Optimization of fluorescence property of the 8-oxodGclamp derivative for better selectivity for 8-oxo-2′-deoxyguanosine

Article abstract of DOI:10.1016/j.tet.2011.03.111

2′-Deoxyguanosine (dG) suffers from oxidation by reactive oxygen species (ROS) to form 8-oxo-2′-deoxyguanosine (8-oxo-dG), which is regarded as a marker of oxidative stress in the cells. In our continuous study for the recognition molecule of 8-oxo-dG, 8-oxoGclamp and its derivatives have been identified as the selective fluorescent probe. However, it is an obstacle for further application that dG also forms a complex with 8-oxoGclamp, resulting in fluorescence quenching in less polar solvents. Quenching of the fluorescence of 8-oxoGclamp is thought to involve photo-induced electron transfer in the complex. It was hypothesized that the energy level of the excited state of 8-oxoGclamp and the HOMO energy of dG are the preliminary determinant of the quenching efficiency. Thus, fluorescence properties of the substituted derivatives at the 7-position of the 1,3-diazaphenoxazine part of 8-oxoGclamp were investigated. Among the new derivatives, fluorescence of the 7-phenyl substituted 8-oxoGclamp was not quenched by dG even in the stable complex, exhibiting the highest selectivity for 8-oxo-dG.

Full text of DOI:10.1016/j.tet.2011.03.111

Tetrahedron 67 (2011) 6746e6752
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Tetrahedron
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Optimization of fluorescence property of the 8-oxodGclamp derivative for better
selectivity for 8-oxo-20-deoxyguanosine
*
Yohei Koga, Yasufumi Fuchi, Osamu Nakagawa, 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
20-Deoxyguanosine (dG) suffers from oxidation by reactive oxygen species (ROS) to form 8-oxo-20-de-
oxyguanosine (8-oxo-dG), which is regarded as a marker of oxidative stress in the cells. In our continuous
study for the recognition molecule of 8-oxo-dG, 8-oxoGclamp and its derivatives have been identified as
the selective fluorescent probe. However, it is an obstacle for further application that dG also forms
a complex with 8-oxoGclamp, resulting in fluorescence quenching in less polar solvents. Quenching of
the fluorescence of 8-oxoGclamp is thought to involve photo-induced electron transfer in the complex. It
was hypothesized that the energy level of the excited state of 8-oxoGclamp and the HOMO energy of dG
are the preliminary determinant of the quenching efficiency. Thus, fluorescence properties of the
substituted derivatives at the 7-position of the 1,3-diazaphenoxazine part of 8-oxoGclamp were in-
vestigated. Among the new derivatives, fluorescence of the 7-phenyl substituted 8-oxoGclamp was not
quenched by dG even in the stable complex, exhibiting the highest selectivity for 8-oxo-dG.
Ó 2011 Elsevier Ltd. All rights reserved.
Article history:
Received 5 March 2011
Received in revised form 29 March 2011
Accepted 29 March 2011
Available online 8 April 2011
Keywords:
Fluorescent probe
8-Oxo-20-deoxyguanosine
8-OxoGclamp
Fluorescence quenching
1. Introduction
resulting in fluorescence quenching in a less polar solvent. In this
study, we attempted to optimize the selectivity for 8-oxo-dG by
Reactive oxygen species (ROS), such as hydroxyl radical, hy-
drogen peroxide, single oxygen, etc., induce oxidative damage to
lipids, proteins, and nucleic acids in living organisms.1 20-Deoxy-
guanosine (dG) is oxidized to form 8-oxo-20-deoxyguanosine (8-
oxo-dG) as a representative oxidative damage. 8-Oxo-dG is a potent
mutagen, which induces G:C to T:A transversion mutations.2 The 8-
oxo-dG level inside and outside cells is regarded as a marker of the
oxidative stress of the cells,3 and its contents in DNA are correlated
with some diseases4 or aging.5 8-Oxo-dG is routinely analyzed by
HPLCeEC,6 HPLC/GCeMS,7 antibodies in an enzyme-linked
immunosorbant assay (ELISA), and immunohistochemical staining
of tissues or cells, and so on.7e,8,9 In an attempt to develop a simpler
method, we have developed a new series of fluorescent recognition
molecules for 8-oxo-dG based on the nucleoside derivatives having
the 1,3-diazaphenoxazine skeleton (Fig. 1A).10 Early studies have
shown that selective fluorescence quenching takes place through
the complex formation by multiple hydrogen bonds between 8-
oxoGclamp (2) and 8-oxo-dG. In the subsequent structureeactivity
investigation, more selective molecules including 8-oxoGclamp (2-
et-naph) have been determined, together with dG-selective
Gclamp (1-urea) (Fig. 1B and C).10b However, it is an obstacle for
further application that dG also forms a complex with 8-oxoGclamp
minimizing fluorescence quenching by dG.
2. Results and discussion
Efficiency of the fluorescence quenching of 8-oxoGclamp by 8-
oxo-dG and dG is highly dependent on the stability of their com-
plex, that is, the distance between them within the ground-state
complexes. The quenching mechanism most likely involves
a photo-induced electron transfer (PET) interaction.11 The differ-
ence in the hydrogen bonds with 8-oxo-dG or dG arises from the
N7-position, where 8-oxoGclamp (2) forms a hydrogen bond with
8-oxo-dG but not with dG, reflecting 10e30 times higher binding
affinity for 8-oxo-dG than for dG. Nevertheless, dG causes fluores-
cence quenching to some extent in less polar organic solvents. In
a previous approach to improve selectivity for 8-oxo-dG in fluo-
rescent quenching, we determined new 8-oxoGclamp derivatives
such as those shown in Fig. 1A with more selective binding affinity
8-oxo-dG. In this study, we adopted a different strategy for higher
quenching selectivity for 8-oxo-dG by minimizing the quenching
efficiency by dG so that dG would not cause efficient quenching
even in the stable complex formation. According to the PET
mechanism, the excited state of 8-oxoGclamp (2) serves as the
electron acceptor from dG as the ground-state electron donor
(Fig. 2A). 8-Oxo-dG has higher HOMO energy than dG and therefore
is a better quencher. We hypothesized that quenching efficiency by
* Corresponding author. E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.03.111

Y. Koga et al. / Tetrahedron 67 (2011) 6746e6752
6747
O
NHX
Gclamp (1)
Gclamp (1-urea)
A
X=H
O
NH
X=CONHCH2CH3
N
O
TBDMSO
8-oxoGclamp (2)
8-oxoGclamp (2-et-naph)
N
X=COOCH2Ph
X=COOCH2CH2Naph
O
TBDMSO
C
B
O
O
O
N
N
O
N
O
O
O
N
H
N
CH3
H
H
H
H
H
N
O
O
N
N
N
N
N
O
H
H
N
dR
N
dR
O
O
N
dR
N
dR
N
N
H
H
N
N
H
H
dG withG-clamp (1-urea)
8-oxodG with 8-oxoGclamp (2-et-naph)
Fig. 1. Structure of Gclamp derivatives (A), a complex with 8-oxo-dG (B) and dG(C).
A
B
derivative 18. The 4-carbonyl group of 12 was activated using PPh3
and CCl4 and subjected to the reaction with 18 to give 13.
