One-pot approach to functional nucleosides possessing a fluorescent group using nucleobase-exchange reaction by thymidine phosphorylase

Article abstract of DOI:10.1039/c3ob41605d

Herein, we describe β-selective coupling between a modified uracil and a deoxyribose to produce functionalized nucleosides catalyzed by thymidine phosphorylase derived from Escherichia coli. This enzyme mediates nucleobase-exchange reactions to convert unnatural nucleosides possessing a large functional group such as a fluorescent molecule, coumarin or pyrene, linked via an alkyl chain at the C5 position of uracil. 5-(Coumarin-7-oxyhex-5- yn)uracil (C4U) displayed 57.2% conversion at 40% DMSO concentration in 1.0 mM phosphate buffer pH 6.8 to transfer thymidine to an unnatural nucleoside with C4U as the base. In the case of using 5-(pyren-1-methyloxyhex-5-yn)uracil (P4U) as the substrate, TP also could catalyse the reaction to generate a product with a very large functional group at 50% DMSO concentration (21.6% conversion). We carried out docking simulations using MF myPrest for the modified uracil bound to the active site of TP. The uracil moiety of the substrate binds to the active site of TP, with the fluorescent moiety linked to the C5 position of the nucleobase located outside the surface of the enzyme. As a consequence, the bulky fluorescent moiety binding to uracil has little influence on the coupling reaction.

Full text of DOI:10.1039/c3ob41605d

Organic&
BiomolecularChemistry
www.rsc.org/obc
Volume 11 | Number 40 | 28 October 2013 | Pages 6865–7028
ISSN 1477-0520
PAPER
Akihiko Hatano et al.
One-pot approach to functional nucleosides possessing a fluorescent group using
nucleobase-exchange reaction by thymidine phosphorylase

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
One-pot approach to functional nucleosides possessing
a fluorescent group using nucleobase-exchange
reaction by thymidine phosphorylase†
Cite this: Org. Biomol. Chem., 2013, 11,
6900
Akihiko Hatano,* Masayuki Kurosu, Susumu Yonaha, Munehiro Okada and
Sanae Uehara
Herein, we describe β-selective coupling between a modified uracil and a deoxyribose to produce
functionalized nucleosides catalyzed by thymidine phosphorylase derived from Escherichia coli. This
enzyme mediates nucleobase-exchange reactions to convert unnatural nucleosides possessing a large
functional group such as a fluorescent molecule, coumarin or pyrene, linked via an alkyl chain at the C5
position of uracil. 5-(Coumarin-7-oxyhex-5-yn)uracil (C4U) displayed 57.2% conversion at 40% DMSO con-
centration in 1.0 mM phosphate buffer pH 6.8 to transfer thymidine to an unnatural nucleoside with
C4U as the base. In the case of using 5-(pyren-1-methyloxyhex-5-yn)uracil (P4U) as the substrate, TP also
could catalyse the reaction to generate a product with a very large functional group at 50% DMSO con-
centration (21.6% conversion). We carried out docking simulations using MF myPrest for the modified
uracil bound to the active site of TP. The uracil moiety of the substrate binds to the active site of TP, with
the fluorescent moiety linked to the C5 position of the nucleobase located outside the surface of the
enzyme. As a consequence, the bulky fluorescent moiety binding to uracil has little influence on the
coupling reaction.
Received 12th July 2013,
Accepted 13th August 2013
DOI: 10.1039/c3ob41605d
www.rsc.org/obc
Introduction
Functionalization of deoxyuridine at the C5 position is of
interest for the purpose of labeling1 and the preparation of
bioactive nucleoside derivatives.2 The C5 position of the pyri-
midine is located in the major groove of DNA and does not
interfere with the stability of the DNA duplex and recognition
by DNA polymerases. Thus, the C5 position of deoxyuridine
was selected for chemical modification.3 Moreover, functiona-
lized appendages will increase the versatility of nucleic acids
for imaging,4 novel materials5 and catalysts.6
Thymidine phosphorylase (TP, EC2.4.2.4) is a unique meta-
bolic control enzyme, which catalyses the conversion of thymi-
dine to deoxyribose-1α-phosphate and a free thymine base in
phosphate buffer (Scheme 1). Additionally, TP can be used to
change the nucleobase moiety of thymidine to a modified
uracil.7,8 It was found that TP can catalyze the coupling reac-
tion of a deoxyribose moiety and many different uracil deriva-
tives substituted at the C5 position. In another case, the
Scheme 1 (a) Catalytic reaction of thymidine phosphorylase and the synthesis
of unnatural nucleosides containing a fluorescent group at the C5 position of
uracil. (b) Illustration of the catalytic intermediate structure between the
modified uracil at the C5 position and the deoxyribose-1α-phosphate in the
active site of thymidine phosphorylase. The functional group linked to the C5
position of uracil projects outwards from the edge of the active site. By contrast,
the uracil moiety is located within the active site itself.
