Synthesis of new derivatives of 8-oxoG-Clamp for better understanding the recognition mode and improvement of selective affinity

Article abstract of DOI:10.1016/j.bmc.2010.04.025

8-Oxoguanosine (8-oxoG) is a representative metabolite derived by the oxidation of guanosine (G) and is regarded as a marker of oxidative stress in the cells. We previously reported the 8-oxoG-clamp as the first fluorescent probe for detection of 8-oxoG. In this study, new 8-oxoG-clamp derivatives having a variety of N-functional groups were synthesized and their recognition properties were investigated. The sp³ oxygen atom of the carbamate unit was revealed to play a significant role in the hydrogen bonding interactions, and the pyrene group produced higher stability with 8-oxoG compared with the original 8-oxoG-clamp.

Full text of DOI:10.1016/j.bmc.2010.04.025

Bioorganic & Medicinal Chemistry 18 (2010) 3992–3998
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new derivatives of 8-oxoG-Clamp for better understanding
the recognition mode and improvement of selective affinity
*
Zhichun Li, Osamu Nakagawa, Yohei Koga, Yosuke Taniguchi, 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
Article history:
8-Oxoguanosine (8-oxoG) is a representative metabolite derived by the oxidation of guanosine (G) and is
regarded as a marker of oxidative stress in the cells. We previously reported the 8-oxoG-clamp as the first
fluorescent probe for detection of 8-oxoG. In this study, new 8-oxoG-clamp derivatives having a variety of
N-functional groups were synthesized and their recognition properties were investigated. The sp3 oxygen
atom of the carbamate unit was revealed to play a significant role in the hydrogen bonding interactions,
and the pyrene group produced higher stability with 8-oxoG compared with the original 8-oxoG-clamp.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 18 March 2010
Revised 6 April 2010
Accepted 7 April 2010
Available online 18 April 2010
Keywords:
8-Oxoguanosine
Fluorescent probe
Molecular recognition
Hydrogen bonding
Stacking interaction
O
N
O
N
1. Introduction
H
7
8
N 7
N
HN
H N
HN
H N
O
Reactive oxygen species oxidize nucleic acid components in liv-
ing organisms to form a variety of oxidized metabolites.1 Among
them 8-oxoguanosine (8-oxoG) is a representative metabolite de-
rived by the oxidation of guanosine (G) and is known to induce
G:C to T:A transversion mutations in DNA.2 The 8-oxoG level inside
and outside cells is regarded as an index of the oxidative damage to
cells3 and is believed to have relevance to some diseases4 or aging5
There are several methods for analysis of 8-oxoG such as HPLC-EC,6
HPLC/GC–MS,7 antibodies in an enzyme-linked immunosorbent as-
say (ELISA) and immunohistochemical staining of tissues or cells,
and so on.8,9,7e Nevertheless, there is a need of more efficient
detecting method for 8-oxoG. Our approach has been directed to
developing small molecules with high specificity for 8-oxoG, espe-
cially those with fluorescence for use in sensors, biological tools
and so on. It has been reported from our group that the Cbz-8-
oxoG-clamp (2), a derivative of G-clamp (1), is a selective fluores-
cent probe for detection of 8-oxoG.10 Cbz-8-oxoG-clamp (2)was
also incorporated into the oligodeoxynucleotide (ODN) probe for
detection of 8-oxo-dG in DNA. In this study, the new 8-oxoG-clamp
derivatives having a variety of N-functional groups were synthe-
sized, and their recognition properties were investigated to obtain
further insight into the recognition mode and development of the
new 8-oxoG-clamp with higher selectivity for 8-oxoG.
N 8
N
2
2
dR
dR
8-Oxoguanosine (8-oxoG)
Guanosine (G)
2. Results and discussion
The formation of five hydrogen bonds has been proved by 1H NMR
study with the complex of Cbz-8-oxoG-clamp (2) and the 8-oxodG
derivative (Fig. 1B). The substituted group (X) was anticipated to pro-
vide boththehydrogen bondandthestacking interaction. However, it
was not clear from the 1H NMR study whether the carbonyl oxygen of
the carbamate group was the acceptor for 7NH and whether the phe-
nyl group of the benzyloxy group provided a stacking interaction.
Therefore, in this study, to clarify these questions as well as to achieve
higher affinity for 8-oxoG, new 8-oxoG-clamp derivatives having a
variety of N-substituted groups (Fig. 1A) have been synthesized and
their recognition properties have been evaluated.
