Synthesis and enzymatic incorporation of photolabile dUTP analogues into DNA and their applications for DNA labeling

Article abstract of DOI:10.1016/j.bmc.2013.04.081

Two novel photolabile nucleotide triphosphate (NTP) analogues were synthesized through Sonogashira coupling and their enzymatic incorporation into DNA was evaluated with three different DNA polymerases (Taq, Vent exo- and T4) by polymerase chain reaction. Both nucleotide triphosphate analogues were recognized by these DNA polymerases as substrates for primer extension. Light irradiation of PCR products removed the photolabile group and released the amino and carboxyl moieties. Further site-specific dual-labeling for oligodeoxynucleotides (ODNs) and random labeling for a long DNA construct with fluorophores were successfully achieved with incorporation of the photolabile amine modified deoxyuridine triphosphate (dUnTP).

Full text of DOI:10.1016/j.bmc.2013.04.081

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and enzymatic incorporation of photolabile dUTP
analogues into DNA and their applications for DNA labeling
⇑
Junzhou Wu, Xinjing Tang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Rd., Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
Two novel photolabile nucleotide triphosphate (NTP) analogues were synthesized through Sonogashira
coupling and their enzymatic incorporation into DNA was evaluated with three different DNA polymer-
ases (Taq, Vent exo- and T4) by polymerase chain reaction. Both nucleotide triphosphate analogues were
recognized by these DNA polymerases as substrates for primer extension. Light irradiation of PCR prod-
ucts removed the photolabile group and released the amino and carboxyl moieties. Further site-specific
dual-labeling for oligodeoxynucleotides (ODNs) and random labeling for a long DNA construct with fluo-
rophores were successfully achieved with incorporation of the photolabile amine modified deoxyuridine
triphosphate (dUnTP).
Keywords:
Photolabile nucleotides
Photoactivation
Enzymatic incorporation
Nucleotide analogues
Fluorescence labeling
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Photocaging strategy is widely used in DNA solid phase synthesis,
but the length of DNA synthesized by solid phase synthesis is lim-
Enzymatic incorporation of modified nucleotide triphosphates
into DNA has a variety of potential applications in modern molec-
ular biology, such as DNA conjugation, sequence analysis, nano-
technology, and DNA sensing.1–9 In literature, nucleotide
triphosphates carrying different functionalities (amine,10,11 car-
boxylic acid,12 alkyne,2,13–15 azide,16,17 etc.) have been enzymati-
cally incorporated into DNA strands, followed by covalent
derivatization with functional tags, such as biotin,16 fluoro-
phores10,18 or peptides.19 This post-modification approach is par-
ticularly useful for large tags that are not suitable substrates for
DNA polymerases. Photocaging strategy is widely used as a pro-
tecting method for modification of oligonucleotides during solid
phase synthesis. Photocaged phosphate, amino, carboxyl, thiol, car-
bonyl in the middle or at 30 or 50 ends of modified oligonucleotides
have been previously achieved by solid phase synthesis.20–24 How-
ever, photolabile nucleotide triphosphates were limited to be
recoverable chain terminators in applications of DNA enzymetic
synthesis.4,5,25–28 We are interested in developing photolabile
deoxyuridine triphosphate analogues for DNA post-modification.
Comparing with post-modification methods reported before, its
advantages are as follows: (1) when DNA is labeled with multiple
functional tags, photocaging strategy may become a good choice in
addition to unprotected or other protected functional groups. (2)
Unprotected functional groups, especially amino group, may have
electrostatic interactions with DNA polymerases, which may lead
to the inhibition of some polymerase activities.1,11,12 (3)
ited. Photolabile nucleotide triphosphates, as substrates of DNA
polymerases would open up the possibility of synthesizing longer
DNA constructs which are usually inaccessible through chemical
synthesis. Here, we synthesized two photolabile nucleotide tri-
phosphate analogues (dUnTP and dUoTP, Scheme 1) that contained
functional substitutions (amine and carboxylic acid, respectively)
protected by a photocleavable group. The position 5 of deoxyuri-
dine is widely used to introduce new functionalities and substitu-
tions that protrude from major groove of DNA duplex and is not
involved in Watson–Crick base-pairing. Nucleotides that modified
at this position are usually well accepted by DNA polymer-
ases.10,12,29 Three different DNA polymerases (Taq, Vent exo- and
T4) were used to evaluate enzymatic incorporation of these two
photolabile nucleotide triphosphate analogues in short oligonucle-
otides. Further labeling of these oligonucleotides and a long DNA
construct were successfully achieved by incorporation of photola-
bile dUnTP, followed by light irradiation and coupling with a fluo-
rescent tag.
