2,6-Diaminopurine nucleoside derivative of 9-ethyloxy-2-oxo-1,3- diazaphenoxazine (2-amino-Adap) for recognition of 8-oxo-dG in DNA

Article abstract of DOI:10.1016/j.bmc.2014.01.024

8-Oxo-2′-deoxyguanosine (8-oxo-dG) is a nucleoside resulting from oxidative damage and is known to be mutagenic. 8-Oxo-dG has been related to aging and diseases, including neurological disorders and cancer. Recently, we reported that a fluorescent nucleoside derivative, adenosine-1,3- diazaphenoxazine (Adap), forms a stable base pair with 8-oxo-dG in DNA with accompanying efficient quenching. In this study, a new Adap derivative having an additional 2-amino group on the adenosine moiety (2-amino-Adap) was designed with the anticipation of additional hydrogen bonding with the 8-oxo group of 8-oxo-dG. The properties of the ODN containing 2-amino-Adap were evaluated by measuring thermal stability and fluorescence quenching. In contrast to the previously designed Adap, the base-pairing and fluorescence quenching properties of 2-amino-Adap varied depending on the ODN sequence, and there was no clear indication of an additional hydrogen bond with 8-oxo-dG. Instead, the base pairing of 2-amino-Adap with dG was significantly destabilized compared with that of Adap with dG, resulting in improved selectivity for 8-oxo-dG in the human telomere DNA sequence. Thus, the telomere-targeting ODN probe containing 2-amino-Adap displayed selective, sensitive and quantitative detection of 8-oxo-dG in the human telomere DNA sequence in a light-up detection system using SYBR Green.

Full text of DOI:10.1016/j.bmc.2014.01.024

Bioorganic & Medicinal Chemistry 22 (2014) 1634–1641
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2,6-Diaminopurine nucleoside derivative of 9-ethyloxy-2-oxo-1,3-d-
iazaphenoxazine (2-amino-Adap) for recognition of 8-oxo-dG in
DNA
⇑
⇑
Yosuke Taniguchi , Keitaro Fukabori, Yoshiya Kikukawa, Yohei Koga, Shigeki Sasaki
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
a r t i c l e i n f o
a b s t r a c t
8-Oxo-20-deoxyguanosine (8-oxo-dG) is a nucleoside resulting from oxidative damage and is known to be
mutagenic. 8-Oxo-dG has been related to aging and diseases, including neurological disorders and cancer.
Recently, we reported that a fluorescent nucleoside derivative, adenosine-1,3-diazaphenoxazine (Adap),
forms a stable base pair with 8-oxo-dG in DNA with accompanying efficient quenching. In this study, a
new Adap derivative having an additional 2-amino group on the adenosine moiety (2-amino-Adap)
was designed with the anticipation of additional hydrogen bonding with the 8-oxo group of 8-oxo-dG.
The properties of the ODN containing 2-amino-Adap were evaluated by measuring thermal stability
and fluorescence quenching. In contrast to the previously designed Adap, the base-pairing and fluores-
cence quenching properties of 2-amino-Adap varied depending on the ODN sequence, and there was
no clear indication of an additional hydrogen bond with 8-oxo-dG. Instead, the base pairing of
2-amino-Adap with dG was significantly destabilized compared with that of Adap with dG, resulting
in improved selectivity for 8-oxo-dG in the human telomere DNA sequence. Thus, the telomere-targeting
ODN probe containing 2-amino-Adap displayed selective, sensitive and quantitative detection of 8-oxo-
dG in the human telomere DNA sequence in a light-up detection system using SYBR Green.
Ó 2014 Published by Elsevier Ltd.
Article history:
Received 24 December 2013
Revised 14 January 2014
Accepted 18 January 2014
Available online 29 January 2014
Keywords:
Oxidative damage
8-Oxo-20-deoxyguanosine
2-Oxo-1,3-diazaphenoxazine
Telomere
Fluorescent nucleoside
Light-up detection
1. Introduction
using a FRET-based Adap-masked probe.11 Based on a plausible
complex structure, shown in Figure 1A, in which Adap forms multi-
8-Oxo-20-deoxyguanosine (8-oxo-dG) is ubiquitous in the
nucleotide triphosphate pool and in DNA after exposure to ultravi-
olet radiation and oxidative stress.1,2 8-Oxo-dG is an inducer of
genotoxicity during gene replication steps, which is in turn pre-
vented by repair enzymes targeting 8-oxo-dGTP and 8-oxo-dG.3
8-oxo-dG is yet unaffected by the repair system to some extent,
and it has been related to aging and diseases, such as neurological
disorders and cancers. Several methods have been developed to
measure 8-oxo-dG levels in biological samples, including HPLC–
EDC,4 HPLC–MS/MS,5 GC–MS,6 and immunoassays,7 among oth-
ers.8 Fluorescent derivatives of 2-oxo-1,3-diazaphenoxazine (8-
oxoG clamp) were developed by our group as the first recognition
molecules for 8-oxo-dG in solution.9 Recently, we reported that the
adenosine derivative of 2-oxo-1,3-diazaphenoxazine (Adap) forms
a selective base pair with 8-oxo-dG in DNA with accompanying
effective fluorescence quenching.10 Subsequently, Adap was ap-
plied to the light-emitting fluorescence detection of 8-oxo-dG by
ple hydrogen bonds on both the Hoogsteen face and the Watson-
Crick face of 8-oxo-dG, we became interested in investigating the
manner in which an additional 2-amino group on the adenosine
moiety (2-amino-Adap in Fig. 1B) affects the recognition of 8-
oxo-dG. It was also expected that the 2-amino group of the aden-
osine moiety might induce steric repulsion between the 2-amino
group of guanosine in the anti-conformation in the minor groove,12
thus potentially improving selectivity for 8-oxo-dG. In this paper,
we describe in detail the synthesis and evaluation of oligonucleo-
tides containing 2-amino-Adap.