PhCH2OCOeNHCH2CH2OH was introduced into 14 under the Mit-
sunobu conditions to produce 14, which was treated with K2CO3 in
MeOH to afford dihydroxyl compound (15) and trihydroxy com-
pound (16).
The synthesis of 3, 4, and 5 is summarized in Scheme 2. The
cyclized compound 19 was obtained by the treatment of the piv-
aloyl protected 15 with TEA in refluxing ethanol, and the hydroxyl
groups were protected with the TBDMS group to produce 3. The
trihydroxy compound 16 was first protected with the TBDMS
group, then cyclized in refluxing methanol in the presence of TEA to
form 4 and 5.
The hydroxyl group of 5 was treated with Tf2NPh in the presence
of TEA to produce the triflate 6, which was used as the common
intermediate for the synthesis of the aromatic group-substituted 8-
oxoGclamp (7e11) (Scheme 3). The triflate group of 6 was
substituted with an aromatic group under SuzukieMiyaura cou-
pling conditions using Pd(OAc)2, phosphine ligand, and the corre-
sponding aromatic boric acid.
Fig. 2. Relative orbital energies for explaining fluorescence quenching by photo-
induced electron transfer mechanism.
dG may be controlled by adjusting the S0 level of the excited state of
8-oxoGclamp derivatives as schematically shown in Fig. 2B. As it is
difficult to theoretically predict the S0 level and quenching effi-
ciency, we investigated the substituent effects of 8-oxoGclamp on
the fluorescent property. To avoid through-space and/or though-
bond interactions with the oxygen atoms at the 5-position or the
substitution at the 9-position, we have chosen the position 7 of the
1,3-diazaphenoxazine skeleton for introduction of a variety of the
compounds (Fig. 3, 3e11).
The UV and fluorescent spectra of the 8-oxoGclamp derivatives
were measured in the presence and absence of the nucleoside
substrates. The quantum yield was obtained using quinine as the
standard compound. The maximum absorbance (Absmax), emission
R=
H
OPiv
OTBDMS
OH
OTf
Phenyl
1-Naphthyl
p-MeOPh
p-CNPh
2-Thiophen
O
8
8-oxoGclamp
2
3
9
R
O
HN
O
7
6
spectra (Emmax), and quantum yield (4) are summarized in Table 1.
O
The fluorescence quenching titration was performed at 25 ꢀC using
the 30-O-, 50-O-di-TBDMS protected nucleoside and the 8-oxoG-
clamp derivative in a CHCl3 solution containing 0.3 mM AcOHeTEA.
The fluorescence spectra were recorded with the excitation wave-
length at 365 nm, and fluorescence quenching was titrated by the
addition of the nucleoside derivative. Some examples are shown in
Fig. 4. The fluorescence spectra of 2 and 7 were almost equally
quenched by 8-oxo-dG (Fig. 4, A and C). In contrast, dG induced
fluorescence quenching for 2, but not for 7 (Fig. 4, B vs D). A bath-
ochromic shift of 10 nm suggests the ground-state complex forma-
tion between 7 and dG (Fig. 4D). The ratio of the fluorescent intensity
at the maximum wavelength in the absence (F0) and presence of
10 equiv of the nucleoside (Fq), Fq/F0, represents an index of the
quenching efficiency. For example, the Fq/F0 values of each combi-
nation are as follows, 2 with 8-oxo-dG, 0.08; 2 with dG, 0.76; 7 with
4
NH
5
O
6
5
N 1
7
4
TBDMSO
8
N 3
9
O
10
11
OTBDMS
Fig. 3. The structure of the 8-oxoGclamp derivatives investigated in this study.
The synthesis started from the diacetate of 20-deoxy-5-bro-
mouridine (12) (Scheme 1). 1,3,5-Trihydroxybenzene (17) was first
nitrated at the 4-position, and the 1-hydroxyl group of the resulting
nitrated compound was protected with the pivaloyl group; then the
nitro group was reduced to 4-amino-1,3,5-trihydroxybenzene

6748
Y. Koga et al. / Tetrahedron 67 (2011) 6746e6752
OPiv
OPiv
O
N
HO
Br
O
O
NH
HO
Br
OH
O
Br
b
a
NH
N
NH
NH
O
O
AcO
O
N
O
AcO
AcO
N
N
O
OAc
O
OAc
12
13
14
OAc
c
OH
OPiv
OH
NH2
OH
PivO
HO
Br
O
O
NH
HO
Br
O
NH
18
OH
NH
N
NH
d
O
O
O
O
N
HO
HO
N
N
O
O
O
HO
OH
OH
OH
17
16
15
Scheme 1. The synthesis of the intermediates 15 and 16. (a) PPh3, CCl4, CH2Cl2, then 18, DBU, rt, 12 h, 83%, (b) PhCH2OCOeNHCH2CH2OH, DIAD, PPh3, CH2Cl2, rt, 25 h, 62%, (c) K2CO3,
MeOH, rt 30 min, 15 (46%), 16 (23%), (d) (1) HNO3, H2SO4, rt, 3 h, 55%, (2) pivaloyl chloride, dry pyridine, (3) H2, Pd/C, MeOH, 0 ꢀC, 2 h, 41% in two steps.
O
O
O
O
H
N
HN
O
HN
O
TfO
O
R
O
PivO
O
O
NH
NH
O
NH
O
O
a
b
N
N
O
15
3
N
TBDMSO
TBDMSO
N
N
a
b
O
O
5
HO
N
O
OTBDMS
6
OTBDMS
7-11
O
19
OH
TBDMSO
Scheme 3. The synthesis of the aromatic ring substituted compounds (7e11). (a)
Tf2NPh, TEA, CH2Cl2, rt, 22 h, 78%, (b) AreB(OH)2, Pd(OAc)2, phosphine ligand,
K3PO4enH2O, dry solvent: 88% for 7, phenyl boric acid, SPhos, toluene, 100 ꢀC, 2 h; 51%
for 8, 1-naphthyl boric acid, SPhos, toluene, 2 h; 59% for 9, 4-methoxyphenyl boric acid,
JohnPhos, dioxane, 6 h; 29% for 10, 4-cyanophenyl boric acid, XPhos, dioxane, 6 h; 12%
for 11, 2-thiophene boric acid, XPhos, dioxane, 100 ꢀC, 2 h.
TBDMSO
Br
O
O
NH
NH
O
O
b
c
substituted group affects the energy level of the excited states. The
Fq/F0 values for 8-oxo-dG of less than 0.1 represent efficient fluo-
rescence quenching of 8-oxodGclamp derivatives by 8-oxo-dG. In
contrast, Fq/F0 values for dG ranged from 0.64 of 3 to 0.98 of 7. There
is no clear relationship between the association constants of the
complex (Ka) and the Fq/F0 values. Forexample, compound 9 showed
a high Ka value of 2.57ꢁ106 Mꢂ1; however, only inefficient fluores-
cence quenching (Fq/F0¼0.84) was observed with dG. It should be
mentioned that phenyl and naphthyl groups enhanced the binding
affinity for 8-oxo-dG without changing the affinity for dG.