preparation of nucleosides of heterocycle pyrimidine and
purine bases using immobilized TP9 and bacterial cells10 was
also reported. TP was immobilized on a solid support in order
to generate a stable and recyclable biocatalyst for nucleoside
synthesis. The immobilized bacterial cells produced nucleo-
sides of several modified purine compounds. The range of
available modified pyrimidines is very limited, and only five
uracil derivatives modified at the C5 position were converted
to unnatural nucleosides. The modeled structure of E. coli TP
was obtained from Protein Data Bank (PDB code: closed form
1TPT, 2TPT, open form 1AZY).11,12 The thymine base is bound
Department of Chemistry, Shibaura Institute of Technology, 307 Fukasaku,
Minuma-ku, Saitama, 337-8570, Japan. E-mail: a-hatano@sic.shibaura-it.ac.jp;
Fax: +81-48-687-5013; Tel: +81-48-687-5035
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob41605d
6900 | Org. Biomol. Chem., 2013, 11, 6900–6905
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
to the thymine binding site and its 4 position O atom, 3 posi-
tion N atom, and 2 position O atom are engaged in direct
interactions with Arg-171, Ser-186 and Lys-190, respectively.
The methyl group at the C5 position of thymine is turned
towards the exit of the active site pocket of TP. We hypo-
thesized that large functional groups attached via an alkyl
chain at the C5 position of uracil would not obstruct the for-
mation of the ES complex between TP and uracil derivatives.
C5-alkynylation and alkenylation of 5-iodo-deoxyuridine
have in the past typically involved transition metal-catalyzed
cross-coupling reactions such as Sonogashira coupling13 and
Heck reaction.14 These cross-coupling reactions are a con-
venient tool for introducing a functional group at the C5
position of uracil. We reasoned that TP and Pd catalyzed
cross-coupling reactions could be used to easily obtain many
Table 1 Nucleobase-exchange reaction from thymidine to unnatural nucleo-
sides using thymidine phosphorylase. Substituted uracils possessing various
groups at the C2 and C5 positions. Thymidine (50 mM, 50 µmol), substituted
uracil (5 mM, 5.0 µmol), phosphate buffer (1.0 mM, pH 6.8, 1.0 mL), thymidine
phosphorylase (1 unit mL−1, 1.0 unit) at 37 °C
R
X
Time/h
Conversion/%
–CH3
–H
–F
–CF3
–CH2OH
–CH2CH3
–CH2CH2CH3
–HCvCH2
–CuC–H
S
S
0.2
0.2
0.2
1
53
43
86
78
89
93
47
86
89
O
O
O
O
O
O
O
2
different functional nucleosides containing
a fluorescent
39
50
0.3
2
group at low cost.
Herein, we report an enzymatic base-exchange reaction to
convert thymidine to unnatural nucleosides possessing a func-
tional group at the C5 position by thymidine phosphorylase.
We also describe the influence of the solvent and alkyl chain
length between the fluorescent group and the uracil on the
enzyme catalyzed reaction.
O
4
56
O
O
O
20
88
5
85
42
57
Results and discussion
We carried out the base-exchange reaction to synthesize un-
natural nucleosides possessing various uracil derivatives modi-
fied at the C2 and C5 positions (Table 1). 2-Thiouracil and
2-thiothymine were reacted with each corresponding nucleo-
side using TP in 53% and 43% yield, respectively. However,
2,4-dithiouracil could not be converted to the corresponding
nucleoside using this enzyme. 5-Fluorouracil and 5-trifluoro-
methyluracil were also prepared. 19F labeled derivatives are
very attractive tools for in vivo molecular imaging such as mag-
netic resonance studies.15 In addition, deoxy-5-fluorouridine,
which was converted from 5-fluorouracil, displays potent anti-
tumor activity. Each 5-fluorine compound showed a high con-
version for base-exchange reactions (–F: 86%, –CF3: 78%).