2. Results and discussion
2.1. Synthesis of 2-amino-Adap and its oligonucleotides
The synthesis of 2-amino-Adap and the oligodeoxynucleotides
(ODNs) are shown in Scheme 1. 20-Deoxyguanosine (1) was pro-
tected by the 30-O- and 50-O-TBDMS groups and converted to the
2,4,6-triisopropylbenzenesulfonyl derivative (2). A substitution
reaction was performed with the phenoxazine unit 3 in the pres-
ence of diisopropylethylamine to give TBS protected 2-amino-Adap
⇑
Corresponding authors. Tel./fax: +81 92 642 6615.
E-mail addresses: taniguch@phar.kyushu-u.ac.jp (Y. Taniguchi), sasaki@phar.
kyushu-u.ac.jp (S. Sasaki).
http://dx.doi.org/10.1016/j.bmc.2014.01.024
0968-0896/Ó 2014 Published by Elsevier Ltd.

Y. Taniguchi et al. / Bioorg. Med. Chem. 22 (2014) 1634–1641
1635
precursor (6) using the conventional method. The synthesized
ODNs were cleaved from the CPG resin, and the Pac groups were
deprotected in 28% aqueous ammonium solution at 55 °C for 6 h.
The products were purified by HPLC and treated with 5% acetic acid
to remove the DMTr-protecting group. The structure and purity of
the synthesized ODNs were confirmed by MALDI-TOF analysis.
O
(A)
N
N
N
H
O
H
O
H
H
N
N
N
N
O
N
O
N
H
O
O
H
N
N
N
O
N
2.2. Thermal denaturing study of ODNs containing 2-amino-
Adap
O
O
Adap
O
(B)
A thermal denaturing study was performed to determine Tm
values using the 13 base pair duplex formed between ODN1 and
ODN2 in a buffer containing 100 mM NaCl and 10 mM sodium
phosphate buffer at pH 7.0. The Tm value at 2 lM of duplex DNA
and thermodynamic parameters were obtained from van’t Hoff
O
N
N
N
H
O
H
O
H
H
N
N
N
N
N
O
N
O
plots with five data points and are summarized in Table 1.
N
H
O
O
H
N
N
The Tm value of the combination of X = 2-amino-Adap and Y = 8-
oxo-dG was 47.6 °C, and the value for X = 8-oxo-dG and Y = 2-ami-
no-Adap was 46.1 °C. These Tm values are equivalent to those for
natural GC and AT base pairs. The Tm values for mismatched com-
binations were lower than those for the 2-amino-Adap and 8-oxo-
dG pairs (X = 2-amino-Adap: Tm = 31.6, 35.6, 39.4 and 35.1 °C for
dA, dC, T and dG, respectively. Y = 2-amino-Adap: Tm = 31.9, 33.0,
37.4 and 35.5 °C for dA, dC, T and dG, respectively.). To confirm a
stabilizing effect of the diazaphenoxazine unit of 2-amino-Adap,
the Tm values of ODN containing 2-amino-20-deoxyadenosine (2-
amino-dA) as a control base were measured. The combination of
2-amino-Adap and 8-oxo-dG gave higher Tm values than that of
2-amino-dA and 8-oxo-dG (40.2 °C vs 47.6 °C and 41.2 vs
46.1 °C), indicating that the diazaphenoxazine interacts with the
Watson-Crick face of 8-oxo-dG in a syn conformation (Fig. 1B).
These results show that 2-amino-Adap discriminates 8-oxo-dG
from other nucleosides in a DNA duplex. However, it has not been
clearly confirmed whether the 8-oxo-dG and 2-amino-Adap base
pair is further stabilized by the additional 2-amino group in a du-
plex with homopyrimidine/homopurine context.
O
N
N
H
O
O
H
2-amino-Adap
O
Figure 1. Expected recognition structure and model structure for base pair
formation. (A) Adap:8-oxo-dG, (B) 2-amino-Adap:8-oxo-dG.
