starting materials in the presence of highly thermostable recombinant purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UP) obtained from hyperthermophilic aerobic crenarchaeon Aeropyrum pernix. Key reaction parameters such as pH, temperature, concentration of buffer and substrates were investigated. At the optimal conditions, 5-methyluridine was achieved in yield 85% with a guanosine conversion of 96% in 10 ml scale. The process can be performed at high temperature, which will highly increase the solubility of substrates, therefore, this process is suitable for the industry application.

Full text of DOI:10.1016/j.bmcl.2011.12.142

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2102–2104
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Two-step efficient synthesis of 5-methyluridine via two thermostable
nucleoside phosphorylase from Aeropyrum pernix
Shaozhou Zhu , Lu Ren , Jianjun Wang , Guojun Zheng a, , Pingwah Tang a
a
a
b,
⇑
⇑
a
State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Methyluridine has been synthesized in high yield using guanosine and thymine as starting materials in
the presence of highly thermostable recombinant purine nucleoside phosphorylase (PNP) and uridine
phosphorylase (UP) obtained from hyperthermophilic aerobic crenarchaeon Aeropyrum pernix. Key
reaction parameters such as pH, temperature, concentration of buffer and substrates were investigated.
At the optimal conditions, 5-methyluridine was achieved in yield 85% with a guanosine conversion of 96%
in 10 ml scale. The process can be performed at high temperature, which will highly increase the
solubility of substrates, therefore, this process is suitable for the industry application.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 31 October 2011
Revised 16 December 2011
Accepted 27 December 2011
Available online 10 January 2012
Keywords:
5
-Methyluridine
Nucleoside phosphorylase
Thermostable
Transglycosylation
5
-Methyluridine is a unique unnatural nucleoside. It is a key
starting material. However, such improvement did not result due
9
,10
intermediate in the synthesis of anti-AIDS drug such as stavudine
to the existing equilibrium in the reaction.
In order to push the
1
,2
(
d4T) and zidovudine (AZT).3
equilibrium forward, increasing the solubility of starting material,
reducing the concentration of released base and carrying out the
reaction at higher temperature were the better choices. For
example, in the presence of xanthine oxidase which reduced the
released base through oxidation the yield of 5-methyluridine
Conventional chemical methods for the synthesis of 5-methylu-
ridine always suffer from the complex protection and deprotection
steps, low stereoselectivity and the environmental concerns of the
process. Biosynthesis of nucleoside analogues has been proven to
be a practicable and favorable option, which are regiospecific, ste-
reospecific and environmentally friendly.
Several processes for enzymatic synthesis of 5-methyluridine
have been developed employing different kinds of enzymes. Using
-deoxyribose-5 -phosphate aldolase (DERA) and phosphopen-
4
1
1,12
increased to 76%.
In the past, we had successfully cloned a purine nucleoside phos-
phorylase and a uridine phosphorylase from Escherichia coli K-12.
The two nucleoside phosphorylases were abundantly expressed
0
0
2
after the induction by isopropyl-thio-b-D-galactoside (IPTG) in
tomutase (PPM), pyrimidine nucleosides were synthesized from
inexpensive sugars, such as glucose in the presence of acetalde-
hyde and nucleobase.
E. coli BL21 and large amount of target proteins were expressed in
a soluble form. Using an auxiliary enzyme such as xanthine oxidase
from Arthrobacter globiformis, the reaction system produces
5-methyluridine from inosine and thymine in yield 70%.
5
,6
An alternative way of obtaining 5-methyluridine is the
biosynthetic process catalyzed by nucleoside phosphorylases from
Compared with inosine, guanosine is a better start material to
synthesize 5-methyluridine because the released guanine in the
reaction (Fig. 1) is less soluble in water than hypoxanthine. Ishii
et al. reported that 5-methyluridine was synthesized in 74% yield
from guanosine and thymine using the whole cell of Erwinia
carotovora AJ-2992 with high nucleoside phosphorylase activity.13
Bode and co-workers developed a high yielding coupled enzymatic
reaction using Bacillus halodurans purine nucleoside phosphorylase
and E. coli uridine phosphorylase for synthesis of 5-methyluri-
7
microorganisms. The process of the enzymatic synthesis of
5
-methyluridine is depicted in Figure 1.
Inosine was first used as starting material to synthesize
-methyluridine. Hori et al. used immobilized purine nucleoside
5
phosphorylase and pyrimidine nucleoside phosphorylase from
Bacillus stearothermophilus JTS 859 to synthesize 5-methyluridine
8
in about 32% yield on a large scale. To improve the conversion yield,
further research was investigated by Hori et al. Various experimen-
tal conditions were conducted to improve the conversion of the
1
4
dine. The drawback of the process using guanosine as the starting
1
5
material was the low solubility of the substrates.