The carbamate derivatives (3–8) were prepared by a coupling
reaction of 1 with the alkoxycarbonylimidazole which was obtained
with the corresponding alcohol and carbonyldiimidazole. The ure-
ido group was similarly introduced using ethylamine (13). The acyl
derivatives (9–12) were obtained using the corresponding acyl
chlorides. The benzyl derivative (14) was synthesized by the reac-
tion of 1 with benzaldehyde in the presence of Mg2SO4 in refluxing
CH2Cl2, followed by reduction with NaBH3CN in MeOH.
* Corresponding author. Tel./fax: +81 92 642 6615.
E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.025

Z. Li et al. / Bioorg. Med. Chem. 18 (2010) 3992–3998
3993
stacking
B
A
O
NHX
O
N H
N
X
N
H
O
NH
hydrogen
bonding
O
H
N
N
O
7
O
O
TBDMSO
N
O
H
H
N
N
N
O
dR
N
dR
TBDMSO
N
H
G-Clamp (1)
8-oxoG
Cbz-8-oxoG-Clamp (2)
X=
X=
H
1 (H)
O
O
8 (OMs)
9 (Ac)
O
2 (OCH2Ph)
O
O
CH3
O
O
O
3 (OEt)
CH3
O
10 (Bz)
O
O
4 (OEtPh)
5 (OPrPh)
O
O
11 (EtPh)
12 (PrPh)
O
O
O
6 (OEtNaph)
7 (OEtPyr)
13 (NHEt)
CH3
N
H
O
O
O
14 (CH2Ph)
Figure 1. The structure of the 8-oxoG-clamp synthesized in this study (A), and the N–X group expected for stacking and hydrogen bonding (B).
The recognition property of the 8-oxoG-clamp derivative was
evaluated by measuring fluorescence quenching phenomena by
the addition of the nucleoside derivative. The 30-O-, 50-O-di-TBDMS
(tertbutyldimethylsilyl) protected nucleoside was added to a solu-
tion of the 8-oxoG-clamp derivative in CHCl3 buffered with10 mM
ETA and 2.7 mM AcOH at 25 °C. The fluorescence spectra were re-
corded with the excitation wavelength at 365 nm, and the change
with the addition of the nucleoside derivative was followed. Figure
2 illustrates an example with the 8-oxoG-clamp (7, OEtPyr), dem-
onstrating the selective quenching by the 8-oxodG derivative and
almost negligible quenching by other nucleoside derivatives.
The titration data thus obtained were analyzed by the curve-fit-
ting method to give the equilibrium binding constants, which are
summarized in Figure 3 (data are listed in Table 1). As the deriva-
tive of dA, dC or dT did not quench efficiently the fluorescence of
the oxoG-clamp derivative as shown in Figure 2, nor did dG to
the 8-oxoGclamp derivatives (2–12 and 14), their data are not
shown. The G-clamp (1) with the amino terminal showed almost
the same binding constants for 8-oxodG and dG. In contrast, the
8-oxoG-clamp (2) was quenched selectively by 8-oxodG as previ-
ously reported. It was anticipated that the benzyloxy group con-
tributed to the complex formation as a stacking unit. However,
the derivative 3 (OEt) without the benzene group exhibited a
slightly higher binding affinity to 8-oxodG.
increased with 4 (OEtPh) but decreased with 5 (OPrPh). As the
ethyl group attached to the benzene ring seemed to be better than
the methyl group of 2 or the propyl group of 5, the naphthalene (6)
and the pyrene unit (7) were introduced as a larger aromatic ring
through the ethyl linker. Eventually, it was found that oxoG-clamp
(7) with the ethyl-linked pyrene produced a great improvement in
binding affinity.
Notably, the acyl derivatives (9–12) showed lower affinity than
the corresponding carbamate derivatives, leading to an assumption
that the sp3 oxygen rather than the carbonyl oxygen of the
carbamate group is included in the hydrogen bond formation with
8-oxo-dG. The low affinity of the carbamate-type 8 may be ratio-
nalized in terms of the steric hindrance of the 2,6-dimethyl groups
of the benzene for the interaction to the sp3 oxygen. The facts that
10 and its reductive form 14 with the N-benzyl group showed al-
most the same binding constants are also indicative of less impor-
tance of the carbonyl oxygen of the carbamate group. On the other
hand, the ureido derivative 13 with the NH group as a H-donor in-
stead of the sp3 oxygen for the H-acceptor in hydrogen bond
formation expressed a 10 times higher affinity for dG than for 8-
oxo-dG. It has been concluded from these results that the sp3
oxygen atom of the carbamate group is involved in the hydrogen
bond formation with 8-oxodG and is responsible for the selectivity
for the 8-oxoG-clamp, as illustrated in Figure 4.