Based on X ray diffraction data of TP11,12 we reasoned that the
C5 position of uracil is not directly involved in the molecular
interaction with the enzyme. Moreover, the C5 position of
uracil is turned to the exit of the active site pocket of the
enzyme. To test this prediction, we investigated the effect of
altering the length of the alkyl chain of the modified uracil at
the C5 position. 5-Ethyluracil was converted to the nucleoside
possessing its base by TP in 93% yield after 39 h. In the case
of 5-propyluracil the corresponding nucleoside was generated
by the enzyme in 47% yield after 50 h. These findings suggest
that elongation of the alkyl chain at the C5 position of uracil
did not drastically interfere with the conversion of the enzy-
matic base-exchange reaction, although the incubation time of
the reaction had to be extended. Substrates possessing a reac-
tive group, such as an alkene (vinyl) or alkyne, displayed good
conversion. The presence of these reactive groups makes
possible the facile introduction of a functional molecule into
DNA or RNA.16 Next, we attempted to introduce the tag moiety
into nucleosides using TP. We also endeavored to couple the
deoxyribose moiety derived from thymidine and the modified
uracil linked hydroxyl alkyne groups (n = 2, 4, and 6) at the C5
position. 5-(1-Hydroxybut-5-yn)uracil (n = 2) was converted to
the corresponding nucleoside in good yield (85%). Elongation
of the alkyl linker (n = 4 and 6) between the uracil and the
hydroxyl group afforded conversion but only in moderate yield,
possibly due to steric repulsion of the extended alkyl chain at
the C5 position of uracil hindering entry of the substrate into
the enzymatic pocket.
Next, we undertook the base-exchange reaction mediated by
TP using the modified uracil linked to a fluorescent group
(Fig. 1). We synthesized three modified uracils linked to a
fluorescent group by different lengths of alkyl chains (n = 2, 4,
and 6). No reaction occurred in phosphate buffer, presumably
due to the decreased nucleophilicity of the uracil derivatives,
although poor substrate solubility may also have played a role.
We then attempted to perform the reaction in buffer contain-
ing a polar organic solvent such as DMSO. The influence
of the mixing ratio of DMSO to buffer is shown in Fig. 1.
5-(Coumarin-7-oxyhex-5-yn)uracil (C4U) displayed the highest
conversion at a DMSO concentration of <40% to transfer
thymidine to unnatural nucleoside with C4U as a base (57.2%
yield). The conversion was drastically enhanced as the concen-
tration of DMSO increased up to 40%. This result indicates
that the contact frequency between a substrate and an enzyme
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 6900–6905 | 6901

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 2 Docking simulation of modified uracils such as C2U and C4U with thymi-
dine phosphorylase as calculated by MF myPresto. (a) Side view of docking
simulation of 5-(coumarin-7-oxyhex-5-yn)uracil (C4U, green) and 5-(coumarin-7-
oxybut-3-yn)uracil (C2U, yellow). (b) Upper view of same complexes by docking
simulation.
Fig. 1 Effect of the ratio of DMSO concentration on conversion of the base-
exchange reaction between the thymidine and the modified uracil catalyzed by
thymidine phosphorylase. The modified uracil is connected to a fluorescent
molecule via an alkyl linker of different chain length (n = 2, 4, and 6). The reac-
tion mixture contained 50 mM thymidine (50 µmol), 5 mM modified uracil
(5.0 µmol) in 1.0 mM phosphate buffer (pH 6.8, 1 mL) and DMSO, 1 unit mL−1
(1.0 unit) of thymidine phosphorylase at 37 °C.
is very important for turnover of the catalytic reaction for C4U.
However, TP appears to be inactivated in solution when the
concentration of DMSO reaches 60% or above. A similar result
was obtained using 5-(coumarin-7-oxyoct-7-yn)uracil (C6U) and
5-(pyren-1-methyloxyhex-5-yn)uracil (P4U) in terms of pro-
duction of unnatural nucleosides upon changing the DMSO
ratio in solution. These substrates showed maximum conver-
sion at
a 50% DMSO ratio. Interestingly, the yield of
5-(coumarin-7-oxybut-3-yn)uracil (C2U) possessing an alkyl
chain n = 2 did not significantly vary across a wide range of
DMSO concentrations (from 30 to 50%) in the reaction solu-
tion. These results seem to show that chain length between
the C5 position of uracil and the fluorescent group influences
the reactivity. Indeed, the distance between the uracil moiety
and the fluorescent group was too close in the molecular struc-
ture of C2U to promote the reaction. We believe a coumarin
group obstructs the binding of the uracil moiety to the active
site of TP.