2. Results and discussion
2.1. Synthesis of photolabile dUoTP and dUnTP
a
-Methyl substituted o-nitrobenzyl moiety has been widely used
as a caging group in nucleic acids due to its higher stability in DNA/
RNA synthesis and enhanced photochemical cleavage than that of its
parent 2-nitrobenzyl group. In our previous study, we applied this
photolabile moiety as a temporary protecting group for the
introduction of functionality at both 50 and 30 of oligonucleotides
⇑
Corresponding author. Tel./fax: +86 10 82805635.
E-mail address: xinjingt@bjmu.edu.cn (X. Tang).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.081
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J. Wu, X. Tang / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Scheme 1. Synthesis of dUoTP and dUnTP.
in solid phase synthesis.20 However, for construction of a longer DNA
or RNA with the incorporation of functional groups, photolabile NTP
analogues are in need. Two photolabile dUTP analogues were then
synthesized with 50-iodo-deoxyuridine as the starting material, as
shown in Scheme 1. In order to introduce amine or carboxylic groups
to deoxyuridine, we first synthesized photolabile ester (2) and car-
bamate (3), followed by coupling to 50-iodo-deoxyuridine through
the palladium-assisted Sonogashira cross-coupling reaction.24,30
These corresponding nucleoside analogues (4 and 5) were obtained
with the yields of 72% and 59%, respectively. If the above reactions
continued overnight, we got the other two nucleoside analogues
which were confirmed to be 6 and 7 (Scheme S1 in Supplementary
data) according to NMR spectra, UV–vis spectra (Figure S1) and liter-
ature.31–34
photocleavage rate than that of dUnTP. (Figure S1) Under our
photolysis conditions, the half-lives of dUnTP and dUoTP
disappearance were 5.0 and 7.5 min, respectively. The uncaged
The resulting nucleoside analogues further reacted with POCl3
to form active intermediates, followed by the addition of tributy-
lammonium pyrophosphate according to the procedure of Yoshik-
awa reaction.35,36 The reaction was quenched with the addition of
triethylammonium bicarbonate (TEAB), and reaction intermediates
were converted to corresponding triphosphate analogues (dUoTP,
dUnTP). After reverse phase HPLC purification, the obtained dUTP
analogues were then characterized by 1H NMR, 31P NMR and mass
spectroscopy (see Supplementary data).
2.2. UV deprotection study of dUoTP and dUnTP
Before enzymatic incorporation of dUoTP and dUnTP into DNA,
we evaluated their photocleavable behaviours. The solutions of
dUoTP (1 mM, 3 mL) and dUnTP (1 mM, 3 mL) were irradiated with
UV light (365 nm, 11 mW/cm2). At different time point, 200
lL
irradiated solution was taken for the analysis by reverse phase
HPLC. Figure 1A shows that the peak intensity of photolabile dUn-
TP (retention time 9.5 min) gradually decreased with almost com-
plete photocleavage in 10 min, while the peak intensity of uncaged
product (dUnTP⁄, retention time 3.2 min) gradually increased. The
loss of starting materials and appearance of uncaged products were
detected in HPLC traces (Table S1) and were used to quantify the
cleavage efficiency (Figure S2). For dUoTP, we observed slower
Figure 1. Photolysis of dUnTP. (a) HPLC traces of dUnTP with different irradiation
time point; (b) percentages of dUnTP disappearance (9.5 min) and dUnTP⁄
production (3.2 min) after integration of each peak in HPLC traces.
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J. Wu, X. Tang / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
products, dUnTP⁄ and dUoTP⁄, were characterized and confirmed
with 1H NMR, 31P NMR and MS (see Supplementary data).
2.3. Enzymatic incorporation of modified nucleotide
triphosphate analogues
Previously, photocaged nucleotide triphosphate analogues were
mainly used for strong temporary chain terminators, which the
caging groups prevented further addition of dNTP upon the incor-
poration of caged NTP analogues by polymerases. To study the ef-
fect of photocleavable groups on enzymatic incorporation of dUoTP
and dUnTP, dUoTP, dUnTP and their corresponding uncaged nucle-
otides (dUoTP⁄ and dUnTP⁄) were utilized in primer extension as-
says. Three commercially available DNA polymerases (Taq, Vent
exo- and T4) were chosen to evaluate enzymatic incorporation of
these modified nucleotide triphosphate analogues. A 33mer tem-
plate and a 24mer Fam-labeled primer were designed for assay
experiments. An adenosine at the 25th position of the template
served as the paired base for the above dUTP analogues.
Primer extension assays were analyzed by determining the
length of extension products via gel electrophoresis. The gel anal-
yses of dUTP analogue incorporation are summarized in Figure 2.