One of the key points of the present work was that two
promising highly thermostable nucleoside phosphorylases (PNP
and UP) from hyperthermophilic aerobic crenarchaeon Aeropyrum
⇑
Corresponding authors. Tel./fax: +86 10 64437507.
E-mail addresses: wangjj@im.ac.cn (J. Wang), zhenggj@mail.buct.edu.cn
(
G. Zheng).
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.142

S. Zhu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2102–2104
2103
Figure 1. Mechanism for synthesis of 5-methyluridine from guanosine.
pernix were found for the first time by our group.16 The two
enzymes were expressed in E. coli BL21 as shown in Figure 2.
Both PNP and UP were purified by one single heat treatment
step. Cells from the expression culture (total 1000 ml) were
suspended in phosphate buffer and sonicated. The cell-free extract
was then heated at 80 °C for 30 min and centrifuged 12000g for
1
5 min. Approximately 100 mg of the recombinant protein was
purified.
Small-scale assays were first performed to optimize the reaction
conditions. As the reaction temperature had significant influences
on the solubility of substrates and productivity of the 5-methyluri-
dine. We first investigated the effect of temperature on the produc-
tivity of 5-methyluridine. Reactions were initialed by adding 100
lg
PNP and 100 g UP into 500 l reaction medium containing 60 mM
l
l
guanosine and 120 mM thymine, with shaking at different temper-
ature. As expected, even at 90 °C, the 5-methyluridne yield still
maintained at a high level of 85% yield. The results showed that
the increasing reaction temperature had a negligible impact on
the productivity from 60 to 90 °C (Fig. 3). This reaction which is
benefited from the increasing solubility of guanosine and thymine,
therefore, can highly improve the productivity.
Next, the optimum reaction pH was studied at 90 °C. As shown
in Table 1, the reaction was carried out at pH 6.0–9.0. We found
that the optimum pH value of the reaction was 9.0. While UP
shows the optimum pH value at 9.0 past studies showed that
PNP exhibited an optimum pH value at 5.0 with a wide range of
solubility at different values of pH. These results suggest that UP
is a key enzyme in the reaction and the solubility of the substrates
increase at the optimum pH value.
Figure 3. Effect of temperature on productivity of 5-methyuridine.
Table 1
Effect of pH on the yields of 5-methyluridine at 90 °C
pH
Guanosine conversion (%)
5-methyluridine yield (%)
6
7
8
9
96.9
97.3
98.0
98.3
97.0
97.1
71.7
78.7
84.1
85
19
17
1
1
0
1
Table 2
Summary of the optimum reaction conditions
Parameters
Optimum
Temperature
90 °C
pH
9.0
Time
48 h
Substrate concentration
Phosphate buffer concentration
Mole ratio of substrate
Enzyme loading
50 g/L
20 mmol/L
Thymine to guanosine (2:1)
UP to PNP (1:2)
The optimum phosphate buffer concentration was investigated
ꢀ1
which showed that optimal concentration was 20 mmol L . The
parameters such as the mole ratio of guanosine and thymine
loading, mole ratio of UP and PNP were also investigated. The
results were summarized in the Table 2.
To demonstrate the overall reaction at a larger scale, an
experiment was conducted at 10 ml using an enzyme loaded with
10 mg PNP and 5 mg UP and a thymine to guanosine mole ratio of
Figure 2. Coomassie-stained gel after SDS–PAGE of expression proteins from
transformed bacteria by gentle lysis (A). M. protein marker; lane 1 E. coli BL21 (DE3)
cells transformed with empty pET30a vector; lane 2. E. coli BL21 (DE3) cells
transformed with pET30a-PnP vector (B). M. protein marker; lane 1. E. coli BL21
2
:1 (177 mM guanosine and 397 mM thymine) performed at 90 °C
over 48 h, with sodium phosphate buffer (20 mM) at pH 9.0. The
result was analyzed by HPLC, showing a high conversion of 85%.
(
DE3) cells transformed with empty pET30a vector; lane 2. E. coli BL21 (DE3) cells
1
7
transformed with pET30a-UP vector.
5-methyluridine was synthesized (Fig. 4).

2
104
S. Zhu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2102–2104
Figure 4. HPLC analysis of the results, 5-methyluridine was synthesized with high yield.
