One-step to get 5-azidomethyl-2′-deoxyuridine from 5-hydroxymethyl-2′-deoxyuridine and detection of it through click reaction

Article abstract of DOI:10.1016/j.tet.2013.08.069

Nowadays a few ways to synthesize 5-azidomethyl-2′-deoxyuridine from 5-hydroxymethyl-2′-deoxyuridine have been reported. But none of them was one-step. And many of them need to protect the hydroxyl group on the pentose ring. The detection of 5-hydroxymethyl-2′-deoxyuridine is also very important in many biological processes. However few fluorescence detection strategies have been tried to do this. Herein, we reported a one-step protocol to synthesize 5-azidomethyl-2′-deoxyuridine, which was then used for detecting 5-hydroxymethyl-2′-deoxyuridine through a click reaction.

Full text of DOI:10.1016/j.tet.2013.08.069

Tetrahedron xxx (2013) 1e5
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Tetrahedron
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One-step to get 5-azidomethyl-20-deoxyuridine from 5-
hydroxymethyl-20-deoxyuridine and detection of it through click
reaction
,
,
Xiaowei Xu a y, Shengyong Yan a y, Jianlin Hu a, Pu Guo a, Lai Wei a, Xiaocheng Weng a,
Xiang Zhou a
,b,
*
a College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University, Hubei, Wuhan
430072, PR China
b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, PR China
a r t i c l e i n f o
a b s t r a c t
Nowadays a few ways to synthesize 5-azidomethyl-20-deoxyuridine from 5-hydroxymethyl-20-deoxy-
uridine have been reported. But none of them was one-step. And many of them need to protect the
hydroxyl group on the pentose ring. The detection of 5-hydroxymethyl-20-deoxyuridine is also very
important in many biological processes. However few fluorescence detection strategies have been tried
to do this. Herein, we reported a one-step protocol to synthesize 5-azidomethyl-20-deoxyuridine, which
was then used for detecting 5-hydroxymethyl-20-deoxyuridine through a click reaction.
Ó 2013 Published by Elsevier Ltd.
Article history:
Received 4 May 2013
Received in revised form 25 August 2013
Accepted 28 August 2013
Available online xxx
Keywords:
5-Hydroxymethyl-20-deoxyuridine
Click reaction
Fluorescence
One-step
Detection
1. Introduction
yields of these routes are not satisfactory and more reagents are
required in these routes.
DNA methylation is related to a variety of diseases, making the
development of good detection methods important. Recently,
much work has been done to study DNA methylation; particularly,
the detection of 5-hmC and 5-mC has attracted enormous
attention.1e10 In addition, 5-hydroxymethyl-20-deoxyuridine (5-
hmdU) is a widely researched form of oxidative damage to
DNA,11,12 and the level of 5-hmdU in DNA from blood is a marker of
breast cancer, as found by Zora and co-workers.13 However, the
detection of 5-hydroxymethyl-20-deoxyuridine, which is involved
in the demethylation of DNA, has attracted little attention.14 Herein,
we provide a simple method to detect 5-hmU in vitro. First, the
hydroxyl group at the C-5 position group of 5-hydroxymethyl-20-
deoxyuridine selectively reacted with sodium azide in trifluoro-
acetic acid to produce 5-azidomethyl-20-deoxyuridine (5-amdU) in
onestep, which was followed by reaction with an alkynyl fluores-
cent probe (Scheme 1). Previously, 5-azidomethyl-20-deoxyuridine
has already been synthesized through several routes15e18 but the
Scheme 1. Schematic illustration of fluorescence turn-on detection of 5-hmdU based
on click reaction.
Our original intention was to detect 5-hydroxymethyl-cytosine,
but interestingly, 5-hydroxymethylcytosine did not react with so-
dium azide in trifluoroacetic acid (TFA). This phenomenon also
demonstrated that the sodium azide did not reacted with the hy-
droxy group on the furan ring. We next tried 5-hydroxymethyl-20-
deoxyuridine and found that the yield was surprisingly high (85%).
This phenomenon may be attributed to the intramolecular hydro-
gen bond formed between the 5-hydroxyl group and the 4-amino
group; this feature can help us distinguish 5-hmdU from 5-hmC.
In order to differentiate 5-hmdU from the four natural nucleosides
* Corresponding author. E-mail address: xzhou@whu.edu.cn (X. Zhou).
y
These authors contributed equally to this work.
0040-4020/$ e see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.tet.2013.08.069
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X. Xu et al. / Tetrahedron xxx (2013) 1e5
in DNA, the four natural bases were treated using the procedure
described and the mixture of the four nucleosides also exhibited
weak fluorescence (Fig. 1). Thus, we can conclude that the selec-
tivity of this strategy is relatively good.