Panel A shows the incorporation of dUnTP and dUnTP⁄, and panel
B shows the incorporation of dUoTP and dUoTP⁄. For Taq and Vent
exo-DNA polymerases, lane ‘control’ shows no extension of primer
with the existence of dATP, dGTP and dCTP. But for T4 DNA poly-
merase, lane ‘control’ shows the disappearance of 24 nt-long pri-
mer and the appearance of a new shorter band due to 30 ? 50
exonuclease activity of this enzyme. In comparison, lane ‘dTTP’ cor-
responds to the use of complete set of dNTPs, which led to the dis-
appearance of 24 nt-long primer due to the full primer extension.
Replacement of dTTP with dUnTP, dUnTP⁄, dUoTP and dUoTP⁄ did
not have an obvious effect on the full extension of the primer,
which indicates that the enzymatic reaction of dUTP analogues
with three different polymerases proceeded successfully under
standard conditions of each enzyme. We then manually reduced
the polymerase activity (Vent exo-polymerase) by lowering the
reaction temperature to 35 °C. We did find the fast initial coupling
of dTTP than that of dUnTP or dUoTP, however, the coupling effi-
ciency reach to the similar level after 30 min incubation time un-
der our conditions (Figure. S4).
Figure 3. Denaturing PAGE analysis of dual-labeled ODN before and after SYBR
Gold DNA stain. Lane 1: Primer only, Lane 2: Primer extension reaction with
sequential addition of dUnTP⁄ and dUnTP, Lane 3: ODN synthesized in lane 2
conditions and labeled with TAMRA alone, Lane 4: ODN synthesized in lane 2
conditions and labeled with TAMRA and FITC. (A) Fluorescence image of gel before
staining; (B) fluorescence image of gel after staining.
2.4. Site-specific single-labeling and dual-labeling of
oligodeoxynucleotides with photolabile deoxyuridine
analogues
A preliminary experiment with a short oligonucleotide was first
tested with Vent exo-polymerase. We applied the same sequences
of the template and primer used previously, except that the primer
was not labeled with fluorescein. After the completion of primer
extension, the reaction solution was irradiated, followed by buffer
exchange to remove tris buffer. Further labeling with FITC was suc-
cessfully achieved. The denaturing PAGE analysis of FITC labeled
ODN before and after staining with SYBR™ Gold DNA stain is
shown in Figure S5. The extended primer labeled with FITC was ob-
served in gel before DNA staining, which was further confirmed to
be the fully extended product by SYBR Gold staining. Furthermore,
the absorption and emission spectra of the enzymatically synthe-
sized DNA post-labeled with FITC were shown in Figure 4A and B.
Another template containing two adenine nucleobases was also
designed for site-specific dual-labeling with a non-fluorescein la-
beled primer (Scheme 2). At the initial primer extension reaction,
dUnTP⁄ was incorporated, but the reaction was stopped due to
no dCTP in solution. After removal of all dNTPs, fresh enzyme with
dUnTP and all other three dNTP was added to the recollected pri-
mer/template duplex in reaction buffer. dUnTP was then incorpo-
rated and fully extended primer was obtained. After buffer
exchange, 5/6-carboxytetramethyl-rhodamine, succinimidyl ester
(5/6-TAMRA, SE) was added into the oligonucleotide solution for
labeling the amine group of dUnTP⁄, and the obtained TAMRA la-
beled ODN was then deprotected by UV irradiation to release the
other amine group of dUnTP. Further labeling with FITC was suc-
cessfully achieved. The denaturing PAGE analysis of dual-labeled
ODN before (Panel A) and after (Panel B) staining with SYBR™ Gold
DNA stain is shown in Figure 3. From the fluorescence images of
the gel, ODN labeled with only TAMRA or both TAMRA and FITC
showed a bright fluorescence band on gel. And fluorescence signal
of dual labeling was more intense than single labeling. Further
staining with SYBR™ Gold confirmed full extension of the primer
with the incorporation of dUnTP⁄ and dUnTP. The absorption and
emission spectra of synthesized ODN with or without dual labeling
were also shown in Figure 4C and D. From fluorescence emission
spectra, FRET between fluorescein and TAMRA was clearly ob-
Figure 2. Denaturing PAGE analysis of primer extension reactions with (A) dUnTP,
dUnTP⁄ or (B) dUoTP, dUoTP⁄ using Taq, Vent exo- or T4 DNA polymerases. Lane
‘marker’ corresponds to the fluorescence labeled 24 nt-long primer. Lane ‘control’
corresponds to PCR reaction with the mixture of template, primer and three dNTPs
(dATP, dGTP and dCTP).
Scheme 2. Flowchart of dual-labeling. PCR-1: PCR with dUnTP⁄ and dATP, then
buffer and dNTPs were removed; PCR-2: PCR with dUnTP, dATP, dCTP and dGTP.
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J. Wu, X. Tang / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Figure 4. Absorption (A) and emission (B) spectra of FITC-labeled ODN. Absorption (C) and emission (D) spectra of dual-labeled ODN with TAMRA and FITC (kex = 488 nm).