To further investigate the influence of the alkyl chain
length of uracil substrates C2U and C4U on reactivity, we
carried out docking simulations using MF myPrest17 for the
modified uracil bound to the active site of TP (Fig. 2).11,12 TP
derived from Escherichia coli is thought to belong to the
induced fit type of enzyme (based on the X-ray diffraction
structure of a protein-substrate complex, 2TPT and 1AZY).12,18
Two substrates whose length of alkyl chain was different (n = 2
and 4) were located approximately in the same position in the
enzyme pocket. The conformer C2U is similar to C4U in a side
view (Fig. 2a). However, an upper view shows the coumarin
Fig. 3 Effect of variation and mixing ratio of the organic solvents on the con-
version of base-exchange reaction of thymidine to 5-(coumarin-7-oxyhex-5-yn)
uracil (C4U, n = 4) using thymidine phosphorylase. The reaction mixture con-
tained 50 mM thymidine (50 µmol), 5 mM modified uracil (5.0 µmol) in 1.0 mM
phosphate buffer (pH 6.8, 1 mL) and DMSO, 1 unit mL−1 (1.0 unit) of thymidine
phosphorylase at 37 °C.
group of C2U is situated closer to the entrance of the active
site crevice of TP than that of C4U (Fig. 2b). This result was
anticipated from the observed reactivity of the two substrates.
In addition, the docking studies explained why the reactivity of
C2U was relatively independent of the ratio of organic solvent
(DMSO concentration from 30 to 50%) in the reaction solution
as shown in Fig. 1.
In order to evaluate the most effective organic solvent, we
attempted the base-exchange reaction with TP in a mixture of
a buffer and a polar organic solvent. These results are shown
in Fig. 3. We reasoned that the presence of a polar solvent,
such as DMSO, DMF, THF or MeOH, would increase the solu-
bility of the modified uracil and facilitate homogeneous
mixing in the phosphate buffer. The production of unnatural
6902 | Org. Biomol. Chem., 2013, 11, 6900–6905
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
nucleosides having C4U as the base was most effective when DE-STR, respectively. ESI-MS were recorded using the Accu
the reaction solvent contained DMSO. In particular, a DMSO TOF TLC JMS-T100TD (JEOL). The UV spectra were measured
content of 40% gave the highest yield of unnatural nucleoside using a JASCO UV 630 BIO spectrometer in a 1 cm quartz cell.
(57.2%). Addition of DMSO drastically enhanced the conver-
Enzymatic reactions
sion of unnatural nucleosides possessing a bulky fluorescent
moiety. Introduction of more polar solvents (methanol, tetra- Incubations generally contained 50 mM thymidine (10 µmol,
hydrofuran or DMF) increased the solubility of the substrate, 0.12 g), 5 mM of various uracil derivatives (1 µmol), 1 unit
but the enzymatic reaction appeared to be inhibited. We mL−1 of thymidine phosphorylase in 10 mL of 1 mM phos-
believe elevated levels of methanol or THF might denature TP. phate buffer (pH 6.8) and 0–60% of DMSO. Mixtures were
The coupling reaction of the modified uracil and deoxyribose- stirred at 37 °C until the reactions reached equilibrium. Product
1α-phosphate was effectively performed with a ratio of DMSO formation was monitored by UV absorption as shown in ESI.†
up to 50%, presumably caused by an increase in the collision After removal of H2O in vacuo, the residue was purified using
frequency between the modified uracil and the TP. However, HPLC by eluting with a H2O–MeCN system to afford a pure
when the ratio of organic solvent was increased to more than unnatural nucleoside possessing a large functional group.
60% the enzyme was denatured. When the ratio of DMSO was
Thymidine phosphorylase (from Escherichia coli, EC 2.4.2.4)
40 to 50%, the productivity of unnatural nucleosides was the was purchased from Sigma-Aldrich Chemical Co. The units of
highest due to a good balance between solubility of the sub- enzymatic activity indicate the transition of native substrates
strate and enzymatic activity in the reaction solution.
as 1.0 µmol each of thymidine and phosphate to thymine and
deoxyribose-1α-phosphate per 1 minute. In this research, we
used the “unit” such as the amount of enzyme activity
although we were using unnatural substrates.