4
+
5
N
16
TBDMSO
N
O
20
TBDMSO
Scheme 2. The synthesis of 3, 4, and 5. (a) TEA, EtOH, reflux, 54 h, 48%, (b) TBDMSCl,
imidazole, DMAP, dry DMF, rt, 13 h, 72% for 3, 82% for 20, (c) TEA, MeOH, reflux, 46 h,
22% (4) and 38% (5).
8-oxo-dG, 0.1; 7 with dG, 0.98. These results have clearly indicated
that 7 is a more selective fluorescence probe than the original 2 for
8-oxo-dG. The equilibrium binding constants (Ka) between the
8-oxoGclamp derivative and 8-oxo-dG or dG were obtained from the
fluorescence titration experiments and are summarized in Table 1.
The quantum yield was not significantly affected by the
substituted group except for the case of 4, 5, and 11. These molecules
have a lone pair of an oxygen or a sulfur atom, which might serve as
an electron donor for the intramolecular quenching. The aromatic
substitution caused an emission shift to a longer wavelength
(438 nm to longer than 446 nm), indicating that the aromatic
It was initially anticipated according to the PET quenching
mechanism that adjusting the S0 level of the excited state of
8-oxoGclamp derivatives might affect the quenching efficiency by
dG. The HOMO of the excited state of the 8-oxoGclamp derivative
serves as an electron acceptor and the HOMO of the ground-state dG
serves as the electron donor. The higher HOMO energy of the excited
state of the 8-oxoGclamp derivative is expected to be a poorer
acceptor, that is, lower quenching efficiency is expected for dG. The
maximum wavelength of UV absorbance reflects the energy differ-
ence between the HOMO and the LUMO of the 8-oxoGclamp
derivative. The energies of the maximum wavelength of the

Y. Koga et al. / Tetrahedron 67 (2011) 6746e6752
6749
Table 1
UV, fluorescent, and binding properties of the 8-oxodGclampa
entry
oxoGclamp
Absmax (nm)
Emmax (nm)
4a
8-oxo-dG
dG
R¼
Fq/F0
Ka (106 Mꢂ1
)
Fq/F0
0.76
Ka (106 Mꢂ1
)
c
c
1
2
2
360
362
360
365
360
371
365
368
372
364
438
442
459
463
430
447
447
456
447
458
0.36
0.29
0.09
0.05
0.32
0.34
0.38
0.28
0.33
0.11
0.08
0.04
0.15
0.24b
0.04
0.10
0.08
0.08
0.08
0.16
4.9
0.48
0.60
0.74
0.51b
1.95
0.80
0.52
2.57
2.80
1.38
H
3
OPiv
4
OTBDMS
5
OH
6
OTf
7
Phenyl
8
Naphthyl
9
MeOPh
10
CNPh
11
12.9
41.0
10.0b
30.7
17.4
19.8
6.7
0.64
0.70
3
4
0.70b
0.39
0.98
0.81
0.84
0.95
0.85
5
6
7
8
9
8.1
10
67.8
Thiophene
a
UV and fluorescent spectra were measured in CHCl3 solution at 25 ꢀC using 30-O- and 50-O-TBDMS protected nucleosides. The fluorescence quenching titration was
performed in the CHCl3 solution containing 0.3 mM AcOHeTEA.
b
Measured in CHCl3 solution containing 10 mM TEA and 2.7 mM AcOH.
The fluorescence quenching ratio, where Fq and F0 represent the fluorescence intensity of the 1:10 mixture of 8-oxoGclamp and 8-oxo-dG or dG and the fluorescence
c
intensity of 8-oxoGclamp, respectively.
Fq/F0
1.0
0.8
0.6
0.4
0.2
0.0
3.32
3.34
3.36
3.38
3.4
3.42
3.44
3.46
λ
hc/e (ev)
Fig. 5. Comparison of the Fq/F0 value and the energy of maximum wavelength. Data
were taken from Table 1. The energy of the maximum wavelength was calculated by
the following equation: E(eV)¼hc/e
l, where h, c, e, and l represent the Planck constant,
the speed of light, the elementary charge, and the wavelength, respectively.
design was based on the assumption that the substitution at the
7-position of its 1,3-diazaphenoxazine part might affect the energy
level of the excited state of 8-oxodGclamp and inhibit quenching
pathway by dG. Among a variety of substituted 8-oxodGclamp de-
rivatives, the phenyl-substituted 8-oxodGclamp (7) was found to
have the best profile in the sense that its fluorescence was not
quenched by dG even when a stable complex was formed. The new
phenyl-substituted 8-oxodGclamp (7) may be a good candidate for
further application to fluorescent detection of 8-oxo-dG.
Fig. 4. Comparison of the quenching behavior of 2 and 7. Quenching experiment was
performed using 1
m mM nucleoside
M 8-oxoGclamp (30O,50O-di-TBDMS), 0 and 10
(30O,50O-di-TBDMS) at 25 ꢀC with excitation at 365 nm in CHCl3 containing 0.3 mM
TEAeAcOH.
8-oxoGclamp derivatives are compared with the Fq/F0 values (Fig. 5).
There is a tendency that the compound with the higher energy, i.e.,
shorter wavelength, produces a higher quenching efficiency by dG.
Although the maximum wavelength does not simply correlate with
the S0 level of the excited state, it is likely that optimizing the
quenching selectivity for 8-oxo-dG has been achieved by adjusting
the electronic property of the 1,3-diazaphenoxazine skeleton.
In conclusion, we have improved the selectivity of 8-oxodGclamp
for detection of 8-oxo-dG in fluorescence quenching. The molecular
3. Experimental
3.1. General
1H NMR (400 MHz) and 13C NMR (125 MHz) spectra were
recorded on a Varian UNITY 400 or 500 spectrometer. Infrared (IR)
spectra were obtained using a PerkineElmer FTIR-SpectrumOne.
The high-resolution mass spectra were recorded by an Applied

6750
Y. Koga et al. / Tetrahedron 67 (2011) 6746e6752
Biosystems Mariner System 5299 spectrometer using nicotinic acid,
bradykinin, neurotensin, and angiotensin as the internal standard.
117.0, 85.9, 85.8, 81.6, 79.1, 74.1, 65.4, 63.6, 26.7, 26.4; IR (cmꢂ1): 2354,
1748; HRMS (ESI) m/z: calcd for C34H39N4O12Br [MþH]þ: 775.1821,
777.1808; found 775.1841, 777.1777.
3.1.1. 4-O-Pivaloyl-2-aminophloroglucinol (18). Under argon atmo-
sphere, pivaloyl chloride (2.0 mL, 13 mmol) was added slowly to
a solution of 2-nitrophloroglucinol (2.0 g, 12 mmol) in pyridine
(20 mL) at 0 ꢀC; then the reaction mixture was allowed to warm to
room temperature and stirred for 2 h. The reaction mixture was
quenched with water and extracted with CH2Cl2. The organic layer
was washed with brine, dried over Na2SO4, and evaporated. The
residue was purified by column chromatography (silica gel, Hex-
ane/AcOEt¼10:1 CHCl3/MeOH¼12:1) to give pale yellow solids.