Absorption (E) and emission (F) spectra of a long multiple FITC-labeled DNA construct.
served, which confirmed the effective uncaging and sequentially
labeling of these two fluorophores.
and DNA without FITC labeling (Lane 2) in panel B. Comparing
Lanes 2 and 3 in panel B, DNA construct without labeling showed
2.5. Random labeling of a long DNA construct
After having successfully achieved the incorporation of dUnTP
and post-modification of short oligodeoxynucleotides, we next
chose a 539 bp template from PUC18 plasmid to investigate
whether dUnTP is capable for effective incorporation of a long
DNA construct with Vent exo-polymerase. In a typical PCR experi-
ment, 50 lM dUnTP together with 250 lM all four natural dNTPs
were added to reaction mixture. After 40 cycles, the reaction mix-
ture was subject to light irradiation and buffer exchange, followed
by DNA labeling with FITC. The agarose gel analyses of FITC labeled
DNA before (Panel A) and after (Panel B) staining with ethidium
bromide (EB) DNA stain are shown in Figure 5. There existed a clear
and intense fluorescence band only for the enzymatically synthe-
sized DNA labeled with FITC (Lane 3). This band corresponded to
the 539 bp DNA construct as indicated for DNA ladder (Lane 1)
Figure 5. Agarose gel electrophoresis of FITC labeled DNA before and after staining
with ethidium bromide DNA stain. Lane 1: 100 bp DNA ladder, Lane 2: 539 bp DNA
construct for PUC18 plasmid, Lane 3: 539 bp DNA construct labeled with FITC. (A)
Fluorescence image of gel before EB staining; (B) fluorescence image of gel after EB
staining.
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J. Wu, X. Tang / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
clear band in the gel, while there existed another faint band of DNA
construct after FITC labeling. This may be due to multiple labeling
with FITC that disrupted the condensed DNA coil structure. Suc-
cessful labeling of a long DNA construct with FITC was further con-
firmed by the absorption and emission spectra of FITC labeled DNA,
as shown in Figure 4E and F. By calibration of absorbance of DNA
(260 nm) and FITC (488 nm), the overall efficiency of uncaging
and labeling [labeled deoxyuracil/(deoxyuracil + thymine)] was
determined to be 12%, which was 60% of the ratio of dUnTP/dTTP
(20%) that were added to the reaction mixture.
whole mixture was stirred at room temperature for 1 h, and was
then poured into ethyl acetate (50 mL). The organic layer was
washed with 8% citric acid aq (2 ꢀ 50 mL), brine (2 ꢀ 50 mL), and
dried over Na2SO4. After removal of ethyl acetate, the organic layer
was concentrated in vacuo, and the residue was purified by short
silica gel column, eluted with petroleum ether:ethyl acetate
(10:1) to give 2 as a clear, yellow oil (970 mg, 3.93 mmol, yield
70%). 1H NMR (400 MHz, CDCl3) d 7.95 (dd, J = 8, 1 Hz, 1H), 7.66–
7.60 (m, 2H), 7.43 (m, 1H), 6.36 (q, J = 6 Hz, 1H), 2.60–2.55 (m,
2H), 2.51–2.46 (m, 2H), 1.95 (t, J = 3 Hz, 1H), 1.65 (d, J = 6 Hz,
3H); 13C NMR (100 MHz, CDCl3) d 170.71 147.88, 137.91, 133.65,
128.56, 127.38, 124.60, 82.41, 69.32, 68.67, 33.55, 22.14, 14.43.
3. Conclusion
4.2. Synthesis of 1-(2-nitrophenyl)ethyl hex-5-yn-1-yl
carbamate (3)
In summary, we have synthesized two new photolabile deoxy-
uridine triphosphate analogues (dUoTP, dUnTP) with temporarily
protected functionalities of amine and carboxylic acid moieties.
These two triphosphates were then evaluated for their incorpora-
tion into oligodeoxynucleotides using three commercially avail-
able DNA polymerases (Taq, Vent exo- and T4). All three DNA
polymerases showed quite effective primer extension with the
existence of dUoTP, dUnTP, or their uncaged dUoTP⁄ and dUnTP⁄.
In addition, dUnTP was effectively incorporated in both short oligo-
deoxynucleotides and a long DNA construct from PUC18 plasmid.
Further labeling of the long DNA construct and site-specific dual-
labeling of oligodeoxynucleotide were successfully achieved with
fluorophores. This work represents the first example of enzymatic
introduction of photocaged functional moieties into DNA for fur-
ther labeling using photolabile nucleotide triphosphate analogues.
