Phosphorylated 5-ethynyl-2′-deoxyuridine for advanced DNA labeling

Article abstract of DOI:10.1039/c5ob00199d

The representative DNA-labeling agent 5-ethynyl-2′-deoxyuridine (EdU) was chemically modified to improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the substrate in DNA polymerization by circumventing the enzymati

Full text of DOI:10.1039/c5ob00199d

Organic &
Biomolecular Chemistry
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Phosphorylated 5-ethynyl-2’-deoxyuridine for
advanced DNA labeling†
Cite this: Org. Biomol. Chem., 2015,
13, 4589
Siyoong Seo,a,b Kazumitsu Onizuka,a Chieko Nishioka,c Eiki Takahashi,c
Satoshi Tsuneda,b Hiroshi Abe*a,d and Yoshihiro Ito*a,b,d
The representative DNA-labeling agent 5-ethynyl-2’-deoxyuridine (EdU) was chemically modified to
improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the sub-
strate in DNA polymerization by circumventing the enzymatic monophosphorylation step that consumes
energy. In addition, to enhance cell permeability, the phosphates were protected with bis-pivaloyloxy-
methyl that is stable in buffer and plasma, and degradable inside various cell types. The phosphorylated
EdU (PEdU) was less toxic than EdU, and had the same or a slightly higher DNA-labeling ability in vitro.
PEdU was also successfully applied to DNA labeling in vivo. In conclusion, PEdU can be used as a less
toxic DNA-labeling agent for studies that require long-term cell survival or very sensitive cell lines.
Received 31st January 2015,
Accepted 6th March 2015
DOI: 10.1039/c5ob00199d
www.rsc.org/obc
(CuAAC) by click chemistry.6–8 The main advantage of using
EdU for DNA detection is that the labeled DNA can be detected
Introduction
Detection of DNA synthesis in growing organisms is important without denaturation because of the much smaller size of the
for biological science including cell proliferation and differen- fluorescent azide allows access to the alkyne group of the
tiation, cell cycle dynamics, and carcinogenesis.1–3 DNA label- incorporated EdU. Despite these advantages, EdU is a highly
ing is conducted for detection using pyrimidine nucleosides toxic antimetabolite that induces cell death.13,14 Its cytotoxicity
such as [3H]thymidine, bromodeoxyuridine (BrdU), and 5- is an impediment to DNA labeling studies that require sub-
ethynyl-2′-deoxyuridine (EdU).
sequent cell survival. Therefore, EdU has been modified to
DNA labeling with [3H]thymidine is a powerful technique reduce its cytotoxicity by substitution of fluoro, hydroxyl and
but it has been replaced by BrdU in many studies because methyl groups with 2′ hydrogen.15 Not only pyrimidine but
autoradiography requires special equipment and is labor also purine nucleoside analogues containing an alkyne group
intensive and very time consuming.4 As an alternative, BrdU have been established.16–18 These compounds were designed
labeling is faster and provides better results in microscopic to bypass the antimetabolic pathway of EdU and maintain the
analyses. However, after incorporation of BrdU into replicated alkyne group for click chemistry with fluorescent azide.
DNA during S phase, the technique requires an anti-BrdU anti-
Here, we applied a different approach by phosphorylation
body that cannot bind to BrdU in double stranded DNA of EdU. Based on the structure of EdU, chemical phosphoryl-
without harsh DNA denaturation for conversion to single ation was carried out on the 5′ hydroxyl group. The chemical
stranded DNA.5
monophosphorylation was expected to increase the efficiency
Recently, DNA labeling techniques using EdU have been of the substrate in DNA polymerization by circumventing the
described as an alternative to both [3H]thymidine and enzymatic monophosphorylation step that consumes energy.
BrdU.9–12 It contains an alkyne group that reacts with fluo- However, a phosphorylated compound is generally ineffective
rescent azide in a copper-catalyzed azide–alkyne cycloaddition at penetrating the cell membrane because of its negative
charges. Therefore, cell permeability was enhanced through
protection of the phosphates by bis-pivaloyloxymethyl [bis-
aNano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako,
(POM)] that is quite stable in buffer and plasma, and reversible
Saitama 351-0198, Japan. E-mail: y-ito@riken.jp
bDepartment of Life Science and Medical Bioscience, Waseda university,
inside various cell types.19–22 After penetrating the cell mem-
brane, progressive degradation of bis(POM)-protecting groups
regenerates EdU monophosphate gradually and can prevent
excessive concentrations of thymidine-like antimetabolites,
resulting in a reduction of cytotoxicity.