In summary, an efficient process to synthesize 5-methyluridine
6. Horinouchi, N.; Ogawa, J.; Kawano, T.; Sakai, T.; Saito, K.; Matsumoto, S.; Sasaki,
M.; Mikami, Y.; Shimizu, S. Appl. Microbiol. Biotechnol. 2006, 71, 615.
Hanrahan, J. R.; Hutchinson, D. W. J. Biotechnol. 1992, 23, 193.
Hori, N.; Watanabe, M.; Sunagawa, K.; Uehara, K.; Mikami, Y. J. Biotechnol. 1991,
17, 121.
was developed which employed a combination of two thermosta-
ble nucleoside phosphorylases in high yields of 85%. Easily enzyme
preparations could be applied directly to a two-step transglycosy-
lation reaction. The most important part of our work was that for
the first time we have successfully employed highly thermostable
enzymes for the preparation of 5-methyluridine. This approach is
promising. By virtue of the excellent thermostability of nucleoside
phosphorylases, this reaction is amenable for industrial prepara-
tion. In fact, the higher the reaction temperature, the higher
solubility of the substrates and the higher speed of the reaction.
As a result, the reaction offers higher yield. To our best knowledge,
this two-step reaction employing highly thermostable nucleoside
phosphorylases was not disclosed in the literature.
7
8
.
.
9.
Hori, N.; Watanabe, M.; Sunagawa, K.; Uehara, K.; Mikami, Y. J. Ferment. Bioeng.
1
992, 73, 326.
0. Hori, N.; Watanabe, M.; Mikami, Y. Biocatalysis Biotransformation 1991, 4, 297.
11. Nobuaki, H.; Kyouko, U.; Yoichi, M. Agric. Biol. Chem. 1991, 55, 1071.
1
12. Pal, S.; Nair, V. Biocatalysis Biotransformation 1997, 15, 147.
1
3. Ishii; M.; Shirae; H.; Yokozeki; K. Enzymatic production of 5-methyluridine
from purine nucleosides and thymine by Erwinia carotovora AJ-2992; Tokyo,
JAPON: Agricultural Chemical Society of Japan, 1989.
1
1
1
4. Gordon, G. E.; Visser, D. F.; Brady, D.; Raseroka, N.; Bode, M. L. J. Biotechnol
2011, 151, 108.
5. Visser, D. F.; Hennessy, F.; Rashamuse, J.; Pletschke, B.; Brady, D. Journal of
Molecular Catalysis B: Enzymatic 2011, 68, 279.
6. The PNP and UP were amplified by PCR. for PNP the primers used were: The
0
0
sense primer APPufwd was5 -gggaattccatatgaggaa gccggttca-3 and the
0
0
antisense primer APPu rwd was 5 -cccaagcttgactcctcctgtgaggac-3 the NdeI
and HindIII sites (underlined) were added to the forward and reverse primers,
respectively for UP the primers used were: The sense primer Appyfwd was
5 -GGGAATTCCATATGGGAGACGAGAGTCT -3 , the NdeI site was underlined.
The antisense primer Appyrwd was 5’-CCCAAGCTTTCATGTGCGTCTGCAC
References and notes
0
0
1
.
Chen, B.-C.; Quinlan, S. L.; Stark, D. R.; Gregory Reid, J.; Audia, V. H.; George, J.
G.; Eisenreich, E.; Brundidge, S. P.; Racha, S.; Spector, R. H. Tetrahedron Lett.
0
1
995, 36, 7957.
GCCAGGCTC -3 , the HindIII site was underlined.
2
3
4
.
.
.
Chen, B.-C.; Quinlan, S. L.; Reid, J. G.; Spector, R. H. Tetrahedron Lett. 1998, 39, 729.
Chen, B.-C.; Quinlan, S. L.; Gregory Reid, J. Tetrahedron Lett. 1995, 36, 7961.
Wilson, L. J.; Hager, M. W.; El-Kattan, Y. A.; Liotta, D. C. Synthesis 1995, 1995,
17. HPLC analysis: sample were analyzed by HPLC on
(interfaced with Shimadzu LC solution Software) equipped with a detector
at 260 nm and Phenomenex Synergi Max-RP 80A column
a Shimadzu HPLC
a
4 l
1
465.
(150 ꢁ 4.60 mm) at 22 °C. The mobile phase was 92% water: 8% methanol,
5
.
Horinouchi, N.; Kawano, T.; Sakai, T.; Matsumoto, S.; Sasaki, M.; Mikami, Y.;
at a flow rate of 1.0 ml/min.
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