15 mL trifluoroacetic acid in a 50 mL bottle, and then sodium azide
(130 mg, 2 mmol) was added slowly into it. The mixture was stirred
at room temperature overnight. Then the mixture was neutralized
by saturated sodium bicarbonate to pH¼7. The mixture was con-
centrated in vacuo to remove the solvent. The residue was purified
by column chromatography (SiO2, CH2Cl2/methanol¼10:1) to get 5-
amdU (241 mg, 0.85 mmol) as white solid, yield¼85%. 1H NMR
60
50
40
30
20
10
(300 MHz, DMSO-d6)
d (ppm): 11.56 (s, 1H), 8.03 (s, 1H), 6.14 (t,
J¼6.3 Hz,1H), 5.28 (d, J¼3.9 Hz,1H), 5.07 (s,1H), 4.23 (s,1H), 4.05 (s,
2H), 3.78 (q, J¼2.7 Hz, 1H), 3.52e3.62 (m, 2H), 2.01e2.11 (m, 2H);
13C NMR (DMSO-d6, 75 MHz)
d (ppm): 162.82,150.11,139.83,108.19,
87.35, 84.11, 70.14, 61.09, 46.77, 40.13; HRMS (ESI) calcd for
C
10H13N5O5 [MꢁH]ꢁ: 282.0838; found: 282.0841.
2.3.2. 7-Ethynyl-2H-chromen-2-one (CA). CA was synthesized
20
according to Melanie Chtchigrovsky and co-workers. 1H NMR
ꢀ
(300 MHz, DMSO-d6)
J¼7.8 Hz), 7.51 (s, 1H), 7.43 (d, 1H, J¼7.5 Hz), 6.52 (d, 1H, J¼9.9 Hz),
4.50 (s, 1H); 13C NMR (CDCl3, 75 MHz)
(ppm): 160.46, 153.74,
142.97, 128.27, 128.01, 125.81, 120.38, 119.31, 117.40, 82.28, 80.97;
HRMS (ESI) m/z: calcd for C11H6O2 [MþNa]þ 193.0265, found:
193.0259.
d
(ppm): 8.06 (d, 1H, J¼9.9 Hz), 7.71 (d, 1H,
0
d
dU
dT
dG
dC
dA
mixture
5-hm
Fig. 1. Fluorescence intensity of selectivity experiments.
2.3.3. 1-((2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-5-((4-(2-oxo-2H-chromen-7-yl)-1H-1,2,3-triazol-1-yl)methyl)py-
rimidine-2,4(1H,3H)-dione (CZ). 5-amdU (28.3 mg, 0.1 mmol) was
dissolved in 5 mL H2O and 5 mL DMF, then catalytic amount of
copper sulfate pentahydrate and sodium ascorbate were added into
the flask. At last, CA (17 mg, 0.1 mmol) was poured into mixture. The
mixture was stirred overnight at room temperature. The solvents
were removed in vacuo and the residue was washed with water and
methanol and then recrystallized from methanol to get CZ (43.5 mg,
0.096 mmol) as white solid. Yield¼96%. 1H NMR (300 MHz, DMSO-
2. Experimental section
2.1. Materials
The following solvents, compounds, and reagents were com-
mercially available: 20-deoxyuridine, 7-hydroxyl coumarine, HPLC/
spectroscopy grade acetonitrile were bought from SigmaeAldrich.
Dimethyl Formamide, triethylamine, copper sulfate pentahydrate,
sodium ascorbate were bought from SCRC (Shanghai, China). The
DNA sequence was bought from Suzhou Ribo Life Science Co. Ltd.
(China).
d6)
d
(ppm): 11.62 (s, 1H), 8.68 (s, 1H), 8.23 (s, 1H), 8.07 (d, J¼9.6 Hz,
1H), 7.70e7.90 (m, 3H), 6.48 (dd, J¼9.6 Hz, 1H), 6.17 (t, J¼3 Hz, 1H),
5.20e5.32 (m, 3H), 5.07 (s,1H), 4.26 (s,1H), 3.80 (s,1H), 3.58 (m, 2H),
2.2. Instruments
2.10e2.20 (m, 2H); 13C NMR (DMSO-d6, 75 MHz)
d (ppm): 162.57,
159.96, 154.08, 150.27, 144.68, 143.97, 141.43, 141.40, 134.25, 129.12,
122.71, 121.18, 118.17, 115.81, 112.15, 107.04, 87.56, 84.54, 70.15, 61.09,
46.58; HRMS (ESI) calcd for C21H19N5O7 [MþH]þ: 454.1363, found:
454.1359, [MþNa]þ: 476.1182, found: 476.1178.