O
O
N
S
O
O
N
N
NH
NH2
N
N
a, b
N
HO
TBDMSO
O
N
NH2
O
OH
OTBDMS
1
2
O
O
N
N
O
N
N
H
N
O
N
H
N
O
N
H
N
O
O
NH2
O
O
2.3. Fluorescence quenching property of 2-amino-Adap in ODNs
NH
NH
3
d,e
c
N
N
N
N
The fluorescence quenching property of 2-amino-Adap in ODNs
was investigated by measuring the fluorescence spectra in the ab-
sence and presence of the complementary ODN using 50 nM of
each ODN in a buffer containing 100 mM NaCl and 10 mM sodium
phosphate at pH 7.0 and 30 °C. These spectra are illustrated in Fig-
ure 2. The fluorescence of 2-amino-Adap in ODN1 was partly
quenched by 8-oxo-dG in ODN2 (Fig. 2A). Surprisingly, the fluores-
cence of 2-amino-Adap in ODN1 was increased by the addition of
dG-containing ODN2 (Fig. 2B). In contrast, the fluorescence spectra
of 2-amino-Adap in ODN2 was effectively quenched by 8-oxo-dG
in ODN1 while retaining the same intensity when base-paired with
TBDMSO
HO
N
N
O
NH2
O
NHPac
N
N
O
OTBDMS
OH
5
4
N
N
H
N
O
O
ODN1
ODN2
X
: 5'-CTTTCT CTCCTT
NH
f, g
h, i
Y
: 5'-AAGGAG AGAAAG
N
N
ODN3: 5'-CCCTAACZCTAACTA
ODN4
Z
: 5'-CCCTAACC TAACTA
DMTrO
N
O
NHPac
N
dG in ODN1 (Fig. 2C vs D). Based on the
DG° values at 30 °C in Ta-
X
Y
Z
and = 2-amino-Adap
,
ble 1, the duplex concentrations for each ODN combinations were
calculated. In cases where the matched 2-amino-Adap and 8-oxo-
dG combination was included (Fig. 2A and C), more than 95% of the
fluorescent ODN1 having 2-amino-Adap was estimated to be in the
duplex form, which represented the fluorescence spectra shown in
the red curve. In cases where the mismatched 2-amino-Adap and
dG combination was included (Fig. 2B and D), 36–40% of the fluo-
rescent ODN2 having 2-amino-Adap was estimated to be in the du-
plex form. The red curves of these spectra contain contributions of
these duplexes. In the previous study, the mismatched dG-Adap
combination in the duplex DNA did not cause significant fluores-
cence quenching similarly with Figure 2D.10 As the fluorescence
intensity of 2-amino-Adap in ODN1 is significantly weaker than
in ODN2 (Fig. 2A vs C), it is likely that 2-amino-Adap might be
stacked within ODN1 in a manner that causes fluorescence
O
P
CN
N
O
6
Scheme 1. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, 73%; (b)
TiPBSCl, Et3N, DMAP, CH2Cl2, rt, 94%; (c) Phenoxazine unit 3, DIPEA, 1-propanol,
reflux, 88%; (d) phenoxyacetylchloride, pyridine, 0 °C, 88%; (e) triethylamine–
trihydrofluoride, THF, 90%; (f) DMTrCl, pyridine, rt, 64%; (g) 2-Cyanoethyl-N,N-
diisopropylchlorophosphoramidite, DIPEA, CH2Cl2, 0 °C, 88%; (h) DNA synthesis; (i)
HPLC purification, DMTr deprotection.
(4). The 2-amino group of 4 was protected with a phenoxyacetyl
group, and the TBS groups were removed to produce compound
5. Finally, 5 was converted to the corresponding phosphoramidite

1636
Y. Taniguchi et al. / Bioorg. Med. Chem. 22 (2014) 1634–1641
Table 1
Thermodynamic parameters of 2-amino-Adap-containing ODNs complexed with complementary ODNsa
ODN1
ODN2
Tm (°C)
D
H°
D
S°cal/K mol
DG°30 °C
X
Y
kcal/mol
kcal/mol
2-Amino-Adap
2-Amino-Adap
2-Amino-Adap
2-Amino-Adap
2-Amino-Adap
dA
dC
T
dG
8-Oxo-dG
dC
dG
T
dA
dA
dC
T
dG
31.6
35.6
39.4
35.1
47.6
31.9
33.0
37.4
35.5
46.1
44.1
48.2
40.9
41.9
34.2
40.2
33.5
41.2
ꢀ111 14
ꢀ336 46
ꢀ278 19
ꢀ228 24
ꢀ9.19 0.3
ꢀ10.7 0.06
ꢀ11.5 0.15
ꢀ10.1 0.02
ꢀ14.0 0.2
ꢀ9.29 0.2
ꢀ11.8 0.1
ꢀ10.5 0.08
ꢀ10.2 0.06
ꢀ14.3 0.3
ꢀ13.4 0.2
ꢀ15.3 0.8
ꢀ11.8 0.2
ꢀ12.3 0.3
ꢀ9.86 0.07
ꢀ11.8 0.09
ꢀ969 0.03
ꢀ12.1 0.1
ꢀ94.9
ꢀ80.6
ꢀ77.2
ꢀ101
6
7
3
6
ꢀ221
8
8-Oxo-dG
2-Amino-Adap
2-Amino-Adap
2-Amino-Adap
2-Amino-Adap
2-Amino-Adap
dG
dC
dA
T
dG
ꢀ287 17
ꢀ405 34
ꢀ269 18
ꢀ224 17
ꢀ240 16
ꢀ306 25
ꢀ288 20
ꢀ328 55
ꢀ259 22
ꢀ266 38
ꢀ215 16
ꢀ269 18
ꢀ202 10
ꢀ276 17
ꢀ132 11
ꢀ93.3
ꢀ78.4
ꢀ82.9
ꢀ107
ꢀ101
ꢀ115
ꢀ90.3
6
5
5
8
6
6
7
ꢀ92.9 10
2-Amino-dA
2-Amino-dA
dG
ꢀ75.0
ꢀ93.3
ꢀ70.9
ꢀ95.7
5
6
3
5
8-Oxo-dG
2-Amino-dA
2-Amino-dA
8-Oxo-dG
a
UV melting profiles were measured using 2
lM of each ODN1 and ODN2. These values were determined by van’t Hoff plots with five data points (1–8
lM) and the curve
fitting method (Meltwin program). Thermodynamic data of natural base pairs are from Ref. 10b.