In this study, bis(POM)-phosphorylated EdU (PEdU) was
synthesized by conjugation of chlorobis(POM) phosphate to
2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan
cSupport Unit for Animal Experiment, Research Resources Center, RIKEN Brain
Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
dEmergent Bioengineering Materials Research Team, RIKEN Center for Emergent
Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob00199d
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EdU.23 The synthesized PEdU was compared with commercial chromatography (MeOH–DCM = 1 : 9), producing 444 mg of
EdU in terms of cytotoxicity and sensitivity in vitro, and its yellow foam. The foam, 5-(trimethylsilyl)ethynyl-2′-deoxyuri-
labeling capability in vivo was evaluated using mice.
dine, was dissolved in THF (4 mL), and TBAF in THF (1.2 mL,
1 M, 1.2 mmol) was added to mixture, followed by stirring at
room temperature for 3 h. Then, the mixture was filtered to
remove the white precipitate that was washed with a small
quantity of THF and eluted (MeOH–DCM = 1 : 9). The resulting
filtrates were concentrated and purified by flash column
chromatography (MeOH–DCM = 1 : 9), producing 181 mg
Materials and methods
Materials
5-Iodo-2′-deoxyuridine (IdU) was purchased from Tokyo
Chemical Industry Co., Ltd. All other reagents were purchased
from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd, or
Wako Pure Chemical Industries, Ltd, and used without further
purification. All non-aqueous reactions were conducted using
super dehydrated solvent in an argon atmosphere. Flash
column chromatography was performed using silica 60
(Spherical, 40–50 nm; Kanto Chemical). Electrospray ioniza-
tion mass spectra (ESI-MS) were obtained on an Agilent 1100
series LC/MSD. Nuclear magnetic resonance (NMR) spectra
were recorded on a JEOL AL-400 (400 MHz). HeLa and 3T3
cells were maintained in Dulbecco’s modified Eagle’s medium
(high glucose with L-glutamine and phenol red; Wako
Pure Chemical Industries, Ltd) supplemented with 10% fetal
bovine serum. Cells were cultured at 37 °C in an atmosphere
with 5% CO2.
1
(69%) of white solid. H NMR (400 MHz, DMSO-d6) 2.12 (2H,
m, J = 6.3 Hz), 3.59 (2H, q, J = 4.5 Hz), 3.80 (1H, d, J = 3.0 Hz),
4.1(1H, s), 4.23(1H, m), 5.13(1H, t, J = 4.8 Hz), 5.24 (1H, d, J =
4.5 Hz), 6.1 (1H, t, J = 6.3 Hz), 8.29(1H, s), and 11.62(1H, s).
ESI-MS: [M + Na]+ calculated for C11H12N2O5Na+ 275.06, found
m/z 275.10.
Synthesis of chlorobis(POM) phosphate (2). Trimethyl phos-
phate (1.50 g, 10.7 mmol) was dissolved in CH3CN (9 mL), fol-
lowed by NaI (4.82 g, 32.1 mmol) and chloromethyl pivalate
(6.28 g, 41.7 mmol). The crude mixture was heated to reflux
and stirred for 1 day. Et2O (100 mL) was added to quench the
reaction, and the organic phase was washed with water three
times and then dried over Na2SO4. After filtration of Na2SO4,
the filtrate was concentrated by the rotary evaporator and puri-
fied by flash column chromatography (EtOAc–Hexane = 1 : 5)
to produce 2.24 g (46%) of viscous oil. The oil was dissolved in
piperidine (20 mL) and stirred at room temperature overnight.
The mixture was concentrated by the rotary evaporator and
dried in a vacuum to a constant weight. The resulting residue
was dissolved in water (30 mL) and then passed through a
column filled with ion exchange resin (Bio-rad AG 50W-X8).