Under H2 atmosphere, the solution of this yellow solid in MeOH
(105 mL) in the presence of 5% Pd/C (170 mg) was stirred for 2.5 h at
room temperature. The reaction mixture was filtered through
a Celite pad, and the filtrate was evaporated. The residue was pu-
rified by column chromatography (silica gel, CHCl3/MeOH¼12:1) to
give 18 as pale yellow solids (1.1 g, 41%, in two steps). 1H NMR
3.1.4. N-[4-(2-N-Benzylcarbamylethoxy-6-hydroxy-4-O-piv-
aloylphenyl)]-20-deoxy-5-bromouridine (15) and N-[4-(2-N-benzyl-
carbamylethoxy-4,6-dihydroxyphenyl)]-20-deoxy-5-bromouridine
(16). A suspension of 14 (3.8 g, 4.91 mmol) and K2CO3 (950 mg
6.9 mmol) in MeOH (190 mL) was stirred for 30 min at room
temperature. The reaction mixture was diluted with water
(200 mL) then MeOH was removed by fractional distillation. Brine
was added to the residue, mixture was extracted with CH2Cl2, and
the organic layer was dried over Na2SO4, then evaporated. The
residue was purified by column chromatography (silica gel, CHCl3/
MeOH¼12:1/CHCl3/MeOH¼8:1) to give 15 as a pale yellow oil
(1.57 g, 46%) and 16 as a pale yellow oil (670 mg, 23%). Compound
15: 1H NMR (400 MHz, CD3OD)
d ppm: 1.33 (9H, s), 2.19e2.13 (1H,
m), 2.40e2.34 (1H, m), 3.47 (2H, t, J¼5.2 Hz), 3.73 (1H, ddd, J¼3.4,
3.6, 11.9 Hz), 3.84 (1H, dd, J¼3.1, 11.9 Hz), 3.93 (1H, dd, J¼3.4,
3.6 Hz), 4.09 (2H, t, J¼5.2 Hz), 4.38e4.34 (1H, m), 5.05 (2H, s), 6.15
(1H, t, J¼6.1 Hz), 6.27e6.33 (2H, m), 7.32e7.25 (5H, m), 8.51 (1H, s);
IR (cmꢂ1): 3328, 1624, 1577; MS (ESI) m/z: [MþH]þ: found 691.05,
(400 MHz, CD3OD)
(125 MHz, CD3OD)
d
ppm: 1.29 (9H, s), 6.02 (2H, s); 13C NMR
d
ppm: 179.2, 147.4, 144.7, 121.1, 101.2, 39.9, 27.4;
IR (cmꢂ1): 1729, 1157; HRMS (ESI) m/z: calcd for C11H15NO4
[MþH]þ: 226.1074; found 226.1088
693.05. Compound 16: 1H NMR (400 MHz, CD3OD)
d ppm:
3.1.2. 30,50-Di-O-acetyl-N-[4-(2,6-dihydroxy-4-O-pivaloylphenyl)]-
20-deoxy-5-bromouridine (13). Under argon atmosphere, a solution
of 30,50-di-O-acetyl-20-deoxy-5-bromouridine (12) (910 mg,
2.3 mmol), triphenylphosphine (1.2 g, 4.7 mmol) in CH2Cl2 (5 mL),
and CCl4 (5 mL) was stirred for 3 h at 70 ꢀC. The reaction mixture
was allowed to cool to room temperature, and then DBU (0.77 ml,
5.1 mmol) and 18 (830 mg, 3.7 mmol) were added. The reaction
mixture was stirred for 12 h at room temperature, and then diluted
with CH2Cl2 (9 mL), hexane (9 mL), and 5% aqueous citric acid
(20 mL). The mixture was extracted with CH2Cl2, and the organic
layer was dried over Na2SO4 and evaporated. The residue was pu-
rified by column chromatography (silica gel, CHCl3/MeOH¼30:1) to
give pale yellow oil, which was purified by column chromatography
(silica gel, Hexane/AcOEt¼2:3) to give 13 as a pale yellow form
2.18e2.12 (1H, m), 2.39e2.33 (1H, m), 3.47 (2H, dd, J¼5.2, 5.5 Hz),
3.73 (1H, dd, J¼3.4,11.9 Hz), 3.83 (1H, dd, J¼2.7,11.9 Hz), 3.92 (1H, q,
J¼3.4 Hz), 4.05 (2H, dd, J¼5.2, 5.5 Hz), 4.36 (1H, dt J¼4.3, 6.1 Hz),
5.05 (2H, s), 6.02e6.06 (2H, m), 6.15 (1H, dd, J¼6.1, 6.4 Hz),
7.32e7.23 (5H, m), 8.45 (1H, s); IR (cmꢂ1): 3330, 2928, 1624, 1573;
MS (ESI) m/z: [MþH]þ: found 691.05, 693.05.
3.1.5. 3-(20-Deoxy-
D-ribofuranosyl)-9-(2-N-benzylcarbamylethoxy)-
1,3-diaza-7-O-pivaloyl-2-oxophenoxadine (19). Under argon atmo-
sphere, a solution of 15 (1.57 g, 2.27 mmol) and Et3N (80 mL) in
EtOH (80 mL) was stirred at 80 ꢀC for 54 h. The reaction mixture
was evaporated to give the residue, which was purified by column
chromatography
(silica
gel,
CHCl3/MeOH¼12:1/CHCl3/
MeOH¼8:1) to give 19 as a pale yellow oil (660 mg, 48%). 1H NMR
(1.2 g, 83%). 1H NMR (400 MHz, DMSO)
d
ppm: 1.27 (9H, s), 2.05 (3H,
(400 MHz, CD3OD) d ppm: 1.31 (9H, s), 2.18e2.11 (1H, m), 2.36e2.30
s), 2.08 (3H, s), 2.34 (1H, ddd, J¼2.7, 3.4, 6.7 Hz), 2.41 (1H, q,
J¼7.3 Hz), 4.20 (1H, dd, J¼2.7, 4.3 Hz), 4.27 (2H, d, J¼4.3 Hz), 5.18
(1H, t, J¼3.4 Hz), 6.08 (2H, s), 6.12 (1H, dd, J¼6.7, 7.3 Hz), 8.00 (1H,
(1H, m), 3.52 (2H, dd, J¼4.9, 5.2 Hz), 3.70e3.75 (1H, m), 3.78e3.83
(1H, m), 3.92 (1H, dd, J¼3.0, 3.4 Hz), 4.03 (2H, dd, J¼4.9, 5.2 Hz),
4.34e4.38 (1H, m), 5.10 (2H, s), 6.21e6.18 (2H, m), 6.35 (1H, s),
s), 8.19 (1H, br), 9.86 (2H, br); 13C NMR(125 MHz, DMSO)
d
ppm:
7.33e7.24 (5H, m), 7.69 (1H, s); 13C NMR (125 MHz, CD3OD)
d ppm:
176.1, 170.1, 170.0, 158.9, 153.8, 152.9, 150.0, 141.3, 110.8, 100.5, 88.1,
85.8, 81.6, 74.1, 63.6, 38.9, 38.5, 26.7, 20.7, 20.6; IR (cmꢂ1): 2977,
1626, 1746; HRMS (ESI) m/z: calcd for C13H15N2O7Br [MþH]þ:
598.1017, 600.0973; found 598.1031, 600.1015.