1H and 13C NMR spectra were recorded on Varian Mercury 300
and 400 spectrometers, respectively. HRMS were recorded on
a Bruker Daltonics, Inc. APEXIII 7.0 TESLA FTMS, and Varian Pro-
MALDI. API-ES were recorded on a Agilent LC/MSD. Fluorescent
emission spectra were collected on PerkinElmer LS 55. UV ab-
sorption spectra were collected on SHIMADZU UV-2550. HPLC
spectra were recorded on a Laballiance Series III. Quartz cuvettes
with 600 mL volume were used for emission measurements. Unless
otherwise specified, all spectra were taken at an ambient
temperature.
2.3.4. 2-Ethyl-6-ethynyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(NP). NP was synthesized according to Sawa and co-workers.21 1
H
NMR (300 MHz, CDCl3)
7.92 (d, 1H, J¼5.7 Hz), 7.82 (m, 1H), 4.23 (m, 2H), 3.73 (s, 1H), 1.33
(m, 3H); 13C NMR (CDCl3, 75 MHz)
(ppm): 163.97, 132.38, 131.86,
d
(ppm): 8.64 (m, 2H), 8.52 (d,1H, J¼5.7 Hz),
d
130.32, 127.91, 126.40, 123.24, 86.75, 80.57, 35.86, 13.60.
2.3. Synthesis
2.3.5. 2-Ethyl-6-(1-((1-(5-hydroxy-4-(hydroxymethyl)tetrahydrofu-
ran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl)-1H-
1,2,3-triazol-4-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(NZ). The synthesis route of NZ was the same as CZ. NZ is dark red
2.3.1. 5-Azidomethyl-20-deoxyurdine (5-amdU).19 20-Deoxyuridine
(5.25 g, 23.0 mmol) and paraformaldehyde (3.11 g, 103.5 mmol)
were added into 250 mL bottle with two necks and dissolved in
80 mL 0.5 mol/L triethylamine aqueous solution. The mixture was
stirred at 60 ꢀC for 4 days. During the reaction, more para-
formaldehyde (4.49 g, 149.5 mmol), triethylamine (1 mL), and
water (10 mL) was added into reaction mixture each day. After
reaction finished, the mixture was concentrated in vacuo to remove
the solvent. The residue was recrystallized in methanol to obtain
4.11 g 5-hmdU as white solid, yield¼62%. 1H NMR (300 MHz,
solid and the yield is 90%. 1H NMR (300 MHz, DMSO-d6)
d (ppm):
11.63 (s, 1H), 9.13 (s, 1H), 8.77 (s, 1H), 8.49 (s, 1H), 8.26 (s, 1H), 8.07
(s, 1H), 7.88 (s, 1H), 6.18 (s, 1H), 5.36 (s, 3H), 5.08 (s, 1H), 4.27 (s, 1H),
4.06 (s, 1H), 3.59 (s, 1H), 2.18 (s, 2H); 13C NMR (DMSO-d6, 75 MHz)
d
(ppm): 163.12, 162.80, 162.66, 150.24, 144.40, 141.31, 133.92,
130.74, 130.30, 128.17, 128.08, 127.50, 127.01, 125.35, 122.35, 121.49,
107.15, 87.53, 84.55, 70.14, 61.22, 46.68, 34.79, 13.08; HRMS (ESI)
calcd for C26H24N6O7 [MþNa]þ: 555.1598, found: 555.1599.
DMSO-d6)
d
(ppm): 11.34 (s, 1H), 7.71 (s, 1H), 6.17 (t, J¼6.8 Hz, 1H),
5.26 (s, 1H), 4.97 (s, 2H), 4.20 (q, J¼3.2 Hz, 1H), 4.10 (s, 2H), 3.75 (q,
2.3.6. 10-(4-Ethynylphenyl)-5,5-difluoro-1,3,7,9-tetramethyl-5H-di-
pyrrolo[1,2-c:20,10-f][1,3,2]diazaborinin-4-ium-5-uide (AB). AB was
synthesized according to Li and co-workers.22 1H NMR (300 MHz,
J¼3.2 Hz, 1H), 3.57e3.47 (m, 2H), 2.09e1.99 (m, 2H); 13C NMR
(DMSO-d6, 75 MHz)
d (ppm): 162.5, 150.2, 136.7, 114.1, 87.1, 83.7,
70.4, 61.3, 55.9, 35.2. 5-hmdU (258 mg, 1 mmol) was dissolved in
CDCl3)
d
(ppm): 7.62 (d, 2H, J¼7.2 Hz), 7.27 (d, 2H, J¼6.9 Hz), 5.98 (s,
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X. Xu et al. / Tetrahedron xxx (2013) 1e5
3
2H), 2.55 (s, 6H), 1.40 (s, 6H). 13C NMR (CDCl3, 75 MHz)
d (ppm):
155.96, 143.12, 140.69, 135.69, 133.03, 131.26, 128.33, 123.08, 121.58,
83.02, 78.83, 14.78.