The column was washed with sufficient water, and the eluent
was collected and lyophilized to produce 2.05 g (93%) of white
solid. After ion exchange, the white solid was dissolved in a
solution of oxalyl chloride (145 μL, 1.53 mmol) in DCM
(3 mL), and then DMF (3 μL) in DCM (3 mL) was added slowly
at 4 °C. After 10 min of stirring, the mixture was removed from
the ice bath and stirred for 2 h at room temperature. The
Synthesis of PEdU
A scheme of the synthesis is shown in Fig. 1.
Synthesis of EdU (1). A round flask was flushed with argon,
and then IdU (371 mg, 1.04 mmol), palladium tetrakis (triphe-
nylphosphine) (113 mg, 98 mmol), and copper iodide (47 mg,
250 mmol) were added to the flask. Anhydrous DMF (12 mL)
was added via a syringe, and the mixture was stirred briefly at
room temperature. After dissolving, triethylamine (0.4 mL,
2.9 mmol) and trimethylsilylacetylene (0.71 mL, 5.0 mmol)
were added to the flask. The crude mixture was stirred at room
temperature for 3.5 h and then concentrated by a rotary evap-
orator. The resulting residue was purified by flash column
Fig. 1 Scheme of PEdU synthesis.
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solvent was removed by the rotary evaporator and dried to a tected from light. After removing the reaction cocktails, the
constant weight, producing 80 mg (75%) of yellowish oil. cells were washed once with 3% BSA in PBS. The labeled cells
Because of its instability, the chlorobis(POM) phosphate was were imaged under a microscope (Axio Observer. Z1, ZEISS).
used immediately for the next step. 1H NMR (400 MHz, CDCl3)
1.24 (18H, s), 5.85 (4H, m), ESI-HRMS: [M − 2CH2O + H]+ cal-
In vivo analysis
culated for C10H19O5PCl 285.0659, found m/z 285.0630.
Six mice (3 weeks old) were injected intraperitoneally (i.p.)
with 0.2, 1, or 5 mg PEdU in 0.1 ml PBS. The negative control
animal was injected i.p. with 0.1 ml PBS only. At 24 and 48 h
after injection, one mouse from each experimental group was
fixed by perfusion with 4% paraformaldehyde. After 24 h, the
tissues were embedded in paraffin, sectioned at 2 μm, and
mounted onto glass slides. After paraffin removal, the sections
were rinsed with PBS and stained with 100 mM Tris (pH 8.5),
1 mM CuSO4, and 10 μM Alexa 488 azide (from a 10 mM stock
solution in DMSO), and 100 mM ascorbic acid (added last
from a 0.5 M stock solution in water) for 30 min. After stain-
ing, the sections were washed twice with PBS and methanol,
washed once with PBS, and then stained with DAPI.
Synthesis of PEdU (3). EdU (50 mg, 0.2 mmol) was dissolved
in pyridine (2 mL) and then cooled to −40 °C. DMAP (13 mg)
was added to the EdU solution followed by stirring for 20 min.
A solution of chlorobis(POM) phosphate (100 mg, 0.29 mmol)
in THF (2 mL) was added to the EdU solution dropwise via a
syringe over 20 min. The mixture was stirred at −40 °C for 4 h
and then at room temperature for 1 day. EtOAc (20 mL) was
added to quench the reaction, and the organic phase was
washed with water (10 mL) twice. The aqueous layer was back-
extracted with EtOAc. The collected organic phase was washed
with brine (10 mL), dried over Na2SO4, and concentrated by
the rotary evaporator. The compound was purified by flash
column chromatography with a gradient (MeOH–DCM = 1 : 45
to 1 : 10), producing 21 mg (18%) of yellowish powder. 1H
NMR (400 MHz, CD3OD) 1.23 (18H, s), 2.13(2H, t, J = 3.0 Hz),
3.54 (1H, s), 3.76 (2H, t, J = 24.1 Hz), 3.92 (1H, d, J = 3.2 Hz),
4.11 (1H, d, J = 4 Hz), 4.40 (1H, d, J = 7.2 Hz), 5.70 (4H, d, J =
9.9 Hz), 6.21 (1H, t, J = 2.85 Hz), 7.98 (1H, s), 8.38(1H, s),
13C NMR (100 MHz, CD3OD) 27.2, 39.8, 41.0, 62.5, 71.7, 76.0,
82.8, 82.8, 84.4, 87.5, 89.2, 100.0, 145.7, 151.3, 164.5, and
178.0. ESI-HRMS: [M + Na]+ calculated for C23H33N2O12PNa+
583.1663 found m/z 583.1625.