178.4, 159.1, 152.2, 148.0, 144.3, 143.5, 129.4, 128.9, 128.7, 114.9,
112.2, 105.2, 103.4, 102.6, 99.2, 89.1, 89.0, 88.2, 87.6, 72.0, 71.4, 69.9,
68.7, 67.6, 62.7, 62.2, 41.9, 41.2, 40.1, 27.4; IR (cmꢂ1): 3323, 2956,
1501; HRMS (ESI) m/z: calcd for C30H34N4O10 [MþH]þ: 611.2348;
found 611.2351.
3.1.3. 30,50-Di-O-acetyl-N-[4-(2-N-benzylcarbamyl-ethoxy-6-hydroxy-
4-O-pivaloylphenyl)]-20-deoxy-5-bromouridine (14). Under argon
3.1.6. 3-(30,50-Di-O-tert-butyl-dimethyl-silane-
D-ribofuranosyl)-9-(2-
atmosphere,
a
solution of 13 (1.15 g, 1.9 mmol), PhCH2O-
N-benzylcarbamyl- ethoxy)-1,3-diaza-7-O-pivaloyl -2-oxophenoxadine
(3). Under argon atmosphere, a solution of imidazole (350 mg,
5.16 mmol), DMAP (63 mg, 0.516 mmol), TBDMSCl (625 mg,
4.13 mmol), and 19 (630 mg, 1.03 mmol) in DMF (7.7 mL) was stirred
at room temperature for 20 h. Water (15 mL) was added to the re-
action mixture, and the mixture was extracted with AcOEt. The or-
ganic layer was dried over Na2SO4 and evaporated. The residue was
purified by column chromatography (silica gel, Hexane/AcOEt¼1:1)
to give 3 as a pale yellow form (620 mg, 72%). 1H NMR (400 MHz,
COeNHCH2CH2OH (480 mg, 2.5 mmol), and triphenylphosphine
(810 mg, 3.1 mmol) in CH2Cl2 (16 mL) was stirred at 0 ꢀC for 15 min
and warmed to room temperature. DIAD (1.6 mL, 3.1 mmol) was
added to the mixture and the whole was stirred for 26 h at room
temperature. The reaction mixture was washed with water and
brine. The organic layer was dried over Na2SO4 and evaporated. The
residue was purified by column chromatography (silica gel, CHCl3/
AcOEt¼2:1) to give pale yellow solids, which were purified by col-
umn chromatography (silica gel, CHCl3/MeOH¼50:1) to give 14 as
CDCl3) d ppm: 0.04 (6H, s), 0.11 (3H, s), 0.13 (3H, s), 0.86 (9H, s), 0.93
a pale yellow form (910 mg, 62%). 1H NMR (400 MHz, CDCl3)
d
ppm:
(9H, s), 1.31 (9H, s), 2.08e2.02 (1H, m), 2.36e2.33 (1H, m), 3.56 (2H,
q, J¼4.9 Hz), 3.74 (1H, d, J¼9.5 Hz), 3.89e3.92 (2H, m), 4.01 (2H, t,
J¼4.9 Hz), 4.37e4.35 (1H, m), 5.10 (2H, s), 5.95 (1H, br), 6.09 (1H, s),
6.19 (1H, s), 6.24 (1H, t, J¼6.1 Hz), 7.34e7.25 (5H, m), 7.57 (1H, s); 13C
1.31 (9H, s), 2.09 (3H, s), 2.11 (3H, s), 2.68 (1H, dd, J¼2.1, 2.7 Hz), 3.64
(2H, d, J¼4.2 Hz), 4.10 (2H, d, J¼4.2 Hz), 4.30 (1H, dd, J¼3.1, 4.0 Hz),
4.36 (2H, t, J¼3.1 Hz), 5.09 (2H, s), 5.19 (1H, dd, J¼2.7, 4.0 Hz),
6.20e6.26 (2H, m), 6.40 (1H, d, J¼2.1 Hz), 7.32e7.31 (5H, m), 7.91
NMR (125 MHz, CDCl3)
d ppm: 176.7, 166.4, 156.6, 152.1, 146.8, 141.7,
(1H, s); 13C NMR (125 MHz, CDCl3)
d
ppm: 176.1, 175.9, 170.1, 170.0,
128.5, 128.1, 128.0, 127.1, 102.5, 87.7, 86.1, 71.1, 68.8, 66.8, 62.4, 41.9,
159.3, 159.0, 156.2, 155.1, 154.6, 153.8, 153.0, 150.4, 150.0, 147.7, 112.0,
40.4, 39.1, 27.1, 26.0, 25.7, 24.9, 18.5, 18.0, ꢂ4.6, ꢂ4.9, ꢂ5.4, ꢂ5.5; IR

Y. Koga et al. / Tetrahedron 67 (2011) 6746e6752
6751
(cmꢂ1): 2930, 1676, 1558, 1499; HRMS (ESI) m/z: calcd for
C42H62N4O10Si2 [MþH]þ: 839.4077; found 839.4090.
ꢂ5.5; IR (cmꢂ1): 2951, 1679, 1561, 1497, 1450, 1425; HRMS (ESI) m/z:
calcd for C38H53F3N4O11Si2 [MþH]þ: 887.2995; found 887.2963.
3.1.7. 30,50-Di-O-tert-butyldimethylsilyl-N-[4-(2-N-benzylcarbamyle-
thoxy-4,6-O-tert-butyldimethylsilylphenyl)]-20-deoxy-5-bromour-
idine (20). The title compound 20 was obtained as described for the
synthesis of 3 using 16 (112 mg, 0.185 mmol). Compound 20 as
3.1.10. 3-(30,50-Di-O-tert-butyldimethylsilyl-
D-ribofuranosyl)-9-(2-N-
benzylcarbamyl-ethoxy)-1,3-diaza-7-phenyl-2-oxophenoxadine (7). A
solution of 6 (50 mg, 0.056 mmol), Pd(OAc)2 (3 mg, 0.012 mmol),
SPhos (12 mg, 0.028 mmol), K3PO4enH2O (45 mg), and phenyl bo-
ronic acid (14 mg, 0.11 mmol) in degassed toluene (5 mL) was heated
at 100 ꢀC for 2 h. The reaction mixture was diluted with AcOEt, fil-
tered through a Celite pad, and then the filtrate was evaporated. The
residue was purified by column chromatography (silica gel, Hexane/
AcOEt¼1:1/Hexane/AcOEt¼1:2) to give 7 as a pale yellow form
a pale yellow oil (162 mg, 82%). 1H NMR (400 MHz, CDCl3)
d ppm:
0.03e0.18 (24H, m), 0.85e0.96 (36H, m), 2.02 (2H, s), 2.37 (1H, br),
3.44 (2H, br), 3.73 (1H, d, J¼11.0 Hz), 3.86e3.89 (2H, m), 4.07e4.13
(3H, m), 4.32 (1H, br), 5.05 (2H, s), 5.97 (1H, s), 6.07 (1H, s), 6.19 (1H,
t, J¼5.8 Hz), 6.64 (1H, s), 7.29e7.30 (5H, m), 8.01 (1H, s); MS (ESI) m/
z: [MþH]þ: found 1063.08, 1065.06.