Conclusions
2-Deoxy-[5-(coumarin-7-oxybut-3-yn)]uridine (dRC2U). 1H
In summary, we have developed an efficient method for gener- NMR (CD3OD-d4): δ 2.23 (1H, J = 6.8, 6.8, 13.6 Hz, ddd), 2.32
ating unnatural nucleosides possessing a functionalized mole- (1H, J = 6.0, 6.4, 13.2 Hz, ddd), 2.93 (1H, J = 6.8 Hz, t), 3.79 (2H,
cule using TP. Recognition of the C5 position of uracil by TP J = 3.4, 11.8, 31.0 Hz, ddd), 3.94 (2H, J = 3.4 Hz, q), 4.27 (2H, J =
was flexible. Thus, the production of unnatural nucleosides 6.6 Hz, t), 4.41 (1H, J = 3.4, 6.8 Hz, dt), 6.26 (1H, J = 6.4 Hz, t),
possessing a fluorescent group such as a coumarin or pyrene 6.27 (1H, J = 9.2 Hz, d), 6.99 (2H, m), 7.56 (1H, J = 8.4 Hz, d),
was possible using this enzyme in a one-pot reaction. We 7.91 (1H, J = 9.6 Hz, d), 8.28 (1H, s); 13C NMR (CD3OD-d4):
believe the uracil moiety of the substrate binds to the active δ 19.8, 40.3, 61.3, 66.5, 70.7, 72.8, 85.6, 87.8, 89.6, 99.3, 101.2,
site of TP, with the fluorescent moiety linked to the C5 posi- 112.2, 112.9, 129.2, 143.5, 144.4, 149.9, 155.7, 162.0, 162.2,
tion of the nucleobase located outside the surface of the 163.4; ESI-MS m/z 441 [M + H]+; Anal. Calcd for C22H20N2O8: C,
enzyme. As a consequence, the bulky fluorescent moiety 60.00; H, 4.58; N, 6.36. Found: C, 60.03; H, 4.53; N, 6.15.
binding to uracil has little influence on the coupling reaction.
2-Deoxy-[5-(coumarin-7-oxyhex-5-yn)]uridine (dRC4U). 1H
Our research describes a novel method for the synthesis of NMR (DMSO-d6): δ 1.61 (1H, J = 7.3 Hz, quin), 1.82 (1H, J =
functionalized nucleosides that can be used as fluorescent 6.9 Hz, quin), 2.06 (2H, J = 5.4, 6.4 Hz, dd), 2.40 (2H, J =
probes. Further chemical modifications at the C5 position of 7.2 Hz, t), 3.74 (1H, J = 3.6 Hz, q), 4.07 (2H, J = 6.4 Hz, t), 4.38
uracil will provide new synthetic approaches to generate many (1H, J = 4.2 Hz, quin), 5.05 (1H, J = 5.0 Hz, t), 5.25 (1H, J =
more functionalized unnatural nucleosides.
4.4 Hz, d), 6.05 (1H, J = 6.4 Hz, t), 6.21 (1H, J = 9.2 Hz, d), 6.90
(2H, m), 7.55 (1H, J = 8.4 Hz, d), 7.91 (1H, J = 9.6 Hz, d), 8.06
(1H, s), 11.5 (1H, br); 13C NMR (DMSO-d6): δ 19.0, 25.2, 28.1,
61.5, 68.4, 70.7, 73.6, 79.6, 85.2, 88.1, 93.6, 99.5, 101.7, 112.8,
112.9, 113.3, 130.0, 143.2, 144.9, 150.0, 155.9, 160.9, 162.3,
162.4; ESI-MS m/z 491 [M + Na]+; HRMS (ESI): calcd for
Experimental section
General
All solvents and reagents were of reagent-grade quality, and C24H24N2O8Na [M + Na]+: 491.1418, found: 491.1430.