Considering that a stacking interaction requires a well-defined
contact between two aromatic groups,11 we further investigated
the effect of the size and the length of the aromatic group (4–7)
with varying the linker from ethyl to propyl and the aromatic
group from benzene to pyrene. Interestingly, the binding affinity
To gain further insight into the effects of the N-substituted
group on the binding constants, binding experiments were per-
formed in the non-buffered CHCl3 solution. When the components
of 10 mM TEA and 2.7 mM AcOH are dissolved in water, the solu-
tion is buffered at pH 7.0. In the meantime, these components also

3994
Z. Li et al. / Bioorg. Med. Chem. 18 (2010) 3992–3998
B: dG
100
A: 8-oxodG
100
80
60
40
80
60
40
20
0
20
0
400
550
Wavelength (nm)
600
450
500
400
550
Wavelength (nm)
600
450
500
D: dT
C: dC
E: dA
100
80
100
80
100
80
60
60
60
40
20
0
40
20
0
40
20
0
400
550 600
450 500
400
550 600
450 500
400
550 600
450 500
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Figure 2. Fluorescence quenching of the 8-oxoG-clamp (7, OEtPyr). The 30-O-, 50-O-di TBS protected 20-deoxynuceoside (0–10
of 7 (1 M) in CHCl3 containing 10 mM ETA and 2.7 mM AcOH at 25 °C. Fluorescence spectra were recorded with excitation at 365 nm.
lM final concentration) was added to a solution
l
100
90
80
70
60
50
40
30
20
10
0
make the organic solutions polar to some extent, namely the non-
buffered CHCl3 solutions provide a less polar medium compared to
the buffered ones. In non-buffered CHCl3, dG also induces fluores-
cence quenching, and the equilibrium binding constants can be ob-
tained accurately (Fig. 5A vs B). The fluorescence quenching is
thought to occur by a photo-induced electron transfer mechanism,
which depends strongly on the distance between the fluorophore
and the quencher.12 Because the hydrogen bond becomes stronger
in non-buffered CHCl3 than that in buffered CHCl3, 8-oxoG-clamp
and dG become closer enough for induction of fluorescence
quenching.
The equilibrium binding constants obtained in non-buffered
CHCl3 are compared in Figure 6 (data are listed in Table 2). The
Ks values obtained in the buffered solution (Fig. 3) overlapped
the bar graphs of the carbamate-type oxoG-clamp (2–8). Interest-
ingly, the effects of the carbamate structure on the binding con-
stants were different from those observed in the buffered CHCl3
solution. The binding constants of 3, 4, 5 and 7 were almost the
same, but the highest was obtained with 6, suggesting possible
involvement of a stacking interaction between the naphthalene
and the 8-oxoguanosine part. Possible complex structures were
simulated by MO calculation (MOPAC, PM6) and some examples
dG
8-oxo-dG
1
2
3
4
5
6
7
8
9
10 11 12 13 14
8-oxoG-clamp
Figure 3. Equilibrium binding constants obtained by fluorescence quenching
experiments in buffered CHCl3 (Ks, 105 Mꢀ1). The bars represent the binding
constants with 8-oxo-dG (blue bards) and those with dG (red bars). Titration
experiments were performed by the method described in the footnote to Figure 2,
and analyzed by the curve-fitting method. Data of 1, 2, 9 and 10 were already
reported in the Ref. 10c.
Table 1
a
Equilibrium binding constants of 8-OxoG-clmap derivatives with dG and 8-oxo-dG derivatives obtained by fluorescence quenching experiments in buffered CHCl3 (Ks, 105 Mꢀ1
)
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
oxodG
dG
7.3
7.1
23
NQ
31
NQ
29
NQ
16
NQ
32
NQ
56
NQ
7.2
NQ
3.0
NQ
2.9
NQ
3.5
NQ
2.1
NQ
9.3
88
3.9
NQ
a
Titration experiments were performed by the method described in the footnote to Figure 1, and analyzed by the least-square method using a Kaleida Graph. Data of 1, 2, 8
and 9 were already reported in Ref. 10c.