To a solution of 1-(2-nitrophenyl)ethyl N-succinimidyl carbon-
ate (see Supplementary data) (1.02 g, 3.0 mmol) in DCM (15 mL),
was added TEA (1.70 ml, 12.0 mmol) and 5-hexyn-1-amine hydro-
chloride (see Supplementary data) (400 mg, 3.0 mmol). The mix-
true solution was stirred at room temperature for 2 h and was
then diluted with DCM (100 mL). The organic layer was washed
with brine (3 ꢀ 50 mL), dried over Na2SO4. After concentration in
vacuo, the residue was purified by short silica gel column with
petroleum ether:ethyl acetate (1:1) to give 3 as a clear, yellow oil
(670 mg, 2.3 mmol, yield 77%).1H NMR (400 MHz, CDCl3) d 7.90
(d, J = 8 Hz, 1H), 7.61 (d, J = 4 Hz, 2H), 7.44–7.34 (m, 1H), 6.22 (q,
J = 6 Hz, 1H), 3.14 (m, 2H), 2.20 (m, 2H), 1.94 (t, J = 6 Hz, 1H),
1.60 (q, J = 6 Hz, 3H), 1.55–1.46 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 155.32, 147.61, 138.78, 133.48, 128.15, 127.05, 124.39, 83.97,
68.71, 68.52, 40.44, 28.95, 25.47, 22.24, 18.05.
4. Experimental section
All chemical solvents used were dried and distilled. Dichloro-
methane (DCM), triethylamine (TEA), tetrahydrofuran (THF), and
acetonitrile were dried over CaH2 and were freshly distilled before
every use. All chemical reagents were purchased from Alfa Aesar,
Sigma–Aldrich, or J&K chemicals, and used without further purifi-
cation. All reactions were monitored by TLC using commercial
Merck plates coated with silica gel GF254 (0.24 mm thick). Flash
column chromatography was performed with silica gel purchased
from Qingdao Haiyang Chemical Company (200–300 mesh). 1H
NMR (400 MHz), 13C NMR (100 MHz) and 31P NMR (162 MHz)
spectra were recorded on a Bruker spectrometer at 25 °C. Chemical
shifts (d, ppm) were quoted relative to the residual solvent and
coupling constants (J) were corrected and quoted to the nearest
1 Hz. MS spectra were measured on Q-TOF spectrometer using
electrospray ionization (ESI) or FT-MS (Bruker APEX IV 7.0T). HPLC
were performed with Alliance e2695 and Agilent XDB C-18 column
4.3. Synthesis of 4
To
a solution of 1-(2-nitrophenyl)ethyl 4-pentynoate (2)
(750 mg, 3 mmol) in acetonitrile (6 mL), was added 5-iodo-20-
deoxyuridine (710 mg, 2.0 mmol), palladium(0)tetrakis(triphenyl-
phosphine) (115 mg, 0.1 mmol), cuprous iodide (40 mg, 0.2 mmol)
and TEA (1.1 mL, 8.0 mmol). The whole mixture was stirred at
60 °C for 2 h under nitrogen atmosphere. The solvents were re-
moved in vacuo, and the remaining crude mixture was purified
by silica gel column with 0–5% MeOH in DCM to give 4 as a yellow
foamy solid (550 mg, 1.16 mmol, yield 58%). 1H NMR (400 MHz,
MeOD) d 8.17 (d, J = 4 Hz, 1H), 7.97–7.85 (m, 1H), 7.78–7.69 (m,
1H), 7.70–7.62 (m, 1H), 7.53–7.42 (m, 1H), 6.29–6.18 (m, 2H),
4.45–4.33 (m, 1H), 3.93 (q, J = 3 Hz, 1H), 3.84–3.68 (m, 2H), 2.70–
2.58 (m, 4H), 2.38–2.12 (m, 2H), 1.63 (d, J = 6 Hz, 3H). 13C NMR
(100 MHz, MeOD)
d 172.69, 164.45, 151.11, 149.10, 144.63,
(50 ꢀ 4.6 mm, 1.8
lm beads). When necessary, the reactions were
conducted in a dark room.
138.58, 134.88, 129.69, 129.69, 128.51, 125.17, 100.64, 93.09,
88.99, 86.91, 73.29, 72.00, 69.63, 69.61, 62.58, 41.54, 34.23,
22.10, 16.00. ESI+ MS: [M+Na]+ 496.28, Calcd [M+Na]+ 496.13.
Oligonucleotides were purchased from Shanghai Sangon Bio-
tech. Deoxyribonucleotide triphosphate (dATP, dCTP, dGTP, and
dTTP) were purchased from Promega. Vent exo-DNA polymerase
was purchased from New England Biolabs. T4 DNA polymerase
was purchased from Fermantas. Taq DNA polymerase and other
biochemical reagent were purchased from TAKARA. G-25 sephadex
column (Nap-10 columns sephadex G-25 DNA Grade) was pur-
chased from GE healthcare. Polymerase chain reaction (PCR) was
performed on ABI Veriti 96-well Thermal Cycler.
e
289 = 11600 L molꢁ1 cmꢁ1
.