2.3.7. 5,5-Difluoro-10-(4-(1-((1-(5-hydroxy-4-(hydroxymethyl)tet-
rahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)
methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,3,7,9-tetramethyl-5H-di-
pyrrolo[1,2-c:20,10-f][1,3,2]diazaborinin-4-ium-5-uide (AZ). The syn-
thesis route of AZ was the same as CZ. AZ is red solid and the yield is
92%. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.59 (s, 1H), 8.60 (s,
1H), 8.24 (s, 1H), 8.04 (d, J¼5.7 Hz, 2H), 7.35e7.55 (m, 2H), 6.19 (s,
3H), 5.27 (s, 3H), 5.06 (s, 1H), 5.07 (s, 1H), 4.27 (s, 1H), 3.81 (s, 1H),
3.60 (m, 2H), 2.46 (s, 6H), 2.17 (s, 2H), 1.41 (s, 6H); 13C NMR (DMSO-
Scheme 2. Synthesis route of CZ, NZ, AZ from CA, NP, AB.
d6, 75 MHz)
d (ppm): 163.30, 155.62, 150.97, 145.99, 143.41, 142.34,
fluorescence emission that was unfavorable for the following
fluorescent detection. We synthesized CA by reported methods,
and CZ was synthesized as described below.16 The yield of the click
reaction was 96%. CZ has a very strong fluorescence emission
compared with CA (Fig. 2a). NZ and AZ were also compared with
142.03, 133.98, 132.26, 131.39, 129.14, 126.50, 122.53, 122.11, 107.90,
88.26, 85.25, 70.84, 61.87, 47.18, 14.86; HRMS (ESI) calcd for
C
31H32N7O5BF2 [MꢁH]þ: 630.2557, found: 630.2557.
2.4. LOD (limit of detection) calculation
The background fluorescence intensity of 500 mM CA (in DMF/
(a)
H2O¼1:1) was tested three times to get three data. Then we calcu-
lated out the standard deviation of the three data. And we multiplied
the standard deviation by three times to get a value. This value was
substituted in to the fitting equation as y (y¼36.1741þ0.20723*x),
and calculated the value of x. And x was the LOD.
1000
800
CZ
CZ
CA
600
400
200
0
2.5. General procedure for selectivity and concentration gra-
dient experiments
All the nucleosides were dissolved in DMSO as the mother li-
quor. And different volumes of them (according to the concentra-
tions needed) were added in to 400 mL TFA and the mixture was
CA
350
400
450
500
550
600
stirred overnight at room temperature. After the reaction, the sol-
vents were removed in vacuo. The residue was neutralized to pH¼7
by adding saturated sodium bicarbonate. The mixture was con-
centrated by centrifuging. Then the residue was redissolved in
/ nm
(b)
100
then concentrated by freeze drying. The residue was dissolved in
200 L DMF and 200 L H2O, then 2 L of 10 mM CA in DMSO,
catalysis amount of copper sulfate pentahydrate and sodium
ascorbate were added, the final volume was 400 L. The mixture
was stirred overnight at room temperature. And then the mixture
was concentrated and dissolved in water (400 L) to investigate the
mL DMSO and centrifuged to remove the salts. The liquid was
400
AB
m
m
m
AZ
AZ
m
200
m
fluorescence propriety. Excitation is 328 nm. Emission is 402 nm.
2.6. General procedure for enzymatic hydrolysis of ss-DNA
A 15nt single strand DNA sequence was dissolved in water and the
AB
0
520 540 560 580 600 620 640 660
/ nm
concentration is 100
hmUAGCCGTA-30. And the control sequence is 50-ACTCAACAGCCGTA-
30. 5
L solution of the DNA was incubated with 2 L S1 nuclease and
L buffer under 37 ꢀC for 16 h. The total volume was 20
L by adding
water into the mixture. Then 2 L alkaline phosphate and 20 L of its
buffer were added. The final volume was 100 L, the exerted volume
m
mol/L. The sequence is 50-GACTCAA5-
(c)
m
m
1000
800
600
400
200
4
m
m
m
m
NZ
m
NP
NZ
was compensated with water. And the mixture was incubated under
37 ꢀC foranother 4 h. At last the solvents were removed bycentrifuging.
