Adenosine-1,3-diazaphenoxazine derivative for selective base pair formation with 8-oxo-2′-deoxyguanosine in DNA

Article abstract of DOI:10.1021/ja200327u

The selective detection of 8-oxo-2′-deoxyguanosine (8-oxo-dG) in DNA without chemical or enzymatic treatment is an attractive tool for genomic research. We designed and synthesized the non-natural nucleoside analogue, the adenosine-1,3-diazaphenoxazine (A

Full text of DOI:10.1021/ja200327u

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Adenosine-1,3-diazaphenoxazine Derivative for Selective Base Pair
Formation with 8-Oxo-2 -deoxyguanosine in DNA
0
Yosuke Taniguchi, Ryota Kawaguchi, and Shigeki Sasaki*
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 Japan, and CREST,
Japan Science and Technology Agency (JST), 4-1-8 Motomachi, Kawaguchi, Saitama 332-0012, Japan
S Supporting Information
b
0
ABSTRACT: The selective detection of 8-oxo-2 -deoxy-
guanosine (8-oxo-dG) in DNA without chemical or enzy-
matic treatment is an attractive tool for genomic research.
We designed and synthesized the non-natural nucleoside
analogue, the adenosine-1,3-diazaphenoxazine (Adap) deri-
vative, for selective recognition of 8-oxo-dG in DNA. This
study clearly showed that Adap has a highly selective
stabilizing effect on the duplex containing the Adapꢀ8-
oxo-dG base pair. Furthermore, the fluorescent property of
Figure 1. Concept of Adenosine-1,3-diazaphenoxazine (Adap) for the
recognition of 8-oxo-dG in DNA.
Adap was shown to be useful for the selective detection of
8
-oxo-dG in the duplex DNA. To the best of our knowledge,
this is the first successful demonstration of a non-natural
nucleoside with a high selectivity for 8-oxo-dG in DNA.
toward 8-oxo-dG in DNA, it was suggested that the 8-oxo-G clamp
hardly formed a selective base pair with 8-oxo-dG in the DNA
duplex. These results led us to design a new nucleoside analogue
12
that recognizes and forms a selective base pair with 8-oxo-dG in
0
DNA. Thus, a 2 -deoxyadenosine derivative having a 1,3-diazaphe-
ellular DNA is continuously exposed to a variety of chemi-
cally reactive species such as alkylating agents, reactive oxygen
noxazine unit (termed “Adap”) that can form multiple H bonds with
8-oxo-dG on both the WatsonꢀCrick face and the Hoogsteen face
was designed (Figure 1). Adap has the useful property of selectively
increasing the thermal stability of duplexes containing an Adapꢀ8-
oxo-dG pair and also results in selective fluorescence quenching with
8-oxo-dG in DNA.
C
species (ROS), etc., resulting in DNA damage that may increase the
0
risk of developing diseases. 8-Hydroxy-2 -deoxyguanosine [or 8-oxo-
0
2
-deoxyguanosine (8-oxo-dG)], the representative damaged nu-
0
cleoside, is generated by the oxidation of 2 -deoxyguanosine tripho-
0
sphate (dGTP) in the triphosphate nucleotide pool or 2 -
deoxyguanosine (dG) in DNA. As 8-oxo-dG forms a base pair
When designing a recognition molecule, we focused on the
conformational isomers around the glycosidic bond of 8-oxo-dG
in DNA (Figure 1). The base of dG adopts the anti conformation,
whereas that of 8-oxo-dG prefers the syn conformation. The
8-oxo-dG base in the syn conformation forms a base pair with dA
between the Hoogsteen face of 8-oxo-dG and the WatsonꢀCrick
face of dA. It was expected that a recognition molecule having the
adenosine skeleton together with the cytosine skeleton might
effectively discriminate 8-oxo-dG from dG. Thus, the new dA
derivative was designed to have the 1,3-diazaphenoxazine unit as
a cytosine unit connected to the 6-amino group of dA through
the ethoxy spacer. The 1,3-diazaphenoxazine skeleton might
form H bonds with the WatsonꢀCrick face of 8-oxo-dG in the
major groove of DNA (Figure 1). The new adenosine-1,3-
diazaphenoxazine analogue has been called Adap.