Z. Li et al. / Bioorg. Med. Chem. 18 (2010) 3992–3998
3995
A
B
O
O
O
O
N
O
O
N
H
O
N
H
N
N
H
CH3
H
H
H
N
O
O
N
N
N
N
N
O
H
H
N
N
dR
dR
O
O
N
N
N
N
H
H
N
N
H
dR
dR
H
8-oxodG with 2
dG with 13
Figure 4. Possible complex structures formed with 8-oxodG (A) and dG (B). (A) The revised mode of the complex with 8-oxodG for inclusion of the hydrogen bond between
the sp3 oxygen of the carbamate and 7-NH of 8-oxodG. (B) The NH of the ureido group may form the hydrogen bond with 7 N of dG.
B: dG
A: 8-oxodG
100
80
100
80
60
60
40
20
0
40
20
0
400
550
Wavelength (nm)
600
450
500
400
550
Wavelength (nm)
600
450
500
Figure 5. Fluorescence quenching experiments in the non-buffered CHCl3 solution. The experiments were performed under the same conditions described in the footnote to
Figure 2, except that TEA and AcOH were not included in CHCl3 solution.
shows close contacts suitable for the formation of the hydrogen
bonds in the complex between 7 (OEtPyr) and the 8-oxoG. This
800
600
400
200
0
400
200
0
molecular modeling also suggests that the ethyl linker between
the sp3 oxygen of the carbamate and the pyrene ring is suitable
for orienting the aromatic ring for such a face-to-face contact.
The complex structures were also calculated with the TBDMS-pro-
tected sugar structure, and have suggested that, as the pyrene ring
has a larger aromatic plane than naphthalene, it causes steric
repulsion toward the sugar parts within a compact complex
(Fig. 7D vs E). The binding affinity of 6 decreased in the buffered
CHCl3 solution (Kbuffered /Knon-buffer = 0.1) to a larger extent than
that of 7 (Kbuffered /Knon-buffer = 0.43). It is likely that stacking inter-
action with the naphthalene ring becomes weaker and that with
the pyrene group is kept in a polar medium.13
Ks in buffer
8-oxo-dG dG
dG
8-oxo-dG
1
2
3
4
5
6
7
8
9
10 11 12 13 14
8-oxoG-clamp
8-oxoG-clamp
3. Conclusion
Figure 6. Equilibrium binding constants obtained by fluorescence quenching
experiments in non-buffered CHCl3 (Ks, 105 Mꢀ1). Titration experiments were
performed by the method described in the footnote to Figure 2, except that TEA and
AcOH were not added to the solution.
This study was aimed at clarifying whether the carbonyl oxygen
or the sp3 oxygen of the carbamate group is involved in the com-
plex formation with 8-oxoguanosine. Quantitative evaluation of
the equilibrium binding constants of the new 8-oxoG-clamp deriv-
atives with a variety of N-functional groups has clearly indicated
that the sp3 oxygen of the carbamate group is responsible for selec-
are summarized in Figure 7. Figure 7B illustrates a face-to-face ori-
entation between the pyrene group and 8-oxoG and Figure 7C
Table 2
a
Equilibrium binding constants obtained by fluorescence quenching experiments in non-buffered CHCl3 (Ks, 105 Mꢀ1
)
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
oxodG
dG
Kbuffer/Knon-buffer
K8oxodG/KdG
36
37
0.20
1.0
71
160
5.1
0.19
31
130
5.4
0.22
24
130
7.6
0.12
17
320
29
0.1
11
130
17
0.43
7.6
59
28
0.12
2.1
13
40
0.23
0.3
25
39
0.11
0.64
12
15
0.29
0.8
11
11
0.19
1.0
34
11
7.1
0.31
10
820
0.27
0.04
6.6
0.35
1.7
b
a
Titration experiments were performed by the method described in the footnote to Figure 1, except that TEA and AcOH were not added into the solution.