Results and discussion
Design and synthesis of PEdU
EdU is a well-known compound for manageable DNA detec-
tion, but is also a cytotoxic antimetabolite. PEdU was designed
to improve the EdU incorporation efficiency and decrease cytotoxi-
city (Fig. 2). By providing a monophosphate, we expected an
improvement in the formation of triphosphate through circum-
venting the enzymatic monophosphorylation step that requires
energy, resulting in efficient incorporation into DNA. However, a
common problem of phosphorylated compounds is that the
negative charges of the phosphates hinder cell membrane per-
meability. Therefore, a monophosphate was added with bis(POM)
protecting groups that are widely used to neutralize negative
charges in prodrug production. A POM protecting group can be
cleaved by esterases inside of cells, resulting in gradual release of
the active form, phosphorylated EdU in this study.19,20 This
gradual regeneration of the thymidine analogue was expected
to prevent an excessive concentration of the antimetabolite.
Cytotoxicity assay
Hela and 3T3 cells were applied to a cytotoxicity assay using
cell counting kit-8 (CCK-8). The cells were seeded in 96-well
plates at a density of 5 × 103 cells per well and incubated over-
night. The medium was removed and various concentrations
(10−1, 100, 101 and 102 μM) of EdU or PEdU in DMSO were
added to the fresh medium. The final concentration of DMSO
in each well was 1%, and the control was treated with 1%
DMSO only. The cells were incubated for 24, 48 and 72 h. After
incubation, a CCK-8 solution was added to each well followed
by incubation for 3 h. Absorbance at 450 nm was recorded
using a microplate reader (Model 680, BIO-RAD).
Cytotoxicity assays
The cytotoxicities of EdU and PEdU were compared at various
concentrations after 24, 48 and 72 h of incubation with mouse
Metabolic labeling of cellular DNA
To analyze metabolic labeling of cellular DNA, we used a Click- embryonic fibroblasts (3T3) and human cervical cancer cells
iT® EdU imaging kit (Invitrogen) according to the manufac- (HeLa) as shown in Fig. 3. After 24 h, only the highest concen-
turer’s protocol. The culture supernatant was removed after tration of EdU, 100 μM, induced some cytotoxicity in both 3T3
incubation with the various concentrations of each nucleoside and HeLa cells. After 48 h, 10 μM EdU induced cytotoxicity,
at 24, 48 and 72 h. The cells were fixed with 4% paraformalde- and many cells were damaged in 100 μM EdU. In contrast,
hyde in phosphate buffered saline (PBS) for 15 min at room PEdU treatment did not lead to any decrease in the viability of
temperature. After washing the cells twice with 3% bovine both cell lines at all concentrations. After 72 h, the cell viability
serum albumin (BSA) in PBS, the cells were permeabilized had decreased rapidly at 10 and 100 μM EdU in both cell lines.
with 0.5% Triton X-100 in PBS for 20 min. The cells were HeLa cells were relatively more affected than 3T3 cells. This
washed twice with 3% BSA in PBS again. Reaction cocktails cytotoxic phenomenon of EdU was also observed with PEdU.
containing Click-iT® reaction buffer, CuSO4, Alexa Fluor® 488 However, PEdU did not induce any cytotoxicity in 3T3 cells
azide, and reaction buffer additive were added to each well fol- until 72 h at all concentrations or in HeLa cells until 48 h.
lowed by incubation for 30 min at room temperature while pro- Cytotoxicity was found only in HeLa cells after 72 h. It is
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Fig. 2 Design of PEdU.