1
not only with dC but also with dA, 8-oxo-dGTP and 8-oxo-dG
induce transversion mutation from the GC to the TA base pair
2
during DNA replication. It has been shown that the intracellular
3ꢀ5
8
-oxo-dG level is relevant to some diseases and aging. Therefore,
it is of significant importance to develop methods for selective
detection of 8-oxo-dG. The 8-oxo-dG nucleoside or its base is
routinely analyzed by HPLCꢀEC, HPLC/GCꢀMS, and
6ꢀ8
ELISA.
Also, recent papers have reported that 8-oxo-dG in
DNA can be detected by the reaction of a biotin-conjugated
9
spermine derivative. Nevertheless, there is a still strong demand
for an efficient detection method for 8-oxo-dG in DNA in its intact
form without degradation. We previously reported the 8-oxo-G
10,11
clamp as a selective fluorescence probe for 8-oxo-G.
TBDMS-protected 8-oxo-G clamp forms multiple H bonds with
-oxo-dG, causing selective fluorescence quenching in organic
The
The synthesis of Adap is summarized in Scheme 1. N1-
Methyl-5-bromouracil (2) was prepared from 5-bromouracil
(1), HMDS, and methyl iodide and then subjected to coupling
with 2,6-dihydroxyaniline to afford 3. N-Fmoc-2-aminoethanol
was connected with 3 by the Mitsunobu reaction to produce 4,
which was treated with 7 M ammonia in methanol to effect the
8
solvents. When the 8-oxo-G clamp was incorporated into an
oligodeoxynucleotide (ODN), selective fluorescence quenching as
a result of hybridization with the complementary DNA incorporating
12
8
-oxo-dG was observed. However, there was a drawback to the use
of the 8-oxo-G clamp in ODNs: the detection selectivity for 8-oxo-
dG was highly dependent on the DNA sequence. Together with the
fact that the 8-oxo-G clamp did not increase the thermal stability
Received: January 13, 2011
Published: April 27, 2011
r 2011 American Chemical Society
7272
dx.doi.org/10.1021/ja200327u
|J. Am. Chem. Soc. 2011, 133, 7272–7275

Journal of the American Chemical Society
COMMUNICATION
a
Scheme 1. Synthesis of Adap and ODNs
Table 1. Thermodynamic Parameters for the Duplex Formed
between ODN1 and ODN2
a
X in
Y in
T
m
ΔH°
ΔS°
310K
ΔG°
(kcal/mol)
ꢀ
1
ꢀ1
ODN1 ODN2 (°C) (kcal/mol) (cal K mol
)
G
C
G
T
A
G
A
44.1 ꢀ100.7 ( 6
48.2 ꢀ114.7 ( 18
40.9 ꢀ90.3 ( 7
41.9 ꢀ92.9 ( 10
33.1 ꢀ94.6 ( 4
35.4 ꢀ71.1 ( 8
ꢀ288 ( 20
ꢀ11.3 ( 0.2
ꢀ13.1 ( 0.8
ꢀ10.1 ( 0.2
ꢀ10.4 ( 0.3
ꢀ7.8 ( 0.04
ꢀ8.7 ( 0.2
ꢀ11.3 ( 0.2
ꢀ9.7 ( 0.05
ꢀ10.6 ( 0.2
ꢀ9.6 ( 0.1
ꢀ7.1 ( 0.4
ꢀ8.8 ( 0.01
ꢀ9.7 ( 0.3
ꢀ8.3 ( 0.1
ꢀ11.7 ( 0.1
ꢀ7.0 ( 0.2
ꢀ8.0 ( 0.2
ꢀ8.8 ( 0.09
ꢀ8.0 ( 0.1
ꢀ12.4 ( 0.4
C
ꢀ328 ( 55
ꢀ259 ( 22
ꢀ266 ( 38
ꢀ280 ( 13
ꢀ201 ( 25
ꢀ281 ( 23
ꢀ263 ( 9
A
T
A
G
C
oxo-G 44.3 ꢀ98.3 ( 8
oxo-G 39.8 ꢀ91.4 ( 3
A
oxo-G
oxo-G
Adap
Adap
Adap
Adap
Adap
A
C
A
A
C
T
G
42.3 ꢀ96.8 ( 8
39.2 ꢀ96.1 ( 7
31.6 ꢀ100 ( 16
36.5 ꢀ124. ( 3
39.6 ꢀ79.7 ( 9
33.9 ꢀ70.8 ( 7
ꢀ278 ( 25
ꢀ279 ( 21
ꢀ299 ( 51
ꢀ372 ( 10
ꢀ226 ( 30
ꢀ201 ( 22
ꢀ241 ( 10
ꢀ267 ( 31
ꢀ180 ( 24
ꢀ247 ( 21
ꢀ201 ( 17
ꢀ363 ( 39
oxo-G 47.2 ꢀ86.5 ( 3
Adap
Adap
Adap
Adap
30.4 ꢀ90.0 ( 10
32.2 ꢀ63.9 ( 8
36.8 ꢀ85.5 ( 6
33.2 ꢀ70.4 ( 5
45.9 ꢀ125 ( 12
C
T
G
oxo-G Adap
a
UV melting profiles were measured using a solution containing each
ODN strand at a concentration of 2 μM in 100 mM NaCl and 10 mM
sodium phosphate buffer at pH 7.0. The data were analyzed by the melt
curve processing program. These values were determined by van’t Hoff
plots with five data points (1ꢀ12 μM) and the curve-fitting method.