used without further purification. The TLC analysis was
2-Deoxy-[5-(coumarin-7-oxyoct-7-yn)]uridine (dRC6U). 1H
carried out using silica gel 60 F254 1.05554 (Merck). Column NMR (CD3OD-d4): δ 1.55 (6H, m), 1.82 (6H, J = 6.4 Hz, quin),
chromatography was performed using Silica gel 60 N (Kanto 2.23 (2H, m), 2.39 (2H, J = 6.8 Hz, t), 3.31 (2H, br), 3.75 (2H, J =
Chemical Co.). The 1H, 13C and COSY NMR spectra were 3.2, 6.8, 6.8 Hz, ddd), 3.92 (1H, J = 3.0, 6.0 Hz, dd), 4.06 (2H,
recorded using a JEOL ECS 400 spectrometer (400.0 MHz for J = 6.4 Hz, t), 4.36 (1H, J = 3.1, 6.0 Hz, tt), 6.22 (2H, m), 6.84
1H; 100.4 MHz for 13C). The spectra were referenced to TMS in (1H, J = 2.4 Hz, d), 6.88 (1H, J = 2.2, 8.8 Hz, dd), 7.48 (1H, J =
chloroform-d3, CD3OD-d4-d4, and DMSO-d6. The chemical 8.8 Hz, d), 7.84 (1H, J = 9.6 Hz, d), 8.17 (1H, s); 13C NMR
shifts (δ) are reported in ppm; multiplicity is indicated by: s (CD3OD-d4: δ 18.7, 25.1, 28.1, 28.6, 40.2, 61.3, 68.3, 70.7, 78.2,
(singlet), d (doublet), t (triplet), q (quartet), quin (quintet), m 85.5, 87.7, 93.7, 99.9, 100.8, 111.9, 112.5, 112.9, 129.1, 129.3,
(multiplet), and br (broad). The coupling constants, J are 142.8, 144.5, 149.9, 142.8, 144.5, 149.9, 155.8, 162.1, 162.8,
reported in Hz. FABMS and MALDI-TOFMS were recorded 163.3; ESI-MS m/z 519 [M + Na]+; HRMS (ESI): calcd for
using a JEOL JMS-700 V, and Applied Biosystems Voyager C26H28N2O8Na [M + Na]+: 519.1732, found: 519.1743.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 6900–6905 | 6903

View Article Online
Paper
Organic & Biomolecular Chemistry
2-Deoxy-[5-(pyrene-1-methyloxyhex-5-yn)]uridine (dRP4U). interaction (EvdW), coulomb interaction (Eelec), hydrogen bond
1H NMR (CD3OD-d4): δ 1.67 (2H, J = 7.2 Hz, quin), 1.81 (2H, (EH-bond) and hydrophobic interaction (EASA). In detail, see
m), 2.19 (2H, m), 2.37 (2H, J = 6.8 Hz, t), 3.69 (1H, m), 3.88 ref. 17.
(1H, J = 3.2, 6.0 Hz, tt), 4.33 (1H, J = 3.2, 6.4 Hz, tt), 5.22 (2H,
Sraw ¼ gðEvdW þ Eelec þ EH-bond þ EASA
Þ
s), 6.20 (2H, J = 6.6 Hz, t), 8.11 (9H, m), 8.38 (1H, J = 9.2 Hz, d);
13C NMR (CD3OD-d4): δ 19.9, 26.4, 30.0, 30.7, 41.6, 62.6, 71.1,
72.1, 72.4, 72.9, 86.9, 89.1, 94.9, 101.3, 124.7, 125.6, 125.9,
126.1, 126.4, 127.2, 128.5, 128.5, 128.8, 130.7, 132.3, 132.7,
132.8, 133.0, 144.3, 151.4, 164.8; ESI-MS m/z 561 [M + Na]+;
Acknowledgements
This work was supported by JSPS KAKENHI Grant number
12874792. This work was supported in part by grants of the
JGC-S Scholarship Foundation and the Amano Institute of
Technology.
HRMS (ESI) calcd for
561.2124.
C32H30N2O6Na: 561.2002, found:
2-Deoxy-[5-(1-hydroxybut-3-yn)]uridine (dRU2OH). 1H NMR
(CD3OD-d4): δ 2.22 (1H, J = 6.8, 13.6 13.6 Hz, ddd), 2.30 (1H, J =
3.6, 6.0, 13.2 Hz, ddd), 2.59 (2H, J = 6.4 Hz, t), 3.31 (1H, br),
3.35(1H, br), 3.69 (2H, J = 6.6 Hz, t), 3.77 (2H, J = 3.2, 11.6,
31.2 Hz, ddd), 3.93 (1H, J = 3.2, 6.4 Hz, dd), 4.40 (1H, J = 3.2, 6.0
Hz, dd), 6.23 (1H, J = 6.8 Hz, t), 8.22 (1H, s); 13C NMR (CD3OD-
d4): δ 23.2, 40.5, 60.2, 61.3, 70.7, 72.3, 85.6, 87.7, 91.0, 99.5,
143.4, 149.9, 163.5; ESI-MS m/z 319 [M + Na]+; HRMS (ESI) calcd
for C13H16N2O6Na: 319.0906, found: 319.0815.