Figure 2. Fluorescence spectra of (A) 50 nM ODN1, 50-CTTTCTXCTCCTT, and (B) 50 nM ODN2, 30-GAAAGAYGAGGAA, containing 2-amino-Adap with excitation at 365 nm in a
buffer containing 100 mM NaCl and 10 mM sodium phosphate buffer at pH 7.0 and 30 °C. The black curves represent the spectra of single stranded ODN, and the red curves
represent the results after the addition of complementary sequences.
quenching. Consequently, the increased fluorescence intensity
shown in Figure 2B may be due to the relief of the intra-strand
stacking by the formation of the duplex.
complementary sequence. It has been reported that dG in the hu-
man telomere TAGTAG(TTAGGG)4 repeat sequence is susceptible
to oxidation to form 8-oxo-dG.13 Examples of thermal melting
curves are illustrated in Figure 3A. The duplex formed between
telomere DNA5 (X = dG, Y = 8-oxo-dG) and ODN3 (Z = 2-amino-
Adap) showed a higher Tm value than that with telomere DNA6
(X = Y = dG) and ODN3 (Z = 2-amino-Adap). The higher stability
of the former duplex was also confirmed by a CD spectrum
2.4. Recognition of 8-oxo-dG in telomere sequence
We next investigated recognition of 8-oxo-dG in the human
telomere sequence using ODN3 and ODN4 with the

Y. Taniguchi et al. / Bioorg. Med. Chem. 22 (2014) 1634–1641
1637
Figure 3. (A) Melting curves of the duplex formed between DNA6 (dG) and ODN3 ant that between DNA5 (8-oxo-dG) and ODN3 (2-amino-Adap). (B) CD spectra of DNA5
(8-oxo-dG) alone (black curve) and the complex with ODN3 (2-amino-Adap) (red curve). (C) CD spectra of DNA6 (dG) alone (black curve) and complex with ODN3 (2-amino-
Adap) (red curve). Telomere DNA, 50-TAGTAGTTA X Y GTTAGGG(TTAGGG)2, in which DNA5 contains X = dG and Y = 8-oxo-dG, DNA6 contains X, Y = dG, and DNA7 contains
X = 8-oxo-dG, Y = dG.
(Fig. 3B, red curve), indicating a typical B-form conformation. The
CD spectrum of telomere DNA6 (X = Y = dG) alone resembled that
of a G-quadruplex (Fig. 3C, black curve),14 which was changed to
a CD spectrum such that both B-type duplex and G-quadruplex
are present (Fig. 3C, red curve).
The measured Tm values are summarized in Table 2. ODN3 (2-
amino-Adap) showed stable duplex formation with the telomere
DNA5 (X = dG, Y = 8-oxo-dG), as described previously (Table 2, En-
try 1 and Fig. 3A). The duplex between ODN3 (2-amino-Adap) and
telomere DNA6 (X = Y = dG) did not show a clear sigmoid curve
(Table 2, Entry 2 and Fig. 3A). The Tm value for ODN3 (2-amino-
Adap) with telomere DNA5 (X = dG, Y = 8-oxo-dG) was almost
equal to that obtained with ODN3 (Adap) (Table 2, Entries 1 and
5). The Tm value for the duplex formed between ODN3 (2-amino-
Adap) and telomere DNA5 (X = dG, Y = 8-oxo-dG) was 19.2 °C high-
er than the value for the duplex formed with the telomere DNA6
(X, Y = dG) when measured in a buffer containing 500 mM NaCl
(Table 2, Entries 3 and 4). It was clear that 2-amino-Adap in
ODN3 formed a stable base pair with 8-oxo-dG in telomere
DNA5, whereas the duplex having the 2-amino-Adap and dG com-
bination did not show a clear melting behavior (Fig. 3A). Appar-
ently, a stabilizing effect of 2-amino-Adap for dG is smaller than
that of Adap in ODN3 (Table 2, Entry 2 vs 6). It was also shown that
2-amino-Adap in ODN4 exhibited a smaller stabilizing effect for
both 8-oxo-dG and dG in telomere DNA7 compared to Adap
(Table 2, Entry 7 vs 11). Interestingly, when the Tm values were
determined in a buffer containing 500 mM NaCl,
DTm obtained
with 2-amino-Adap for 8-oxo-dG and dG became significantly lar-
ger than that obtained with Adap in a buffer containing 100 mM
NaCl (Table 2, entry 3 vs 4; 19.2 vs 7.1 °C, entry 9 vs 10; 9.6 vs
7.1 °C). These results indicate that 2-amino-Adap in ODN3 and 4
better discriminates 8-oxo-dG over dG in the telomere DNA.
Table 2
Thermal denaturing results for ODN3 and 4 with the telomere DNA (5–7) containing 8-oxo-dG or dGa
Entry
ODN
Z
DNA
X
Y
Tm
DTm
1
2
ODN3
2-Amino-Adap
DNA5
DNA6
DNA5
DNA6
DNA5
DNA6
DNA7
DNA6
DNA7
DNA6
DNA7
DNA6
dG
dG
dG
dG
dG
dG
8-Oxo-dG
dG
8-Oxo-dG
dG
8-Oxo-dG
dG
8-Oxo-dG
dG
8-Oxo-dG
dG
dG
dG
dG
43.4
<30
>13.4
19.2
7.1
3b
4b
5
2-Amino-Adap
Adapc
50.1
30.9
44.5
37.4
37.7
<30
44.3
34.7
44.1
37.0
6
7
8
ODN4
2-Amino-Adap
2-Amino-Adap
Adapc
>7.7
9.6
9b
10b
11
12
dG
dG
dG
8-Oxo-dG
dG
7.1
a
b
c
UV-melting profiles measured using 1
The buffer here contains 500 mM NaCl and 10 mM sodium phosphate at pH 7.0.
The Tm values of ODNs containing Adap are from Ref. 10b.
Tm = Tm (8-oxo-dG) ꢀ Tm (dG). The sequences of DNA5–7 are shown in the footnote to Figure 3.
lM ODN strand in a buffer containing 100 mM NaCl and 10 mM sodium phosphate at pH 7.0.
D

1638
Y. Taniguchi et al. / Bioorg. Med. Chem. 22 (2014) 1634–1641
Figure 4. Fluorescence spectra of (A) 50 nM ODN3 containing 2-amino-Adap and (B) 50 nM ODN3 containing Adap in the same buffer conditions as in Figure 2. The black
curves represent the spectra of single stranded ODN, the red curves show those after the addition of an 8-oxo-dG-containing sequence, and the blue curves show those after
the addition of a dG-containing sequence.