4.4. Synthesis of 5
To a solution of 1-(2-nitrophenyl)ethyl hex-5-yn-1-yl carba-
mate (3) (667 mg, 2.3 mmol) in acetonitrile (6 mL), was added
5-lodo-20-deoxyuridine (750 mg, 2.1 mmol), palladium(0)tetra-
kis(triphenylphosphine) (115 mg, 0.1 mmol), cuprous iodide
(40 mg, 0.2 mmol), TEA (1.1 mL, 8 mmol). The mixture solution
was stirred at 60 °C for 1.4 h under nitrogen atmosphere. After re-
moval of solvents in vacuo, the remaining residue was purified by
silica gel column with 0–5% MeOH in DCM to give 5 as a yellow
foamy solid (780 mg, yield 72%). 1H NMR (400 MHz, MeOD) d
8.20 (d, J = 3.0 Hz, 1H), 7.94 (d, J = 8 Hz, 1H), 7.74–7.66 (m, 2H),
7.51–7.44 (m, 1H), 6.24 (m, 1H), 6.12 (q, J = 6 Hz, 1H), 4.43–4.37
4.1. Synthesis of 1-(2-nitrophenyl)ethyl 4-pentynoate (2)
To
5.6 mmol) in DCM (12 mL) was added 4-pentynoic acid (500
5.6 mmol), (DCC, 2.06 g,
N,N0-dicyclohexylcarbodiimide
10.0 mmol) and 4-dimethylamiopryidine (240 mg, 2.0 mmol). The
a
solution of 1-(2-nitrophenyl) ethanol (1) (940 mg,
lL,
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J. Wu, X. Tang / Bioorg. Med. Chem. xxx (2013) xxx–xxx
(m, 1H), 3.94–3.91 (m, 1H), 3.83–3.63 (m, 2H), 3.06 (t, J = 6 Hz, 2H),
2.39–2.23 (m, 4H), 1.61 (d, J = 6 Hz, 3H), 1.58–1.50 (m, 4H). 13C
NMR (100 MHz, MeOD) d 164.68, 157.93, 151.21, 149.04, 144.31,
139.98, 134.85, 129.52, 128.16, 125.26, 101.12, 94.72, 89.04,
86.85, 72.94, 72.02, 69.42, 62.58, 41.62, 41.14, 29.85, 26.63,
22.47, 19.79. ESI+ MS: [M+Na]+ 539.62, Calcd [M+Na]+ 539.18.
primer and 1.2 lM template, 50 lM dATP, dGTP and dCTP, 50 lM
dUTP analogues, 0.4 U Taq or 0.2 U Vent exo- or 0.2 U T4 DNA poly-
merase in 1ꢀ polymerase buffer as given by the suppliers. The
incubation procedure included steps of a 4 min denaturation at
94 °C, 8ꢀ (94 °C, 30 s; 55 °C, 30 s; 68 °C, 2 min) for Taq and Vent
exo-polymerases, steps of 37 °C, 20 min and 72 °C, 10 min for T4
polymerase. Reaction temperature was returned to 4 °C after each
e
289 = 12,000 L molꢁ1 cmꢁ1
reaction. Then an aliquot (5
l
L) of the unpurified reaction was
4.5. Synthesis of dUoTP
mixed with 1
l
L loading buffer (10ꢀ DNA loading buffer: Formam-
ide = 1:4). After denaturation at 92 °C for 2 min, primer extension
products were electrophoresed on a denaturing 20% (v/v) poly-
acrylamide gel containing 7 M urea (1ꢀ TBE buffer; pH = 8.2). Gels
were imaged with Molecular Imager ChemiDoc™ XRS+ before and
after SYBR™ Gold staining.
To a cooled solution (0 °C) of 4 (120 mg, 0.25 mmol) and 1,8-
bis(dimethylamino) naphthalene (proton sponge, 107 mg,
0.50 mmol) in trimethyl phosphate (2 mL), was added phospho-
rous oxychloride (38 lL, 0.40 mmol) dropwise over 10 min under
nitrogen atmosphere. The reaction mixture was stirred for
40 min at 0 °C. A solution of tributylammonium pyrophosphate
(250 mg, 0.50 mmol) in dry DMF (2.0 mL) and TEA (140 lL,
4.8. Enzymatically synthesis and dual-fluorophore labeling of
oligodeoxynucleotide with photolabile dUnTP
1.0 mmol) were added. The reaction mixture was stirred for an-
other 30 min, followed by quenching with triethylammonium
bicarbonate (TEAB, 0.1 M, 10 mL). The mixture was left to warm
to room temperature whilst stirring for 2 h. Reverse phase HPLC
purification (A, 0.05 M TEAA in H2O; B, acetonitrile; B, 0–60% in
25 min, 60% in 5 min 60–0% in 5 min) yielded dUoTP as the trieth-
ylammonium salt, further desalting was performed with ion-
exchange resin (H+ form). (Yield 46%, determined by the
absorbance at 260 nm) 1H NMR (400 MHz, D2O) d 7.93–7.76 (m,
2H), 7.69 (m, 1H), 7.56 (d, J = 7 Hz, 1H), 7.44–7.31 (m, 1H),
6.26–6.09 (m, 2H), 4.51–4.48 (m, 1H), 4.22–4.01 (m, 3H), 2.69–
2.53 (m, 4H), 2.40–2.21 (m, 2H), 1.56 (t, J = 9 Hz, 3H). 31P NMR
(162 MHz, D2O) d ꢁ10.304 (br), ꢁ11.012 (br), ꢁ22.719 (br).