Notes and references
1 Recent examples: J. Riedl, P. Ménová, R. Pohl, P. Orsá,
M. Fojta and M. Hocek, J. Org. Chem., 2012, 77, 8287–8293;
L. Wicke and J. W. Engels, Bioconjugate Chem., 2012, 23,
627–642; S. Ikeda and A. Okamoto, Chem.–Asian J., 2008, 3,
958–968; P. Kaden, E. M.-Enthart, A. Trifonov, T. Fiebig and
H.-A. Wagenknecht, Angew. Chem., Int. Ed., 2005, 44, 1636–
1639.
2 M. M. Masud, T. Masuda, Y. Inoue, M. Kuwahara, H. Sawai
and H. Ozaki, Bioorg. Med. Chem. Lett., 2011, 21, 715–717;
S. M. Park, H. Yang, S.-K. Park, H. M. Kim and B. H. Kim,
Bioorg. Med. Chem. Lett., 2010, 20, 5831–5834.
3 C. Höbartner, G. Sicoli, F. Wachowius, D. B. Gophane and
S. T. Sigurdsson, J. Org. Chem., 2012, 77, 7749–7754;
A. Roychowdhury, H. Illangkoon, C. L. Hendrickson and
S. A. Benner, Org. Lett., 2004, 6, 489–492; A.-M. Chacko,
W. Qu and H. F. Kung, J. Org. Chem., 2008, 73, 4874–4881.
4 H. Yamakoshi, K. Dodo, M. Okada, J. Ando, A. Palonpon,
K. Fujita, S. Kawata and M. Sodeoka, J. Am. Chem. Soc.,
2011, 133, 6102–6105; S. Ikeda, T. Kubota, D. O. Wang,
H. Yanagisawa, T. Umemoto and A. Okamoto, ChemBio-
Chem, 2011, 12, 2871–2880.
5 D. J. Hurley and Y. Tor, J. Am. Chem. Soc., 2002, 124, 3749–
3762; I. Bouamaied and E. Stulz, Synlett, 2004, 9, 1579–
1583.
6 D. M. Perrin, T. Garestier and C. Hélène, J. Am. Chem. Soc.,
2001, 123, 1556–1563; C. H. Lam and D. M. Perrin, Bioorg.
Med. Chem. Lett., 2010, 20, 5119–5122.
7 A. Hatano, A. Harano and M. Kirihara, Chem. Lett., 2006,
232–233.
8 A. Hatano, A. Harano, Y. Takigawa, Y. Naramoto, K. Toda,
Y. Nakagomi and H. Yamada, Bioorg. Med. Chem., 2008, 16,
3866–3870.