2.5. Detection and quantification of 8-oxo-dG in the telomere
DNA sequence
ODN3 (2-amino-Adap) and telomere DNA5 (8-oxo-dG), whereas a
duplex is only slightly formed between ODN3 (2-amino-Adap) and
telomere DNA6 (dG) under the same conditions. SYBR Green in
buffer emits strong fluorescence by binding with a duplex; thus,
light-up detection of 8-oxo-dG in telomere DNA5 is possible. Telo-
mere DNA5 containing 8-oxo-dG was added to a mixture of ODN3
(2-amino-Adap) and SYBR Green in buffer at 30 °C, resulting in in-
creased fluorescence intensity (Fig. 5A). In contrast, the fluores-
cence intensity of SYBR Green did not increase in a solution with
the dG-containing telomere DNA6 and ODN3 (2-amino-Adap),
indicating that no duplex had formed (Fig. 5B). Similar phenomena
were observed with ODN3 (Adap), except that fluorescence had
slightly increased with telomere DNA6 (dG) because ODN3 (Adap)
formed duplex DNA under the same conditions (Fig. 5C and D). The
calibration curve was obtained using a constant ODN3 (2-amino-
Adap) concentration and a varied concentration of telomere
In our previous study, 8-oxo-dG in telomere DNA5 was detected
by the fluorescence quenching method, as shown in Figure 4B.
Similarly, the fluorescence of 2-amino-Adap in ODN3 (2-amino-
Adap) was quenched by telomere DNA5 containing 8-oxo-dG
(Fig. 4A, red line) but was unresponsive to telomere DNA6 contain-
ing dG (Fig. 4A, blue line). However, the quenching efficiency of 2-
amino-Adap in ODN3 was smaller than that of Adap in ODN3
(Adap) (Fig. 4B, red line). Analogous to the discussion of the fluo-
rescence intensity of ODN1 and 2, the fluorescence of 2-amino-
Adap tends to be quenched in this ODN, most likely due to intra-
strand stacking. Therefore, we focused on the large
DTm value be-
tween 8-oxo-dG and dG when complexed with 2-amino-Adap
(Table 2, entries 1, 3 and 2, 4). Namely, a duplex is formed between
Figure 5. Light-up detection of 8-oxo-dG in telomere DNA5 using SYBR Green. The combination of (A) 2-amino-Adap and 8-oxo-dG, (B) 2-aminoAdap and dG, (C) Adap and
8-oxo-dG and (D) Adap and dG. The fluorescence spectra were measured using 2 nM of each ODN with excitation at 494 nm in a buffer containing 100 mM NaCl and 10 mM
sodium phosphate at pH 7.0 and 30 °C.

Y. Taniguchi et al. / Bioorg. Med. Chem. 22 (2014) 1634–1641
1639
isopropylphenylsulfonyl chloride (9.2 g, 30.3 mmol) and DMAP
(154 mg, 1.3 mmol) were added to the reaction mixture at room
temperature. After stirring for 48 h, the reaction mixture was di-
luted with EtOAc. The organic layer was washed with water, dried
over Na2SO4, and evaporated under vacuum. The residue was puri-
fied by silica gel column chromatography (CH2Cl2/MeOH = 100/0–
99/1) to give compound 2 (7.3 g, 9.6 mmol, 94%) as a white
powder. 1H NMR (400 MHz, CD3OD) d (ppm) 8.13 (1H, s), 7.29
(2H, s), 6.28 (1H, t, J = 6.2 Hz), 4.64 (1H, dt, J = 5.5, 3.7 Hz), 4.24
(2H, q, J = 6.7 Hz), 3.96–3.93 (1H, m), 3.82 (1H, dd, J = 11.0,
3.7 Hz), 3.76 (1H, dd, J = 11.0, 3.7 Hz), 2.95 (1H, q, J = 6.7 Hz), 2.73
(1H, ddd, J = 13.3, 6.7, 6.2 Hz), 2.39 (1H, ddd, J = 13.3, 6.2, 4.0 Hz),
1.26 (6H, d, J = 6.7 Hz), 1.24 (12H, d, J = 6.7 Hz), 0.92 (9H, s), 0.87
(9H, s), 0.13 (6H, s), 0.05 (3H, s), 0.03 (3H, s). 13C NMR (125 MHz,
Figure 6. The calibration line of 8-oxo-dG in telomere DNA5 using SYBR Green. The
conditions are the same as in Figure 5 except that the concentrations of telomere
DNA5 are 0.2, 0.4, 0.6, 1.0 and 2.0 nM.
DMSO-d6)
d (ppm) 159.1, 156.3, 155.6, 144.5, 141.0, 140.7,
133.2, 132.3, 117.3, 88.5, 84.5, 72.6, 63.5, 41.5, 26.6, 26.4, 23.4,
21.8, 19.1, 18.7, ꢀ4.0, ꢀ4.1, ꢀ4.7, ꢀ4.8. HRMS (m/z) calcd for
C
37H64N5O6SSi2 (M+H)+ 762.4110, found 762.4150.
DNA5 (8-oxo-dG), indicating a linear correlation in the range be-
tween 0.4 and 2.0 nM (Fig. 6, close diamond) with a detection limit
of 0.2 nM (Fig. 6, open triangle).