The ratio of the binding constant (Ks) with 8-oxodG to dG in the non-buffered solution.
b

3996
Z. Li et al. / Bioorg. Med. Chem. 18 (2010) 3992–3998
A
E
B
D
O
N
O
O
H
H
O
N H
O
N
C
O
N
O
N
H N
N
CH3
CH3
N
H
N
H
Figure 7. A simulated complex structure between 7 (OEtPyr) and the 8-oxoG. The 20-deoxyribose part was replaced by the methyl group for simplification in MO calculation
in A–C, and the protected derivatives were used in simulation of D and E (MOPAC2009, PM6). (A) The dashed lines represent a distance less than 2.5–2.6 Å. Gray lines show
the structure at the back side. (B) The side view of the complex illustrates a face-to-face orientation between the pyrene group and 8-oxoG. (C) The top view of the complex
shows the close contact for the formation of hydrogen bonds. (D) The view from the side of the pyrene ring shown in green. (E) The view from the side of the naphthalene ring
shown in green of the complex between 6 (OEtNaph) and the 8-oxoG.
tive complexation with 8-oxoguanosine. Another purpose of this
study was to determine the appropriate structure of the N-func-
tional group, especially in terms of the stacking interaction, for
obtaining better recognition molecules for 8-oxoguanosine. As a
result, the new oxoG-clamp derivative (7) with the 2-pyren-1-yl-
ethoxycarbonyl unit has been determined to be a better recogni-
tion molecule for 8-oxo-dG. These new oxoG-clamp derivatives
and the clarified binding mode will be useful for further design
of the molecules that may be used for recognition of 8-oxoG in nu-
cleic acids as well as for the purpose of 8-oxoG detection in water.
for 24 h. The reaction mixture was quenched with aqueous NaH-
CO3 (10 mL) and extracted with CHCl3 (10 mL). The organic layers
were evaporated to give a crude oil, which was chromatographed
on a silica gel column (AcOEt/n-hexane = 1:1 to give 4 (8 mg,
34%). 1H NMR (400 MHz, CDCl3) d: 7.65 (1H, s), 7.25 (3H, m),
7.18 (2H, m), 6.78 (1H, t, J = 8 Hz), 6.44 (1H, d, J = 9 Hz), 6.35 (1H,
d, J = 8 Hz), 6.26 (1H, t, J = 6 Hz), 5.93 (1H, br), 4.37 (1H, m), 4.26
(2H, t, J = 7 Hz), 4.02 (2H, m), 3.91 (3H, m), 3.75 (1H, m), 3.55
(2H, m), 2.91 (2H, t, J = 7 Hz), 2.35 (1H, m), 2.08 (1H, m), 0.94
(9H, s), 0.86 (9H, s), 0.14 (3H, s), 0.12 (3H, s), 0.04 (6H, s). 13C
NMR (125 MHz, CDCl3) d: 156.8, 143.2, 129.0, 128.4, 126.4, 108.3,
107.3, 87.8, 9.1, 70.8, 68.5, 65.4, 62.3, 42.0, 40.4, 35.6, 26.0, 25.7,
18.5, 18.0, ꢀ4.6, ꢀ4.9, ꢀ5.5, ꢀ5.5. IR mmax (film): 2929, 1701,
1473, 1276 cmꢀ1. High-resolution MS (ESI): calcd for C38H57N4O8-
Si2 (M+H)+: 753.3710. Found 753.3719.
4. Experimental section
4.1. OEt-8-oxoG-clamp (3)
Under Ar atmosphere, N,N0-carbodiimidazole was added into a
solution of ethanol (250 lL) in dry CH2Cl2 (10 mL), and the mixture
4.3. OprPh-8-oxoG-clamp (5)