The following steps were the same as mentioned above.
3. Results and discussions
NP
600
We chose coumarin as the fluorophore because the precursor,
7-acetylene coumarin (CA), has little or no fluorescence. Several
other fluorophores were investigated, such as derivatives of
BODIPY and naphthalic anhydride that also contain an alkynyl
group (Scheme 2), but both of them had a strong background
0
400
450
500
550
650
/ nm
Fig. 2. Fluorescent properties of CZ, NZ, AZ compared with CA, NP, AB.
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4
X. Xu et al. / Tetrahedron xxx (2013) 1e5
NP and AB. The insets were photos taken under UV lamp (excita-
tion is 365 nm). The fluorescence quantum yield of CZ is
We found that the fluorescence intensity increased as the con-
centration of 5-hmdU increased, with a reasonable linear re-
lationship between the concentration of 5-hmdU and the
fluorescence intensity (Fig. 3). We calculated the limit of detection
was not a potential problem under these conditions. As there are
a lot of peaks in the spectrum, we used MS to confirm the result
(Fig. S20). We could not figure out the other peaks. We calculated
the final yield of all these steps, which was 54%. We propose two
reasons for this lower yield. First, we may have lost some of the 5-
hmdU during the enzymatic hydrolysis of the DNA. Second, the
reaction efficiency decreased as the concentration of 5-hmdU de-
creased, in accordance with the concentration gradient experiment
described above.
F
¼8.4%.
(LOD) of 5-hmdU, which was 2.78 mM. The LOD was higher than
the reported methods, in which the order of magnitude was pmol
or even lower.10 This is the disadvantage because the amount of 5-
hmdU in practical samples is very low.
(a)
100
80
60
40
20
0
400
450
500
550
Fig. 4. The UV spectrum of CZ and CA.
nm
4. Conclusions
(b)
90
80
70
60
50
40
30
y = 36.1741 + 0.20723*x
In summary, we provide a one-step approach to replace the 5-
hydroxyl group of 5-hydroxymethyl-20-deoxyuridine with an
azide group, so that the azide-modified deoxyuridine can be linked
with a fluorophore via a click reaction. The UV and fluorescence
proprieties of the click reaction product were determined, and we
also tried to detect 5-hmdU from DNA using this strategy. As the
clinical samples are very complicated and the detection sensitivity
of our method is not very good, it is still very difficult to detect 5-
hmdU from clinical samples. Further optimization of the reaction
conditions is on-going.
2
R =0.93
0
50
100
150
200
250
Acknowledgements
Concentration M
Fig. 3. Concentration gradient and linear results.
This work was supported by the National Basic Research Pro-
gram of China (973 Program) (2012CB720600, 2012CB720603), the
National Science Foundation of China (Nos. 91213302, 21272181,
21072115). Supported by ‘Program for Changjiang Scholars and
Innovative Research Team in University (IRT1030)’. Supported
by the National Grand Program on Key Infectious Disease
(2012ZX10003002-014). Supported by ‘the Fundamental Research
Funds for the central Universities’ (2012203020212).
The mixture of the four bases is a model of DNA that is used in
the following experiments. However, when considering the sta-
bility of DNA in TFA, we found that DNA was unstable and that the
structure of DNA was destroyed under this condition. When we
tried this method without considering the integrity of the DNA we
observed the change in fluorescence (Fig. S3), which was far from
satisfactory. Thus, the application of this method under biological
conditions was severely limited. We tried other approaches to
produce 5-amdU under milder conditions, but all of them were
unworkable. Finally, we attempted to hydrolyze single-strand DNA
containing 5-hmdU into nucleosides using S1 nuclease and alkaline
phosphatase, and then performed our detection strategy as de-
scribed above (Fig. S5).
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2013.08.069.
References and notes
We analyzed the reaction mixture with high-performance liquid
chromatography. 5-hmdU has a maximal UV absorbance at 254 nm,
and the product of the click reaction, CZ, has a maximal UV ab-
sorbance at 328 nm (Fig. 4). Under the same elution conditions, the
retention times of the four natural nucleosides, 5-hmdU and CA
(Fig. S5) were all longer than those of CZ, so their co-eluting with CZ
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Please cite this article in press as: Xu, X.; et al., Tetrahedron (2013), http://dx.doi.org/10.1016/j.tet.2013.08.069