4.1.2. 30,50-Bis-O-tert-butyldimethylsilyl-20-deoxy-6-N-{2-[(1,3-
diaza-3-methyl-2-oxo-phenoxazine-9-yl)oxy]-ethyl}-2-amino-
adenosine (4)
3. Conclusion
A reaction mixture of 2 (870 mg, 1.14 mmol), phenoxazine unit
3 (209 mg, 0.76 mmol) and DIPEA (0.20 mL, 1.14 mmol) in 1-pro-
panol (3.5 mL) was refluxed at 100 °C for 12 h. The reaction was
quenched by saturated aqueous NaHCO3. The 1-propanol was re-
moved under reduced pressure. The aqueous layer was extracted
with EtOAc. The organic layer was repeatedly washed with water
and brine, dried over Na2SO4, and evaporated under vacuum. The
residue was purified by silica gel column chromatography (Hex-
ane/EtOAc = 10/1 to 1/1) to give compound 4 (503 mg, 0.67 mmol,
88%) as a yellow powder. 1H NMR (400 MHz, CD3OD) d (ppm): 7.92
(1H, s), 7.06 (1H, br s), 6.78 (1H, t, J = 8.0 Hz), 6.59 (1H, d,
J = 8.0 Hz), 6.37 (1H, d, J = 8.0 Hz), 6.28 (1H, t, J = 6.4 Hz), 4.62–
4.59 (1H, m), 4.30–4.20 (2H, m), 3.96 (1H, dd, J = 7.6, 3.6 Hz),
3.87–3.77 (4H, m), 3.22 (3H, s), 2.59–2.55 (1H, m), 2.41–2.36
(1H, m), 0.94 (9H, s), 0.87 (9H, s), 0.10 (6H, s), 0.06 (6H, d,
J = 8.4 Hz). 13C NMR (125 MHz, CD3OD) d (ppm) 161.8, 156.3,
137.0, 129.9, 129.4, 124.5, 114.9, 109.6, 103.9, 89.2, 84.9, 73.6,
64.2, 42.1, 30.8, 27.5, 26.5, 26.3, 25.4, 19.2, 18.9, 1.50, ꢀ4.02,
ꢀ4.43, ꢀ4.56, ꢀ5.27, ꢀ5.32. HRMS (m/z) calcd for C35H54N9O6Si2
(M+H)+ 752.3730, found 752.3745.
In this study, we developed the 2-aminoadenosine derivative of
2-oxo-1,3-diazaphenoxazine (2-amino-Adap) for recognition of 8-
oxo-dG in DNA in the anticipation of the formation of an additional
hydrogen bond with the 8-oxo group of 8-oxo-dG. The recognition
properties of ODNs containing 2-amino-Adap were investigated by
measuring the Tm values and the fluorescence quenching. In con-
trast to the originally designed Adap, the base-pairing and fluores-
cence quenching properties of 2-amino-Adap were varied
depending on the ODN sequence, and there was no clear indication
that additional hydrogen bond formation with the 8-oxo group of
8-oxo-dG had occurred. Instead, the base-pairing of 2-amino-Adap
with dG was significantly destabilized compared with the base-
pairing of Adap with dG, resulting in a better selectivity for 8-
oxo-dG in the human telomere DNA sequence. Thus, ODN3 (2-ami-
no-Adap) displayed selective, sensitive and quantitative detection
of 8-oxo-dG in the human telomere DNA sequence, as indicated
by the light-up detection system using SYBR Green.
4. Experimental Section
4.1.3. 20-Deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxo-
phenoxazine-9-yl)oxy]-ethyl}-2-phenoxyacetylamino-
adenosine (5)
1H NMR, 13C NMR and 31P NMR spectra were recorded on
400 MHz and 500 MHz NMR instruments using CDCl3, CD3OD or
DMSO-d6 as a solvent. High resolution mass (HRMS) was recorded
on an EIS-TOF mass instrument in positive mode. MALDI-TOF data
were collected in negative mode.
Phenoxyacetyl chloride (0.17 mL, 0.50 mmol) was added to a
solution of 4 (376 mg, 0.50 mmol) in pyridine (5.0 mL) at 0 °C. After
stirring for 24 h at room temperature, the reaction was quenched
by saturated aqueous NaHCO3 and extracted with CH2Cl2. The or-
ganic layer was washed with brine, dried over Na2SO4, and evapo-
rated under vacuum. The residue was then washed with Et2O to
give a yellow powder (324 mg, 0.44 mmol, 88%). This yellow pow-
der (300 mg, 0.41 mmol) was treated with triethylamine-trihydro-
fluoride (0.11 mL, 0.70 mmol) in THF (3.5 mL). After stirring for 2 h,
the organic layer was evaporated under vacuum. The residue was
purified by silica gel column chromatography (CHCl3/
MeOH = 100/1 to 10/1) to give compound 5 (243 mg, 0.33 mmol,
90%) as a yellow powder. 1H NMR (400 MHz, DMSO-d6) d (ppm)
10.12 (1H, s), 9.92 (1H, br s), 8.49 (1H, br s), 8.28 (1H, s), 7.47
(1H, br s), 7.29 (2H, t, J = 8.0 Hz), 6.93 (3H, m), 6.74 (1H, t,
J = 8.2 Hz), 6.56 (1H, d, J = 8.2 Hz), 6.40 (1H, d, J = 8.2 Hz), 6.28
(1H, t), 5.27 (1H, d), 5.04 (2H, br s), 4.91–4.87 (1H, m), 4.40–4.36
(1H, m), 4.08–4.05 (2H, m), 3.88 (1H, br s), 3.84–3.80 (1H, m),
3.60–3.47 (2H, m), 3.18 (3H, s), 2.65–2.63 (1H, m), 2.49–2.47
(1H, m). 13C NMR (125 MHz, DMSO-d6) d (ppm) 167.6, 157.9,
4.1. Synthesis of 2-amino-Adap phosphoramidite precursor
4.1.1. 30,50-Bis-O-tert-butyldimethylsilyl-20-deoxy-O6-[(2,4,6-
triisopropylphenyl)sulfonyl]-guanosine (2)
To a solution of 20-deoxyguanosine (1) (5.0 g, 18.7 mmol) and
imidazole (12.8 g, 187 mmol) in DMF (50 mL) was added tert-
butyldimethylsilyl chloride (11.5 g, 74.8 mmol). After stirring for
24 h, the reaction mixture was diluted with EtOAc. The organic
layer was washed with water and evaporated. The residue was
purified by recrystallization from MeOH to provide 2 (6.8 g,
13.6 mmol, 73%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d
(ppm) 7.85 (1H, s), 6.46 (1H, s), 6.09 (1H, t, J = 6.8 Hz), 4.48 (1H,
br), 3.80 (1H, br), 3.61–3.91 (2H, m), 2.59–2.66 (1H, m), 2.20–
2.25 (1H, m), 0.88 (9H, s), 0.86 (9H, s), 0.09 (6H, s), 0.03 (6H, s). This
white solid
2 (5.0 g, 10.1 mmol) and triethylamine (2.2 mL,
30.3 mmol) were dissolved in CH2Cl2 (50 mL), and then 2,4,6-tri-

1640
Y. Taniguchi et al. / Bioorg. Med. Chem. 22 (2014) 1634–1641
154.3, 152.4, 149.4, 149.4, 142.3, 138.4, 129.4, 125.8, 122.8, 120.8,
115.0, 114.5, 107.7, 106.7, 87.7, 83.0, 70.7, 67.4, 61.7, 59.9, 36.5,
14.0. HRMS (m/z) calcd for C31H32N9O8 (M+H)+ 658.2368, found
658.2334.