8-Oxoguanosine is abbreviated as oxo-G.
a
Conditions: (a) HMDS, reflux, and then CH
b) PPh , CCl , CH Cl , reflux, then 2,6-dihydroxyaniline, DBU, 45%.
c) N-Fmoc-2-aminoethanol, PPh , DIAD, CH Cl , 96%. (d) Ammonia
3
I, MeCN, r.t. to reflux, 68%.
(
(
(
3
4
2
2
3
0
2
2
0
7 M in MeOH), 100%. (e) 2 O,5 O-Di-TBS-6-chloroadenosine, DIPEA,
1
-propanol, reflux, 44%. (f) TBAF, THF, 100%. (g) (1) DMTrCl,
0
pyridine; (2) 2-cyanoethyl-N,N -diisopropylchlorophosphorodiamidite,
The combination of dC and 8-oxo-dG showed a T of 44.3 °C,
m
DIPEA, CH Cl ; 73% for two steps. (h) DNA automated synthesizer.
2
2
which is 3.9 °C lower than the value for the corresponding natural
CG pair (48.2 °C). This is probably because WatsonꢀCrick base-
pair formation involving dC requires the unfavorable anti conforma-
tion of 8-oxo-dG. On the other hand, the dAꢀ8-oxo-dG combina-
removal of the Fmoc protecting group from the amino group
together with the cyclization reaction, yielding the 1,3-diazaphe-
0
0
13
noxazine derivative 5. 2 O,5 O-Di-TBS-6-chloroadenosine was
tion provided a T value similar to that for the dAꢀdT base pair
m
coupled with 5 in the presence of diisopropylethylamine
(39.8 vs 40.9 °C) and higher than that for the mismatched dAꢀdG
pair (39.8 vs 33.1 °C) because dA forms the base pair with the
favorable syn conformation of 8-oxo-dG. It should be noted that
selective stabilization was observed for the combination of Adap in
(DIPEA) under reflux conditions to give 6. After removal of
the TBDMS groups of 6 with TBAF, the resulting diol com-
pound 7 was transformed into the amidite precursor 8 according
to the conventional method. Amidite 8 was applied to the
automated DNA synthesizer to incorporate Adap into the
ODNs. Adap was incorporated in the middle of the pyrimidine
strand of ODN1, in the middle of the purine strand of ODN2,
ODN1 and 8-oxo-dG in ODN2 (T = 47.2 °C for 8-oxo-dG vs
m
31.6, 36.5, 39.6, and 33.9 °C for dA, dC, T, and dG, respectively).
The high selectivity is represented by the significant difference of
13.3 °C between the T values for dG and 8-oxo-dG. Interestingly,
m
0
and at the 5 end of ODN3. ODNs ODN4ꢀ7 were designed for
Adap formed a base pair with T at a slightly lower stability than the
natural dAꢀT pair, while a greater stabilizing effect was obtained
with the Adapꢀ8-oxo-dG combination. This result indicates the
possible and expected formation of additional H bonds of Adap with
the WatsonꢀCrick face of 8-oxo-dG. In the different combination
in which Adap was incorporated in the purine strand of ODN2, a
highly selective stabilizing effect was observed for the 8-oxo-
biologically relevant DNA sequences. After cleavage from the
resin and purification by reversed-phase HPLC, the structures of
the ODNs were confirmed by MALDIꢀTOF MS analysis.