(40 mg, 88%). 1H NMR (400 MHz, CD3OD)
d ppm: 0.11 (6H, s), 0.16
(3H, s), 0.18 (3H, s), 0.91 (9H, s), 0.97 (9H, s), 2.06e2.13 (1H, m),
2.28e2.35 (1H, m), 3.55 (2H, t, J¼4.9 Hz), 3.67e3.71 (1H, m),
3.80e3.83 (1H, m), 3.92 (2H, dd, J¼2.7, 7.0 Hz), 4.12 (2H, t, J¼4.9 Hz),
4.45e4.46 (1H, m), 5.10 (2H, s), 6.16 (1H, t, J¼6.1 Hz), 6.57 (1H, s), 6.81
(1H, s), 7.22e7.32 (6H, m), 7.39 (2H, t, J¼7.6 Hz), 7.49 (2H, d, J¼7.6 Hz),
3.1.8. 3-(30,50-Di-O-tert-butyldimethylsilyl-
(2-N-benzylcarbamylethoxy)-1,3-diaza-7-O-tert-butyldimethylsilyl-
2-oxophenoxadine (4) and 3-(30,50-di-O-tert-butyldimethylsilyl-
-ri-
D-ribofuranosyl)-9-
D
bofuranosyl)-9-(2-N-benzylcarbamylethoxy)-1,3-diaza-7-hydroxy-2-
oxophenoxadine (5). Under argon atmosphere, a solution of 20
(85 mg, 0.08 mmol) and Et3N (4 mL) in MeOH (4 mL) was stirred at
80 ꢀC for 47 h. The reaction mixture was evaporated, and the res-
idue was purified by column chromatography (silica gel, CHCl3/
MeOH¼25:1) to give pale yellow oil, which was purified by column
7.59 (1H, s); 13C NMR(125 MHz, CD3OD)
d ppm: 171.1, 155.4, 144.5,
141.0, 138.6, 129.9, 129.5, 129.4, 128.9, 128.7, 127.5, 123.5, 107.6, 107.1,
89.4, 87.6, 73.1, 69.8, 67.6, 63.8, 42.7, 41.3, 26.6, 26.3, 19.4, 18.9, ꢂ4.4,
ꢂ4.6, ꢂ5.2; IR (cmꢂ1): 2925, 2354,1673,1563,1493,1421; HRMS (ESI)
m/z: calcd for C43H58N4O8Si2 [MþH]þ: 815.3866; found 815.3889.
chromatography
(silica
gel,
Hexane/AcOEt¼4:1/Hexane/
AcOEt¼2:1) to give 4 as a pale yellow form (15 mg, 22%) and 5 as
3.1.11. 3-(30,50-Di-O-tert-butyldimethylsilyl-
D-ribofuranosyl)-9-(2-N-
a pale yellow form (23 mg, 38%). Compound 4: 1H NMR (400 MHz,
benzylcarbamyl-ethoxy)-1,3-diaza-7-(1-naphthyl)-2-oxophenoxadine
(8). A solution of 6 (40 mg, 0.045 mmol), Pd(OAc)2 (3 mg,
0.012 mmol), SPhos (9 mg, 0.023 mmol), K3PO4enH2O (36 mg), and
1-naphthalene boronic acid (16 mg, 0.09 mmol) in degassed toluene
(4 mL) was heated at 100 ꢀC for 2 h. The reaction mixture was diluted
with AcOEt, filtered through a Celite pad, and then the filtrate was
evaporated. The residue was purified by column chromatography
(silica gel, Hexane/AcOEt¼3:2/Hexane/AcOEt¼1:2) to give pale
yellow form, which was purified by column chromatography (silica
gel, CHCl3/acetone¼10:1) to give 8 as a pale yellow form (20 mg,
CDCl3) d ppm: 0.04 (6H, s), 0.12 (3H, s), 0.14 (3H, s), 0.16 (6H, s), 0.86
(9H, s), 0.94 (18H, d, J¼2.1 Hz), 2.02e2.09 (1H, m) 2.32e2.35 (1H,
m), 3.57 (2H, t, J¼4.5 Hz), 3.75 (1H, d, J¼9.5 Hz), 3.88 (1H, d,
J¼9.5 Hz), 3.93 (1H, s), 3.97 (2H, t, J¼4.5 Hz), 4.34e4.38 (1H, m),
5.10 (2H, s), 5.87 (1H, d, J¼2.2 Hz), 5.97 (1H, s), 6.21 (1H, t, J¼5.8 Hz),
6.27 (1H, br), 7.25e7.35 (5H, m), 7.61 (1H, s); 13C NMR (125 MHz,
CDCl3)
d ppm: 163.8, 156.7, 153.5, 151.3, 147.6, 143.4, 136.7, 128.4,
128.0, 127.9, 127.0, 100.4, 100.1, 87.7, 86.0, 70.7, 68.6, 66.6, 62.3, 41.9,
40.4, 29.7, 26.0, 25.7, 25.6, 18.5, 18.2, 17.9, ꢂ1.3, ꢂ4.5, ꢂ4.6, ꢂ4.9,
ꢂ5.5, ꢂ5.6; IR (cmꢂ1): 2928, 2856, 1716, 1555, 1503; HRMS (ESI) m/
z: calcd for C43H68N4O9Si3 [MþH]þ: 869.4367; found 869.4406.
51%). 1H NMR (400 MHz, CD3OD)
d ppm: 0.10 (6H, s), 0.11 (3H, s), 0.12
(3H, s), 0.91 (18H, s), 2.07e2.14 (1H, m), 2.30e2.36 (1H, m), 3.51 (2H,
t, J¼4.9 Hz), 3.79 (1H, d, J¼9.2 Hz), 3.89e3.92 (2H, m), 4.03 (2H, t,
J¼4.9 Hz), 4.44e4.47 (1H, m), 5.10 (2H, s), 6.17 (1H, t, J¼6.1 Hz), 6.42
(1H, s), 6.64 (1H, s), 7.22e7.34 (6H, m), 7.39 (1H, dd, J¼7.3, 7.6 Hz),
7.46 (2H, dd, J¼7.3, 8.2 Hz), 7.60 (1H, s), 7.85 (3H, dd, J¼7.6, 8.2 Hz);
Compound 5: 1H NMR (400 MHz, CDCl3)
d ppm: 0.02 (3H, s), 0.04
(3H, s), 0.13 (6H, s), 0.84 (9H, s), 0.94 (9H, s), 2.18 (1H, br), 2.31 (1H,
br), 3.45 (2H, br), 3.71 (1H, d, J¼9.1 Hz), 3.85 (4H, s), 4.37 (1H, s),
4.97 (2H, s), 5.85 (1H, d, J¼7.1 Hz), 6.32 (2H, br), 6.27 (1H, br), 7.14
(5H, br); 13C NMR (125 MHz, CDCl3)
d
ppm: 156.8, 143.3, 136.8,
13C NMR(125 MHz, CD3OD)
d ppm: 169.8, 159.1, 155.5, 147.8, 144.0,
136.6, 128.3, 128.0, 127.8, 121.8, 96.4, 87.8, 86.0, 71.8, 69.6, 68.1, 66.9,
66.5, 62.8, 41.4, 40.3, 26.1, 25.7, 18.5, 17.9, ꢂ4.6, ꢂ4.9, ꢂ5.5, ꢂ5.5; IR
(cmꢂ1): 3219, 2930, 2858, 1671, 1556, 1505, 1472; HRMS (ESI) m/z:
calcd for C37H54N4O9Si2 [MþH]þ: 755.3502; found 755.3545.