FT-MSꢁ: [MꢁH]ꢁ 712.0345, [Mꢁ2H+Na]ꢁ 734.0167, [MꢁH2PO3]ꢁ
632.0668; Calcd [MꢁH]ꢁ 712.0346, [Mꢁ2H+Na]ꢁ 734.0165,
[MꢁH2PO3]ꢁ 632.0683.
Primer extension reactions were carried out in PCR tubes
(200
M non-Fam labeled primer (50-ATG GGA CTA ACT AAT CTT
TGC TTA-30) and 1.2 M template (50-TCC TAG TTA TAA GCA AAG
AAGA TTA GTT AGT CCC AT-30), 50 M dATP and dUnTP⁄, 1 U Vent
lL) in a total volume of 50 lL. Each reaction contained
1
l
l
l
exo- in 1ꢀ Vent exo-polymerase buffer as given by the suppliers.
After the RCR reaction steps of 4 min denaturation at 94 °C and
8ꢀ (94 °C, 30 s; 55 °C, 30 s; 68 °C, 2 min), reaction temperature
was returned to 4 °C. The mixture was passed through Millipore
Amicon™ Ultra (Ultracel-3 Membrane, 3.5 kDa) to remove tris buf-
fer and dNTP, and the detained residue was redissolved in 1ꢀ Vent
exo-polymerase buffer. Then 50 lM dATP, dCTP, dGTP, 50 lM dUn-
TP and 1 U Vent exo- were added. After the RCR reaction steps of
4 min denaturation at 94 °C and 8ꢀ (94 °C, 30 s; 60 °C, 30 s;
68 °C, 2 min), reaction temperature was returned to 4 °C. The mix-
ture was passed through Millipore Amicon™ Ultra (Ultracel-3
Membrane, 3.5 kDa) to remove dNTP and tris buffer and the de-
4.6. Synthesis of dUnTP
tained residue was redissolved in 50
pH = 8.3). 5/6-Carboxytetramethylrhodamine, succinimidyl ester
(10 mM in DMF, 5 L) was added into the mixture and vortexed
at 60 °C for 12 h. After sedimentation and desalting (NAP-10), the
TAMRA labeled oligonucleotide was redissolved in 50 L NaHCO3
lL NaHCO3 (0.1 mM,
To a cooled solution (0 °C) of 5 (80 mg, 0.16 mmol) and 1,8-
bis(dimethylamino) naphthalene (proton sponge, 60 mg,
0.20 mmol) in trimethyl phosphate (2 mL) was added phosphorous
oxychloride (19 lL, 0.20 mmol) dropwise over 10 min under nitro-
gen atmosphere. The reaction mixture was stirred for 40 min at
0 °C. A solution of tributylammonium pyrophosphate (160 mg,
0.29 mmol) in dry DMF (2.0 mL) and TEA (69 lL, 0.50 mmol) were
l
l
(0.1 mM, pH = 8.3) and irradiated by UV light (365 nm, 11 mW/
cm2) for 10 min to remove the photolabile group. FITC (10 mM in
DMSO, 5 lL) was added into the mixture and it was vortexed at
then added. The reaction mixture was stirred for another 5 min,
followed by quenching with triethylammonium bicarbonate
(0.1 M, 10 mL). The reaction solution was left to warm to room
temperature whilst stirring for 2 h. RP-HPLC purification (A,
0.05 M TEAA in H2O; B, acetonitrile; B, 0–60% in 25 min, 60% in
5 min 60–0% in 5 min) yield dUnTP as the triethylammonium salt,
further desalting was performed with ion-exchange resin (H+
form). (Yield 21%, determined by the absorbance at 260 nm) 1H
NMR (400 MHz, D2O) d 7.99–7.82 (m, 2H), 7.68 (s, 2H), 7.44 (s,
1H), 6.22 (s, 1H), 6.02 (s, 1H), 4.13 (m, 3H), 4.56 (s, 1H), 2.99 (s,
2H), 2.80 (s, 1H), 2.53 (s, 1H), 2.33 (s, 2H), 1.56 (d, J = 6 Hz, 3H),
1.37 (m, 4H). 31P NMR (D2O, 162 MHz): d ꢁ8.926 (br), ꢁ12.115
(br), ꢁ22.764 (br). FT-MSꢁ: [MꢁH]ꢁ 755.0763, [MꢁH2PO3]ꢁ
675.1149; Calcd [MꢁH]ꢁ 755.0768, [MꢁH2PO3]ꢁ 675.1105.