The thermal stabilization effects of Adap were investigated
using the 13-mer duplex formed with ODN1 and ODN2
by measuring the T values at different duplex concentrations
m
in a buffer containing 100 mM NaCl and 10 mM sodium
phosphate at pH 7.0 (representative melting curves are shown
in Figure S1 in the Supporting Information). The thermody-
namic parameters were obtained from van’t Hoff plots (Figure
S2) using different ODN concentrations and are summarized in
Table 1. The data obtained for the natural ODNs are also shown
for comparison.
dGꢀAdap combination (T = 45.9 °C for 8-oxo-dG vs 30.4,
m
32.2, 36.8, and 33.2 °C for dA, dC, T, and dG, respectively). From its
large, planar and hydrophobic structure, one may expect stacking
interactions for Adap. To check whether such effects were included,
thermodynamic parameters were obtained using OND3 containing
0
Adap or dA at the 5 dangling end. It was shown that Adap produced
no significant stabilizing effect on ODN3, indicating that
7
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Journal of the American Chemical Society
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Figure 3. SternꢀVolmer plots for ODN1 (X = Adap) duplexed with
ODN2 containing Y = 8-oxo-dG (9, red line) and dG (2, black line).
The fluorescence spectra were measured using 10 nM ODN1 with
excitation at 365 nm under the same buffer conditions as in Figure 2. F0
and F are the fluorescence intensities at 452 nm without and ODN2 (0,
0.1, 0.2, 0.5, 1, 2 nM), respectively. The inset shows plots at lower
ODN2 concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 nM).
control ODN substrates having 8-oxo-dG at the positions other than
the complementary site of ODN2 (Figure S5). ODN2 (Y = Adap)
containing Adap in the purine strand also exhibited selective
fluorescence quenching when duplexed with ODN1 (X = 8-oxo-
dG) containing 8-oxo-dG in the pyrimidine strand (Figure S6).
These results indicate that the formation of a tight complex between
Adap and 8-oxo-dG plays a key role in the efficient and selective
fluorescence quenching. We also tested the fluorescence quenching
properties of TBDMS-protected Adap (6) in organic solvents. In
contrast to the quenching in the duplex DNA, the fluorescence of 6
was nonselectively quenched by the addition of the TBDMS-
protected 8-oxo-dG and dG (Figure S7). These quenching results
indicate that the selective quenching of Adap by 8-oxo-dG in the
duplex DNA may require a restricted environment of the duplex
DNA that enables Adap to form a base pair with the syn conforma-
tion of 8-oxo-dG. The plots of quenching efficiency of ODN1 were
linear at low concentrations of ODN2 (Figure 3). This graph
indicates that the probe having Adap can accurately detect 8-oxo-
dG at ∼100 pM concentrations of DNA under these conditions.
To test whether Adap can detect 8-oxo-dG in biologically
relevant DNA sequences, a preliminary investigation was performed
using the telomere sequence. It has been reported that the oxidation
of dG in the telomere sequence induces 8-oxo-dG more efficiently
Figure 2. Fluorescence quenching of ODN1 containing Adap. (AꢀF)
Fluorescence spectra of 50 nM ODN1 (X = Adap) and 50 nM ODN2
with Y = (A) 8-oxo-dG, (B) dG, (C) dC, (D) T, (E) dA, and (F) dAP [a
stable abasic site (tetrahydrofuran ring)]. Excitation at 365 nm was used,
and the solvent was 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 ODN1, and the red curves show those after
the addition of ODN2. (G) Photos of solutions of the ODNs (5 μM
each) under the above conditions.
nonselective hydrophobic interactions or stacking interactions are
not provided by Adap (Table S1). The circular dichroism spectrum
showed that typical B-type DNA was not disturbed in the presence
of the Adapꢀ8-oxo-dG pair (Figure S3). Thus, it has been clearly
demonstrated that Adap is the first nucleoside analogue having a
highly selective stabilizing effect with 8-oxo-dG in DNA.