140.1, 138.4, 138.0, 135.4, 132.6, 129.5, 129.4, 129.1, 128.9, 128.7, 127.7,
127.2,127.0,126.4,126.4,123.6,110.9,110.3, 89.3, 87.5, 72.9, 69.8, 67.6,
63.7, 42.8, 41.3, 26.6, 26.3, 19.3, 18.9, ꢂ4.4, ꢂ4.6, ꢂ5.2, ꢂ5.3; IR
(cmꢂ1): 2952, 2366, 1696, 1676, 1559, 1493, 1422; HRMS (ESI) m/z:
calcd for C47H60N4O8Si2 [MþH]þ: 865.4022; found 865.4010.
3.1.9. 3-(30,50-Di-O-tert-butyldimethylsilyl-
D-ribofuranosyl)-9-(2-N-
benzylcarbamyl-ethoxy)-1,3-diaza-7-O-triflate-2-oxophenoxadine
(6). Under argon atmosphere, Et3N (0.3 mL, 2.27 mmol) was added
3.1.12. 3-(30,50-Di-O-tert-butyldimethylsilyl-
D-ribofuranosyl)-9-(2-N-
benzylcarbamyl-ethoxy)-1,3-diaza-7-(4-methoxyphenyl)-2-ox-
ophenoxadine (9). A solution of 6 (50 mg, 0.056 mmol), Pd(OAc)2
(3 mg, 0.012 mmol), JohnPhos (9 mg, 0.03 mmol), K3PO4 (35 mg,
0.165 mmol), and 4-methoxyphenyl boronic acid (18 mg,
0.118 mmol) in degassed 1,4-dioxane (1.5 mL) was heated at 100 ꢀC
for 6 h. The reaction mixture was diluted with AcOEt, filtered
through a Celite pad, and then the filtrate was evaporated. The
residue was purified by column chromatography (silica gel, Hex-
ane/AcOEt¼1:1) to give 9 as a pale yellow form (28 mg, 59%). 1H
to
a solution of 5 (570 mg, 0.757 mmol) and N-phenyl-
bis(trifluoromethanesulfonimide) (406 mg, 1.14 mmol) in CH2Cl2
(9.6 mL) and the mixture was stirred at room temperature for 22 h.
The reaction mixture was washed successively with saturated
aqueous NaHCO3, brine, and water. The organic layer was dried over
Na2SO4 and evaporated. The residue was purified by column chro-
matography (silica gel, Hexane/AcOEt¼1:1/Hexane/AcOEt¼1:2) to
give 6 as a pale yellow form (520 mg, 78%).1H NMR (400 MHz, CDCl3)
d
ppm: 0.04 (6H, s), 0.12 (3H, s), 0.13 (3H, s) 0.86 (9H, s), 0.93 (9H, s),
NMR (400 MHz, CD3OD) d ppm: 0.11 (6H, s), 0.17 (3H, s), 0.19 (3H, s),
2.08e2.11 (1H, m), 2.33e2.38 (1H, m), 3.59 (2H, d, J¼4.3 Hz), 3.75
(2H, d, J¼10.1 Hz), 3.89e3.94 (2H, m), 4.01 (2H, d, J¼10.1 Hz), 4.36
(1H, d, J¼4.9 Hz), 5.08 (2H, s), 6.24 (1H, t, J¼5.5 Hz), 6.26 (1H, s), 6.37
(1H, s), 7.26e7.30 (5H, m), 7.71 (1H, s); 13C NMR(125 MHz, CDCl3)
0.92 (9H, s), 0.97 (9H, s), 2.06e2.12 (1H, m), 2.32e2.38 (1H, m), 3.55
(2H, t, J¼4.9 Hz), 3.80 (3H, s), 3.83 (1H, d, J¼3.1 Hz), 3.91e3.93 (2H,
m), 4.11 (2H, t, J¼4.9 Hz), 4.45e4.49 (1H, m), 5.11 (2H, s), 6.16 (1H, t,
J¼6.1 Hz), 6.51 (1H, s), 6.75 (1H, s), 6.94 (2H, d, J¼8.8 Hz), 7.23e7.32
(6H, m), 7.42 (2H, d, J¼8.5 Hz), 7.57 (1H, s); 13C NMR(125 MHz,
d
ppm: 156.7, 152.4, 151.3, 147.3, 145.3, 143.3, 136.6, 128.4, 128.0,
128.0, 126.6, 123.8, 122.5, 119.9, 117.4, 114.8, 102.3, 101.3, 87.7, 86.2,
CD3OD) d ppm: 160.9, 159.1, 155.2, 144.4, 138.1, 133.3, 130.0, 129.5,
70.6, 69.2, 66.7, 62.2, 41.8, 40.7, 40.1, 29.7, 27.1, 26.0, 25.7, ꢂ4.6, ꢂ4.9,
129.4, 128.9, 128.7, 128.5, 127.6, 123.2, 115.3, 107.0, 106.4, 89.3, 87.5,

6752
Y. Koga et al. / Tetrahedron 67 (2011) 6746e6752
73.1, 69.6, 67.6, 63.9, 55.8, 42.7, 41.3, 27.8, 26.7, 26.3, 19.4, 18.9, ꢂ4.4,
ꢂ4.6, ꢂ5.2, ꢂ5.2; IR (cmꢂ1): 2929, 1717, 1676; HRMS (ESI) m/z: calcd
for C44H60N4O9Si2 [MþH]þ: 845.3972; found 845.3978.
in the fluorescence quenching ratio represent the fluorescence in-
tensity of the 1:1 mixture of 8-oxoGclamp and 8-oxo-dG or dG and
the fluorescence intensity of 8-oxoGclamp, respectively. Fluores-
cence titration was performed by the addition of a stock solution of
3.1.13. 3-(30,50-Di-O-tert-butyldimethylsilyl-
D
-ribofuranosyl)-9-(2-N-
the 30-O-, 50-O-di-TBDMS protected 20-deoxynucleoside (0e10
mM
benzylcarbamyl-ethoxy)-1,3-diaza-7-(4-cyanophenyl)-2-oxophenox-
adine (10). A solution of 6 (25 mg, 0.028 mmol), Pd(OAc)2 (2 mg,
0.0082 mmol), XPhos (7.5 mg, 0.014 mmol), K3PO4 (18 mg,
0.084 mmol), and 4-cyanophenyl boronic acid (8 mg, 0.056 mmol)
in degassed 1,4-dioxane (0.5 mL) was heated at 100 ꢀC for 17 h. The
reaction mixture was diluted with AcOEt, filtered through a Celite
pad, and then the filtrate was evaporated. The residue was purified
by column chromatography (silica gel, CHCl3/MeOH¼49:1) to give
10 as a pale yellow form (10 mg, 43%). 1H NMR (400 MHz, CD3OD)
final concentration) to a solution of the 8-oxoGclamp analog (1 mM)
under the following conditions, A: CHCl3 solution containing
10 mM TEA and 2.7 mM AcOH, B: CHCl3 solution containing
0.33 mM TEAeAcOH, C: CHCl3 solution without addition of
AcOHeTEA. The titration curve was analyzed by the non-linear
curve fitting method based on the 1:1 complex ratio.