4.4. Fluorescence measurements
The fluorescence spectra displayed in Figures 2 and 4 were mea-
sured using 50 nM of each ODN in the buffer containing 100 mM
NaCl with 10 mM sodium phosphate at pH 7.0 and 30 °C with exci-
tation wavelength at 365 nm.
4.1.4. 20-Deoxy-5’-O-dimethoxytrityl-6-N-{2-[(1,3-diaza-3-
methyl-2-oxo-phenoxazine-9-yl)oxy]-ethyl}-2-
phenoxyacetylamino-adenosine-30-(2-cyanoethyl-N,N-
4.5. CD measurements
diisopropylphosphoramidite) (6)
To a solution of the corresponding diol 5 (190 mg, 0.29 mmol)
in pyridine (3 mL) was added DMTrCl (118.6 mg, 0.35 mmol). After
stirring for 1 h, the reaction mixture was diluted with EtOAc. The
organic layer was repeatedly washed with water and a satd NaCl
solution, dried over Na2SO4, and then evaporated under reduced
pressure. The residue was purified by flash column chromatogra-
phy (CHCl3/MeOH = 100/0 to 20/1) to give the DMTr-protected
compound as a yellow foam (178.4 mg, 0.19 mmol, 64%). The
DMTr-protected compound was dissolved in CH2Cl2 (3.5 mL) and
diisopropylethylamine (0.19 mL, 1.10 mmol) at 0 °C. After stirring
for 15 min at the same temperature, N,N-diisopropylchlorophosph-
oramidite (0.12 mL, 0.55 mmol) was added to the reaction mixture,
which was stirred for 1 h at room temperature. The reaction mix-
ture was diluted with EtOAc, and the organic layer was washed
with a sat. NaHCO3 solution, dried over Na2SO4, and then evapo-
rated under reduced pressure. The residue was purified by flash
column chromatography (CHCl3/MeOH = 100/0 to 20/1) to give a
pale yellow foam. This foam was dissolved in CH2Cl2 and poured
into hexane at ꢀ78 °C. The resulting white powder was collected
and dried under vacuum to give the corresponding amidite precur-
sor 6 (190.4 mg, 0.16 mmol, 88%). 1H NMR (400 MHz, CDCl3) d
(ppm) 8.36 (1H, br), 8.18 (1H, br), 7.37–7.35 (2H, m), 7.32–7.25
(4H, m), 7.24–7.10 (4H, m), 7.01–6.97 (3H, m), 6.78–6.71 (6H,
m), 6.43 (1H, d, J = 7.6 Hz), 6.34–6.31 (2H, m), 4.89 (1H, br), 4.65
(1H, br), 4.23–4.07 (5H, m), 3.82 (2H, s), 3.71 (6H, t, J = 2.0 Hz),
3.60–3.44 (4H, m), 3.37–3.27 (2H, m), 3.15 (3H, s), 2.79–2.72
(3H, m), 2.67–2.52 (2H, m), 2.41 (1H, t, J = 6.4 Hz), 1.56 (12H, s).
31P NMR (162 MHz, CDCl3) d (ppm) 148.7. HRMS (m/z) calcd for
CD spectra were measured using 5.0 lM of each ODN in the buf-
fer containing 100 mM NaCl and 10 mM sodium phosphate at pH
7.0 and 25 °C. The black line shows the single stranded target
DNA5 and 6 containing 8-oxo-dG and dG, respectively. The red line
shows the mixture of corresponding target DNA and ODN3 con-
taining 2-amino-Adap.
4.6. Detection and quantification of duplex formation by
fluorescence measurements using SYBR Green I
A mixture of ODN3 (2-amino-Adap or Adap) (2 nM) and SYBR
Green was stirred for 10 min in buffer containing 100 mM NaCl
and 10 mM sodium phosphate at pH 7.0 and 30 °C. After addition
of telomere DNA containing 8-oxo-dG or dG (2 nM) and stirring
for 10 min at 30 °C, the fluorescence spectrum was recorded with
excitation at 494 nm. The construction of a calibration curve was
plotted for fluorescence intensity values at 520 nm using different
concentrations of the telomere DNA5 from 0.2 to 2.0 nM. The
straight line and the R2 values were calculated using linear regres-
sion for the data between 0.4 and 2.0 nM, and are shown in
Figure 6.