Fig. 3 3T3 and HeLa cells were treated with various concentrations of EdU or PEdU in DMSO. The control was treated with DMSO only. n = 4.
known that the cytotoxicity of EdU is mediated via two path- antimetabolite that inhibits dTMP synthesis.15 Although it is
ways (Fig. S1†). One is hydrolysis of glycosidic bonds, resulting not known how the protective groups in PEdU affected the
in the generation of the 5-ethynyluracil antimetabolite24–26 as cytotoxicity via the second mechanism, the first mechanism
well as EdUMP from EdU by phosphorylation, resulting in an must be considered to evade the cytotoxic pathways.
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In vitro metabolic DNA labeling
found that the fluorescence intensity of PEdU incorporation
was almost the same as that of EdU or slightly higher. Impor-
To evaluate the ability of PEdU to incorporate into DNA, we tantly, even though PEdU was less toxic than EdU, its DNA
conducted CuAAC with a fluorescent azide (Alexa Fluor 488). labeling ability was similar to EdU.
Various concentrations (0.1–100 μM) of EdU or PEdU were
Salic and Mitchison10 confirmed DNA labeling by EdU using
incubated with 3T3 and HeLa cells for 24 h. After CuAAC with hydroxyurea as an inhibitor. They reported that EdU labeling is
Alexa Fluor 488 azide, the incorporated DNA was detected by greatly reduced in cells with DNA replication blocked by
fluorescence microscopy (Fig. 4A). In 3T3 cells, fluorescence hydroxyurea.10 Considering the similar structures of PEdU and
was observed at 1, 10 and 100 μM EdU and PEdU, but fluo- EdU, we expected that PEdU would also label DNA. It has been
rescence was observed rarely at 0.1 μM. In HeLa cells, fluo- reported that EdU is fully replaced with dT during PCR.27 In the
rescence was observed clearly at 10 and 100 μM EdU and case of BrdU, CsCl density gradient ultracentrifugation has
PEdU, but hardly observed at 0.1 and 1 μM. There were no sig- shown that the ratios of BrdU/dT are about 28.5% and 68% in
nificant differences between EdU and PEdU. Fluorescence cells treated for 2d and 3d, respectively, after subculture.28
intensities were measured by image analysis software Image J Because of the structural similarity of EdU released from PEdU
(Fig. 4B) to compare the incorporation abilities. As a result, we and BrdU, we expected a similar incorporation ratio.
Fig. 4 Metabolic labeling of DNA in 3T3 and HeLa cells at various concentrations (0.1–100 μM) of EdU or PEdU. After 24 h, incorporated DNA was
stained with Alexa Fluor 488 azide (A). Scale bar: 50 μm. The relative fluorescence intensity was measured using image analysis software (B). n = 4.
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Fig. 5 Mice were injected i.p. with 0.2, 1 or 5 mg PEdU. After 24 h, ileums (A) and colons (B) were harvested. Sections from the organs were fixed
and stained with Alexa Fluor 488 azide. Total DNA was stained with DAPI. Scale bar: 100 μm.
In vivo metabolic DNA labeling
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To evaluate the ability of PEdU to label DNA in animals,
3-week-old mice were injected i.p. with various concentrations
of PEdU in PBS. After 24 h, their tissues were harvested and
stained with Alexa Fluor 488 and DAPI. Alexa Fluor 488 stain-
ing indicated newly synthesized DNA after PEdU injection, and
DAPI staining showed total DNA. Even at the lowest amount of
PEdU (0.2 mg), PEdU-incorporated DNA was detected clearly
(Fig. 5A and B, ileum and colon, respectively). A total of 0.2 mg
PEdU was also sufficient for DNA labeling of the liver, kidney,
and thymus (Fig. S7†). These results showed that PEdU could
be used to detect DNA synthesis in animals.
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PEdU was designed as a low toxic and efficient DNA labeling
agent. PEdU exhibited a remarkable reduction in cytotoxicity
compared with EdU. Nevertheless, PEdU was incorporated as
efficiently as EdU in metabolic DNA labeling at the same con-
centration and time. Accordingly, PEdU can be used as a less
toxic DNA labeling agent in studies that require subsequent
cell survival over long time periods or a very sensitive cell line
or animal. This study indicates the possibility for modification
of nucleoside analogues and prodrugs to enhance their bio-
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