References and notes
1. (a) Risom, L.; Moller, P.; Loft, S. Mutat. Res. 2005, 592, 119e137; (b) Knaapen, A.
d
ppm: 0.11 (6H, s), 0.16 (3H, s), 0.18 (3H, s), 0.92 (9H, s), 0.97 (9H, s),
€
€
M.; Gungor, N.; Schins, R. P. F.; Borm, P. J. A.; Van Schooten, F. J. Mutagenesis
2006, 21, 225e236; (c) Valavanidis, A.; Vlahogianni, T.; Dassenakis, M.; Scoullos,
M. Ecotoxicol. Environ. Saf. 2006, 64, 178e189; (d) Halliwell, B. Biochem. J. 2007,
401, 1e11.
2.08e2.14 (1H, m), 2.30e2.36 (1H, m), 3.57 (2H, t, J¼5.2 Hz), 3.83
(1H, d, J¼11.0 Hz), 3.91e3.94 (2H, m), 4.17 (2H, t, J¼5.2 Hz), 4.46
(1H, br), 5.10 (2H, s), 6.16 (1H, t, J¼6.0 Hz), 6.65 (1H, s), 6.92 (1H, s),
7.32e7.23 (5H, m), 7.61 (1H, s), 7.70e7.76 (2H, m); 13C NMR
2. Examples, (a) Shibutani, S.; Takeshita, M.; Grollaman, A. P. Nature 1991, 349,
431e434; (b) A recent report Damsma, G. E.; Cramer, P. J. Biol. Chem. 2009, 284,
(125 MHz, CD3OD)
d ppm: 179.2, 170.3, 160.0, 155.4, 145.5, 144.5,
ꢀ
31658e31663; (c) Bregeon, D.; Peignon, P.-A.; Sarasin, A. PLoS Genet. 2009, 5,
136.2, 133.8, 129.4, 128.9, 128.6, 128.4, 125.0, 109.2, 108.4, 89.3, 87.5,
72.9, 69.7, 67.5, 63.7, 42.8, 41.3, 26.6, 26.3, 19.4, 5.7, ꢂ4.5, ꢂ4.7, ꢂ5.3;
IR (cmꢂ1): 2952, 2352, 1718, 1676; HRMS (ESI) m/z: calcd for
C44H60N5O8Si2 [MþH]þ: 840.3818; found 840.3845.
e1000577.
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3.1.14. 3-(30,50-Di-O-tert-butyldimethylsilyl-
D-ribofuranosyl)-9-(2-N-
benzylcarbamyl-ethoxy)-1,3-diaza-7-(thiophenyl-2-yl)-2-ox-
ophenoxadine (11). A solution of 6 (25 mg, 0.028 mmol), Pd(OAc)2
(2 mg, 0.0082 mmol), cyclohexyl JohnPhos (5 mg, 0.014 mmol),
K3PO4 (18 mg, 0.084 mmol), and 2-thiophene boronic acid (7 mg,
0.056 mmol) in degassed 1,4-dioxane (2 mL) was heated at 100 ꢀC
for 24 h. The reaction mixture was diluted with AcOEt, filtered
through a Celite pad, and then the filtrate was evaporated. The
residue was purified by column chromatography (silica gel, Hex-
ane/AcOEt¼2:1) to give dark yellow oil crude, and purified by
preparative thin layer chromatography (silica gel, Hexane/
AcOEt¼1:1) to give 11 as a pale yellow form (2.7 mg, 12%). 1H NMR
(400 MHz, CD3OD) d ppm: 0.05 (6H, s), 0.13 (3H, s), 0.15 (3H, s), 0.87
(9H, s), 0.95 (9H, s), 2.21e2.12 (1H, m), 2.39e2.28 (1H, m), 3.57 (2H,
t, J¼5.2 Hz), 3.86e3.80 (1H, m), 3.97e3.93 (2H, m), 4.16 (2H, t,
J¼5.2 Hz), 4.49 (1H, br), 5.12 (2H, s), 6.19 (1H, t, J¼6.0 Hz), 6.60 (1H,
s), 6.90 (1H, s), 7.04e7.07 (1H, m), 7.17e7.36 (8H, m), 7.70 (1H, s);
13C NMR(125 MHz, CDCl3)
d ppm: 133.4, 131.0, 130.3, 128.9, 128.3,
9. (a) Soultanakis, R. P.; Melamede, R. J.; Bespalob, I. A.; Wallace, S. S.; Beckman, K.
B.; Ames, B. N.; Taatjes, D. J.; Janssen-Heininger, W. M. W. Free Radical Biol. Med.
2000, 28, 987e998; (b) Chiou, C. C.; Chang, P. Y.; Chan, E. C.; Wu, T. L.; Tsao, K.
C.; Wu, J. T. Clin. Chim. Acta 2003, 334, 87e94; (c) Machella, N.; Regoli, F.;
Cambria, A.; Santella, R. M. Mar. Environ. Res. 2004, 58, 725e729.
10. (a) Nakagawa, O.; Ono, S.; Li, Z.; Tsujimoto, A.; Sasaki, S. Angew. Chem., Int. Ed.
2007, 46, 4500e4503; (b) Li, Z.; Nakagawa, O.; Koga, Y.; Taniguchi, Y.; Sasaki, S.
Bioorg. Med. Chem. 2010, 18, 3992e3998; (c) Nasr, T.; Li, Z.; Nakagawa, O.;
Taniguchi, Y.; Ono, S.; Sasaki, S. Bioorg. Med. Chem. Lett. 2009, 19, 727e730.
11. (a) Heinlein, T.; Knemeyer, J.-P.; Piestert, O.; Sauer, M. J. Phys. Chem. B 2003,
107, 7957e7964; (b) Seidel, C. A. M.; Schulz, A.; Sauer, M. H. M. J. Phys. Chem.
1996, 100, 5541e5553; (c) Torimura, M.; Kurata, S.; Yamada, K.; Yokomaku, T.;
Kamagata, Y.; Kanagawa, T.; Kurane, R. Anal. Sci. 2001, 17, 155e160.
127.6, 126.8, 126.6, 126.1, 125.5, 124.9, 124.3, 105.5, 105.3, 95.0, 89.8,
88.6, 86.8, 71.1, 68.8, 66.3, 42.1, 40.3, 29.7, 27.5, 26.0, 14.1, ꢂ4.6, ꢂ4.9,
ꢂ5.5; IR (cmꢂ1): 2927, 1711, 1561, 1494; HRMS (ESI) m/z: calcd for
C41H56N4O8SSi2 [MþH]þ: 821.3430; found 821.3470.
3.2. UV and fluorescence spectra, and fluorescence quenching
experiments
UV and fluorescent spectra were measured in CHCl3 solution at
25 ꢀC using 30-O- and 50-O-TBDMS protected nucleosides. Fq and F0