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Re-
search (S) (Grant Number 21229002 for S.S.) and Challenging
Exploratory Research (Grant Number 24659047 for Y.T.) from the
Japan Society for the Promotion of Science (JSPS). Y.T. acknowl-
edges support from a Kyushu University Interdisciplinary Program
in Education and Projects in Research Development (P&P).
C
61H67N11O11P (M+H)+ 1160.4754, found 1160.4718.
4.2. Synthesis of ODNs containing 2-amino-Adap
Supplementary data
ODNs were synthesized by standard DNA synthesis procedures.
After synthesis of ODN, the resin was treated with 28% ammonium
solution at 55 °C for 6 h. The crude product was purified by HPLC to
obtain pure DMTr-ODN. The DMTr-group was then cleaved in 5%
acetic acid at room temperature, and the resulting DMTr-OH was
removed by washing with ether. The purities and structures of
each synthesized ODN were identified by MALDI-TOF analysis.
ODN1 (2-amino-Adap): calcd 4095.67, found 4095.49, ODN2
(2-amino-Adap): calcd 4358.86, found 4358.58, ODN3 (2-amino-
Adap): calcd 4733.85, found 4733.74, ODN4 (2-amino-Adap): calcd
4733.85, found 4733.64. Other ODNs and target DNAs were pur-
chased from Japan Bio Services Co., LTD.
Supplementary data (1H NMR spectrum of new compound 2, 4,
5 and 6) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2014.01.024.
References and notes
1. (a) Lindahl, T. Nature 1993, 362, 709; (b) Cooke, M. S.; Evans, M. D.; Dizdaroglu,
M.; Lunec, J. FASEB J. 2003, 17, 1195.
2. Neeley, W. L.; Essigmann, J. M. Chem. Res. Toxicol. 2006, 19, 491.
3. Lu, A.-L.; Li, X.; Gu, Y.; Wright, P. M.; Chang, D.-Y. Cell Biochem. Biophys. 2001,
35, 141.
4. Hofer, T.; Seo, A. Y.; Prudencio, M.; Leeuwenburgh, C. Biol. Chem. 2006, 387, 103.
5. Simoeki, A.; Rytarowska, A.; Poplawska, A. S.; Gackowski, D.; Rozalski, R.;
Dziaman, T.; Szaflarska, M. C.; Olinski, R. Carcinogenesis 2006, 27, 405.
6. Malayappa, B.; Garrett, T. J.; Segal, M.; Leeuwenburgh, C. J. Chromatogr. A 2007,
1167, 54.
4.3. Tm measurements
7. (a) Yoshida, R.; Ogawa, Y.; Kasai, H. Cancer Epidemiol. Biomarkers Prev. 2002, 11,
1072; (b) Shimoi, K.; Kasai, H.; Yokota, N.; Toyokuni, S.; Kinae, N. Cancer
Epidemiol. Biomarkers Prev. 2002, 11, 767.
Duplex DNA was heated to 90 °C for 5 min and annealed by
slow cooling in 10 mM sodium phosphate buffer containing
100 mM NaCl or 500 mM NaCl at pH 7.0 prior to the Tm measure-
ment. The temperature was raised from 10 °C to 80 °C at a rate of
1.0 °C/min and the UV absorbance was monitored at 260 nm. The
Tm values were obtained by the analysis of the melting curve with
MeltWin(v3.5). Thermodynamic parameters summarized in Table 1
were calculated from the Tm values obtained using the different
8. (a) Poulsen, H. E.; Nadal, L. L.; Broedbaek, K.; Nielsen, P. E.; Weimann, A.
Biochim. Biophys. Acta 2014, 1840, 801; (b) Michel, C.; Vincent-Hubert, F. Mutat.
Res. 2012, 741, 1; (c) Hah, S. S.; Mundt, J. M.; Kim, H. M.; Sumbad, R. A.;
Turteltaub, K. W.; Henderson, P. T. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 11203.
9. (a) Nakagawa, O.; Ono, S.; Li, Z.; Tsujimoto, A.; Sasaki, S. Angew. Chem., Int. Ed.
2007, 46, 4500; (b) Li, Z.; Nakagawa, O.; Koga, Y.; Taniguchi, Y.; Sasaki, S. Bioorg.
Med. Chem. 2010, 18, 3992; (c) Nasr, T.; Li, Z.; Nakagawa, O.; Taniguchi, Y.; Ono,
S.; Sasaki, S. Bioorg. Med. Chem. Lett. 2009, 19, 727.
concentration of duplex DNA at 1, 2, 3, 5 and 8
summarized in Table 2 were obtained using 1
l
M. The Tm values
10. (a) Taniguchi, Y.; Kawaguchi, R.; Sasaki, S. J. Am. Chem. Soc. 2011, 133, 7272; (b)
Erratum J. Am. Chem. Soc. 2011, 133, 10322.
l
M of duplex DNA.

Y. Taniguchi et al. / Bioorg. Med. Chem. 22 (2014) 1634–1641
1641
11. Taniguchi, Y.; Koga, Y.; Fukabori, K.; Sasaki, S. Bioorg. Med. Chem. Lett. 2012, 22,
13. (a) Oikawa, S.; Tada-Oikawa, S.; Kawanishi, S. Biochemistry 2001, 40, 4763; (b)
Oikawa, S.; Kawanishi, S. FEBS Lett. 1999, 453, 365.
14. Vorlickova, M.; Tomasko, M.; Sagi, A. J.; Bednarova, K.; Sagi, J. FEBS J. 2012, 279,
29.
543.
12. Cheong, C.; Tinoco, I., Jr.; Chollet, A. Nucleic Acids Res. 1988, 16, 5115.