2-Deoxy-[5-(1-hydroxyhex-5-yn)]uridine (dRU4OH). 1H NMR
(CD3OD-d4): δ 1.63 (4H, m), 2.24 (2H, m), 2.39 (2H, J = 6.4 Hz,
t), 2.56 (2H, J = 6.0 Hz, t), 3.74 (2H, J = 3.2, 11.8, 32.2 Hz, ddd),
3.90 (1H, J = 2.8, 5.6 Hz, dd), 4.39 (1H, J = 3.2, 6.0 Hz, tt), 6.22
(1H, J = 6.4 Hz, t), 8.20 (1H, s); 13C NMR (CD3OD-d4): δ 18.6,
4.7, 31.4, 47.4, 61.1, 70.7, 71.6, 85.5, 87.7, 93.5, 99.9, 142.9,
150.2, 163.7; Anal. Calcd for C15H20N2O6: C, 55.55; H, 6.22; N,
8.64. Found: C, 55.58; H, 6.16; N, 8.46.
2-Deoxy-[5-(1-hydroxyoct-7-yn)]uridine (dRU6OH). 1H NMR
(DMSO-d6): δ 1.27 (4H, br), 1.36 (2H, J = 6.4 Hz, quin), 1.56
(2H, J = 7.2 Hz, quin), 2.00 (1H, J = 6.2, 14.0 Hz, tt), 2.34 (1H,
m), 2.59 (2H, J = 7.4 Hz, t), 3.33 (1H, J = 6.4, 11.6 Hz, dd), 3.87
(1H, J = 3.6, 7.2 Hz, dd), 4.18 (2H, J = 4.4, 9.6 Hz, tt), 4.36 (1H,
J = 5.2 Hz, t), 5.10 (1H, J = 5.2 Hz, t), 5.29 (1H, J = 4.0 Hz, d),
6.12 (1H, J = 6.2 Hz, t), 6.37 (1H, s), 8.61 (1H, s); 13C NMR
(DMSO-d6): δ 25.6, 26.9, 27.8, 28.7, 32.8, 41.7, 61.2, 70.2, 79.6,
87.9, 88.6, 100.2, 107.0, 137.3, 154.4, 159.0, 171.7; Anal. Calcd
for C17H24N2O6: C, 57.94; H, 6.86; N, 7.95. Found: C, 57.73;
H, 6.78; N, 7.79.
Docking studies
Docking studies were performed using the MF myPresto siev-
gene program (FiatLux) with an AMBER-type molecular force
field. This simulation was performed using two basic criteria
as follows. Firstly, a global minimum search was carried out
that assumes contact between three atoms of the substrate
and three atoms at the surface of the protein pocket. This
simulation evaluates the distance and similarities between the
protein pocket and substrate. Secondly, a local minimum
search was performed that is reinitiated with different con-
formers. The receptor–substrate interactions accounted for van
der Waals, coulomb, hydrogen bond, and hydrophobic inter-
actions. The dimensionless raw docking score that was used
9 S. Immacolata, S. C. Daniela, R. Silvia, U. Daniela and
T. Marco, Enzyme Microb. Technol., 2011, 49, 52–58;
I. Serraa, T. Bavaroa, D. A. Cecchinia, S. Dalyc,
A. M. Albertinib, M. Terrenia and D. Ubiali, J. Mol. Catal. B:
Enzym., 2013, 95, 16–22.
for receptor–substrate docking is below. The “g” was a para- 10 I. Votruba, A. Holý, H. Dvořáková, J. Günter, D. Hocková,
meter and it was set to 0.01 mol kcal−1. “E” value showed the
potential functions for each interaction, van der Waals
H. Hřebabecký, T. Cihlar and M. Masojídková, Collect.
Czech. Chem. Commun., 1994, 59, 2303–2330.
6904 | Org. Biomol. Chem., 2013, 11, 6900–6905
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
11 W. J. Cook, G. W. Koszalka, W. W. Hall, C. L. Burns and 16 E. F. V. Scriven and K. Turnbull, Chem. Rev., 2009, 109,
S. E. Ealick, J. Biol. Chem., 1987, 262, 3788–3789;
M. R. Walter, W. J. Cook, L. B. Cole, S. A. Short,
G. W. Koszalka, T. A. Krenitsky and S. E. Ealick, J. Biol.
Chem., 1990, 265, 14016–14022.
4207–4220; A. H. El-Sagheerab and T. Brown, Chem. Soc.
Rev., 2010, 39, 1388–1405; K. Fujimoto, S. Matsuda,
N. Takahashi and I. Saito, J. Am. Chem. Soc., 2000, 122,
5646–5647; H. Xiong and F. Seela, Bioconjugate Chem.,
2012, 23, 1230–1243.
12 M. J. Pugmire, W. J. Cook, A. Jasanoff, M. R. Walter and
S. E. Ealick, J. Mol. Biol., 1998, 281, 285–299.
13 F. W. Hobbs Jr., J. Org. Chem., 1989, 54, 3420–3422.
14 H. Li, L. Czuchajowski and W. R. Trumble, J. Heterocycl.
Chem., 1997, 34, 999–1003.
17 Y. Fukunishi, Y. Mikami and H. Nakamura, J. Mol.
Graphics Modell., 2005, 24, 34–45; Y. Fukunishi,
Y. Mikami, K. Takedomi, M. Yamanouchi, H. Shima
and H. Nakamura, J. Med. Chem., 2006, 49, 523–
533.
15 S. Mizukami, R. Takikawa, F. Sugihara, Y. Hori, H. Tochio,
M. Wälchli, M. Shirakawa and K. Kikuchi, J. Am. Chem. 18 A. Greube, K. Müller, E. T.-Petersen, A. Herrmann and
Soc., 2008, 130, 794–795.
P. Müller, Biochemistry, 2004, 43, 464–472.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 6900–6905 | 6905