was stirred for 2.5 h at room temperature. Evaporation of the sol-
vents gave a crude product, which was crystallized from n-hexane
to give a white powder (imidazole/1H-imidazole-1-carboxylic acid
ethyl ester = 12:1 by NMR). The above power (36 mg, 0.036 mmol)
was added into a solution of 1 (20 mg, 0.033 mmol) in dry CH3CN
(0.5 mL), and the mixture was stirred for 24 h. The reaction mix-
ture was quenched with aqueous NaHCO3 (10 mL) and extracted
with CHCl3 (20 mL). The organic layers were evaporated to give a
crude oil, which was chromatographed on a silica gel column
(AcOEt/n-hexane = 1:1 to give 3 (13 mg, 57%). 1H NMR (400 MHz,
CDCl3) d: 7.61 (1H, s), 7.77 (1H, t, J = 9 Hz), 6.45 (1H, d, J = 8 Hz),
6.35 (1H, d, J = 8 Hz), 6.25 (1H, t, J = 6 Hz), 5.79 (1H, br), 4.37 (1H,
m), 4.11 (2H, m), 4.03 (2H, t, J = 5 Hz), 3.90 (3H, m), 3.75 (1H, d),
3.55 (2H, q), 2.37 (1H, m), 2.09 (1H, m), 1.24 (3H, m), 0.94 (9H,
s), 0.86 (9H, s), 0.14 (3H, s), 0.12 (3H, s), 0.04 (6H, s). 13C NMR
(125 MHz, CDCl3) d: 156.9, 146.7, 143.2, 127.3, 124.1, 122.6,
108.4, 107.3, 87.6, 86.0, 70.8, 68.6, 62.3, 61.0, 41. 9, 40.4, 26.0,
25.7, 18.5, 18.0, 14.6, ꢀ4.6, ꢀ4.9, ꢀ5.5, ꢀ5.5. IR mmax (film): 2929,
Under Ar atmosphere, N,N0-carbodiimidazole was added into a
solution of 3-phenyl-1-propanol (500 mg) in dry CH2Cl2 (10 mL),
and the mixture was stirred for 2.5 h at room temperature. Evapora-
tion of the solvents gave a crude product, which was crystallized
from n-hexane to give a white powder (imidazole/1H-imidazole-
1-carboxylic acid 3-phenyl propyl ester = 3:1 by NMR). The above
power (14 mg, 0.027 mmol) was added into a solution of 1 (15 mg,
0.025 mmol) in dry CH3CN (0.5 mL), and the mixture was stirred
for 24 h. The reaction mixture was quenched with aqueous NaHCO3
(10 mL) and extracted with CHCl3 (10 mL). The organic layers were
evaporated to give a crude oil, which was chromatographed on a sil-
ica gel column (AcOEt/n-hexane = 1:1to give 5 (12 mg, 63%). 1H NMR
(400 MHz, CDCl3) d: 7.67 (1H, s), 7.25 (2H, m), 7.15 (3H, m), 6.80 (1H,
t, J = 9 Hz), 6.47 (1H, d, J = 9 Hz), 6.37 (1H, d, J = 8 Hz), 6.25 (1H, t,
J = 6 Hz), 5.75 (1H, br), 4.38 (1H, m), 4.10 (2H, m), 4.05 (2H, m),
3.92 (2H, m), 3.77 (1H, dd), 3.57 (2H, m), 2.67 (2H, m), 2.37 (1H,
m), 2.07 (1H, m), 1.94 (2H, m), 0.94 (9H, s), 0.86 (9H, s), 0.14 (3H,
s), 0.12 (3H, s), 0.05 (6H, s). 13C NMR (125 MHz, CDCl3) d: 157.95,
146.8, 143.2, 128.4, 128.3, 127.1, 125.8, 124.6, 108.4, 107.3, 87. 8,
86.1, 70.8, 68.5, 64.4, 62.3, 42.0, 40.4, 32.1, 30.6, 26.0, 25.7, 18.5,
18.0, ꢀ4.6, ꢀ4.9, ꢀ5.5, ꢀ5.5. IR mmax (film): 3082, 2955, 1696,
1474 cmꢀ1. High-resolution MS (ESI): calcd for C39H59N4O8Si2
(M+H)+: 767.3866. Found 767.3913.
1695, 1473, 1099 cmꢀ1
. High-resolution MS (ESI): calcd for
C32H53N4O8Si2 (M+H)+: 677.3397. Found: 677.3410.