We next applied Adap to the fluorescent detection of 8-oxo-dG in
DNA. In our previous study, the fluorescence of the 1,3-diazaphe-
noxazine unit of the 8-oxo-G clamp was selectively quenched by
complexation with 8-oxo-dG. Therefore, quenching of the fluores-
cence spectra of ODN1 containing Adap was measured in the
absence and the presence of ODN2 having 8-oxo-dG, dG, dC, dT,
dA, or dAP. Effective fluorescence quenching was observed only for
the duplex containing ODN2 with Y = 8-oxo-dG (Figure 2A). No
quenching was observed in the presence of ODN2 incorporating dG,
dC, T, dA, or dAP (Figure 2BꢀF). The quenching efficiency was so
high that the presence of 8-oxo-dG in the duplex DNA could be
detected visually (Figure 2G). Higher quenching efficiencies were
observed at lower temperatures and higher ODN concentrations
14
than that in non-telomere sequences. Chemical oxidation by
H O /CuCl induced 8-oxo-dG formation at the underlined posi-
2
2
2
0
tions in the telomere sequence of 5 -d(TAG TAG TTA GGG TTA
GGG TTA GGG TTA GGG)-3 . Accordingly, ODN4, -5, and -6
were designed for the detection of 8-oxo-dG at the positions of dG ,
dG , and dG , respectively, where the superscript number repre-
0
6
1
0
11
0
sents the position starting from the 5 end. Each ODN showed
selective fluorescence quenching for 8-oxo-dG at the respective
target site (Figure S8AꢀC). Another selective detection was
0
demonstrated using ODN7 for 8-oxo-dG in the sequence 5 -d(ATA
0
ATG ACT GAA CC 8oxo-G AAG GCC GGT TC)-3 , which was
reported to induce efficient G-to-T transversion mutation in a yeast
15
strain lacking the rad30 gene (Figure S8D). These preliminary
data demonstrate the potential utility of Adap for the analysis of
8-oxo-dG in biologically relevant sequences.
It should be noted that 8-oxo-dG in the long 80-mer DNA was
detected by ODN1 containing Adap with a similar efficiency. No
quenching was observed with the 80-mer DNA that did not
contain 8-oxo-dG (Figure S9). The 80-mer ODN8, which
(Figure S4). Also, no or much less quenching was induced by the
7
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Journal of the American Chemical Society
COMMUNICATION
non-natural nucleoside with a high selectivity for 8-oxo-dG in DNA.
Further application of the new Adapꢀoxo-dG base pair, such as the
use of the Adapꢀtriphosphate derivative, is now in progress.
’
ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures,
b
thermodynamic results, and fluorescence data. This material is
available free of charge via the Internet at http://pubs.acs.org.
’
AUTHOR INFORMATION
Corresponding Author
sasaki@phar.kyushu-u.ac.jp
’
ACKNOWLEDGMENT
We are grateful for the support by a Grant-in-Aid for Scientific
Research (S) from the JSPS and by CREST from JST.
Figure 4. Fluorescence quenching of ODN1 by 80-mer ODNs. The
conditions were described in Figure 2, except for the length of the ODN.
’
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dG in the complex.
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its large, planar, hydrophobic nature, Adap did not show significant
stacking or hydrophobic interactions. To obtain insight into the
complex structure, duplex DNA having the Adapꢀ8-oxo-dG com-
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formation, which showed that the complex is stabilized by multiple H
bonds (Figure S10A). Upon optimization, duplex DNA containing
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the B-DNA conformation, in which the 1,3-diazaphenoxazine part of
the Adapꢀ8-oxo-dG base pair is located in the major groove without
causing any steric repulsions (Figure S10B,C). In contrast to Adap,
the previous 8-oxo-G clamp did not show thermal stabilization effects
toward 8-oxo-dG in the duplex DNA, probably because it can form
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(
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1
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In conclusion, we have shown that Adap has a highly selective
stabilizing effect on the duplex containing the Adapꢀ8-oxo-dG base
pair. Furthermore, the fluorescent property of Adap has been shown
to be useful for the selective detection of 8-oxo-dG in DNA. To the
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