60 °C for 12 h. After sedimentation, desalting and concentration,
the dual-fluorophore labeled oligonucleotide was obtained. The
obtained residue was redissolved in 50
lL H2O, and a aliquot
(5 lL) of above mixture was mixed with 1
lL loading buffer and
electrophoresed on 20% (v/v) polyacrylamide gel containing 7 M
urea (1ꢀ TBE buffer, pH = 8.2). The same gel was then imaged be-
fore and after SYBR™ Gold staining by Molecular Imager Chemi-
Doc™ XRS+.
4.9. Synthesis and random labeling of a long DNA construct
with photolabile dUnTP
4.9.1. Template preparation
PCR was carried out in PCR tubes (200
100 L. Each reaction contained 1
M forward primer (50-GAG
GCG GTT TGC GTA TTG G-30), 1 M reverse primer (50-GTC GTG
TCC TAC GGG GTT GGA-30), 1 ng PUC18 plasmid, 250
M dNTP,
lL) in a total volume of
l
l
4.7. Primer extension assay
l
Template sequence: 50-TCC TTG TGA TAA GCA AAG ATT AGT
TAG TCC CAT-30.
l
2 U Taq in 1ꢀ Taq polymerase buffer as given by the supplier. The
incubation steps consisted of a 4 min denaturation at 94 °C, and
30ꢀ (94 °C, 15 s; 55 °C, 15 s; 72 °C, 1 min). The reaction temperature
was returned to 4 °C after the reaction was completed. The obtained
DNA was recovered by TAKARA™ PCR product recovery kit,
Primer sequence: 50-Fam-ATG GGA CTA ACT AAT CTT TGC TTA-
30.
Primer extension reactions were carried out in PCR tubes (200
lL) in a total volume of 10 lL. Each reaction contained 1 lM
Please cite this article in press as: Wu, J.; Tang, X. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.081

J. Wu, X. Tang / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
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l
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Cicero, R.; Clark, S.; Dalal, R.; Dewinter, A.; Dixon, J.; Foquet, M.; Gaertner, A.;
Hardenbol, P.; Heiner, C.; Hester, K.; Holden, D.; Kearns, G.; Kong, X.; Kuse, R.;
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4.9.2. Preparation and labeling of a long modified DNA
construct
PCR was carried out in PCR tubes (200
L. Each reaction contained 1
M forward primer (50-GAG
GCG GTT TGC GTA TTG G-30), 0.01
M long DNA template as pre-
pared before, 250 M dNTP, 50
M dUnTP, 1 U Vent exo- in 1ꢀ
lL) in a total volume of
50
l
l
l
l
l
Vent exo-polymerase buffer as given by the supplier. The incuba-
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(94 °C, 15 s; 55 °C, 15 s; 68 °C, 2 min). The reaction temperature
was returned to 4 °C after the reaction was completed. The reaction
mixture was irradiated by UV light (365 nm, 11 mW/cm2) for
10 min to remove the photolabile group. According to the same
working procedure as the synthesis and labeling of the above oli-
godeoxynucleotide, the obtained residue was redissolved in
50 lL H2O, and a aliquot (5 lL) of above mixture was mixed with
1
l
L loading buffer and electrophoresed on 1% agarose gel (1ꢀ
TAE buffer, pH = 8.0). The same gel was then imaged before and
after staining with EB by Molecular Imager ChemiDoc™ XRS+
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Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 21072015), the National Basic Research
Program of China (973 Program; Grant No. 2012CB720600), Pro-
gram for New Century Excellent Talents in University (Grant No.
NCET-10-0203), and Scientific Research Foundation for the Re-
turned Overseas Chinese Scholars (Grant No. JWSL44-9).
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M. L. Nucleic Acids Res. 2011, 39, e39.
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Hersh, M. N.; Metzker, M. L. Nucleic Acids Res. 2012, 40, 7404.
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Supplementary data
Supplementary data associated (supporting figures, synthetic
procedure of 5-hexyn-1-amine and 1-(2-nitrophenyl)ethyl N-succ-
inimidyl carbonate and characterization of intermediates and
products) with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2013.04.081.
References and notes
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Please cite this article in press as: Wu, J.; Tang, X. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.081