4.2. OEtPh-8-oxoG-clamp (4)
Under Ar atmosphere, N,N0-carbodiimidazole was added into a
solution of 2-phenylethanol (500 mg) in dry CH2Cl2 (10 mL), and
the mixture was stirred for 2.5 h at room temperature. Evaporation
of the solvents gave a crude product, which was crystallized from
n-hexane to give a white powder (imidazole/1H-imidazole-1-car-
boxylic acid 2-phenylethyl ester = 4:1 by NMR). The above power
(20 mg, 0.036 mmol) was added into a solution of 1 (20 mg,
0.033 mmol) in dry CH3CN (0.5 mL), and the mixture was stirred
4.4. OEtNap-8-oxoG-clamp (6)
Under Ar atmosphere, N,N0-carbodiimidazole was added into a
solution of 1-naphthaleneethanol (700 mg) in dry CH2Cl2 (10 mL),
and the mixture was stirred for 1.5 h at room temperature. Evapo-
ration of the solvents gave a crude product, which was crystallized

Z. Li et al. / Bioorg. Med. Chem. 18 (2010) 3992–3998
3997
from ether to give a white powder (imidazole/1H-imidazole-1-car-
boxylic acid 2-(2-naphthyl) ethyl ester = 3:1 by NMR). The above
power (13 mg, 0.027 mmol) was added into a solution of 1
(15 mg, 0.025 mmol) in dry CH3CN (1 mL), and the mixture was
stirred for 24 h. The reaction mixture was quenched with aqueous
NaHCO3 (10 mL) and extracted with CHCl3 (15 mL). The organic
layers were evaporated to give a crude oil, which was chromato-
graphed on a silica gel column (CHCl3/MeOH = 100:1to give 6
(15 mg, 74%). 1H NMR (400 MHz, CDCl3) d: 8.07 (1H, m), 7.80
(1H, d, J = 8 Hz), 7.69 (1H, dd), 7.60 (1H, s), 7.45 (2H, m), 7.36
(2H, m), 6.76 (1H, t, J = 8 Hz), 6.43 (1H, d, J = 8 Hz), 6.36 (1H, d,
J = 8 Hz), 6.28 (1H, t), 5.80 (1H, br), 4.39 (4H, m), 4.01 (1H, m),
3.91 (2H, m), 3.76 (1H, m), 3.54 (1H, m), 3.38 (3H, m), 2.37 (1H,
m), 2.09 (1H, m), 0.94 (9H, s), 0.86 (9H, s), 0.14 (3H, s), 0.12 (3H,
s), 0.05 (6H, s). 13C NMR (125 MHz, CDCl3) d: 156.8, 153.1, 152.1,
146.7, 143.2, 133.8, 132.1, 128.7, 127.3, 127.0, 126.1, 125.5,
125.5, 123.7, 108.4, 107.3, 87.6, 86.0, 70.8, 68.5, 65.0, 62.3, 41.9,
40.4, 32.7, 26.1, 25.7, 18.5, 18.0, ꢀ4.6, ꢀ4.9, ꢀ5.5, ꢀ5.5. IR mmax
(film): 2857, 1693, 1474, 1255. High-resolution MS (ESI): calcd
for C42H59N4O8Si2 (M+H)+: 803.3866. Found 803.3862.
and the mixture was stirred for 2.5 h at room temperature. Evapo-
ration of the solvents gave a crude product, which was crystallized
from n-hexane to give a white powder (imidazole/1H-imidazole-1-
carboxylic acid 2,4,6-trimethyl phenyl ester = 2:1 by NMR). The
above power (12 mg, 0.043 mmol) was added into a solution of 1
(16 mg, 0.026 mmol) in dry CH3CN (0.5 mL), and the mixture was
stirred for 24 h. The reaction mixture was quenched with aqueous
NaHCO3 (10 mL) and extracted with CHCl3 (10 mL). The organic
layers were evaporated to give a crude oil, which was chromato-
graphed on a silica gel column (AcOEt/n-hexane = 1:1) to give 8
(2 mg, 10%). 1H NMR (400 MHz, CDCl3) d: 7.64 (1H, s), 6.81 (2H,
m), 6.78 (1H, t), 6.48 (1H, d, J = 8 Hz), 6.39 (1H, d), 6.27 (1H, t),
4.38 (1H, m), 4.10 (2H, m), 3.89 (2H, m), 3.76 (2H, m), 3.58 (2H,
m), 2.38 (1H, m), 2.23 (3H, s), 2.11 (7H, m), 0.95 (9H, s), 0.86
(9H, s), 0.15 (3H, s), 0.13 (3H, s), 0.05 (6H, s). IR mmax (film):
2952, 2322, 1712, 1474 cmꢀ1. High-resolution MS(ESI): calcd for
C39H59N4O8Si2 (M+H)+: 767.3866. Found 767.3841.
4.8. Et-Ph-8-oxoG-clamp (11)
Under Ar atmosphere, 3-phenylpropionyl chloride (7
lL,
4.5. 1-Pyrenyl-2-ethanol
0.05 mmol) and Et3N (12 L, 0.08 mmol) were added to a solution
l
of compound 1 (25 mg, 0.04 mmol) in anhydrous THF (0.5 mL) at
0 °C and the mixture was stirred at room temperature for 1 h.
The reaction mixture was quenched by addition of saturated NaH-
CO3 solution. The mixture was extracted with CHCl3.The organic
phase was washed with water and brine and dried over Na2SO4
and concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography (CHCl3/MeOH = 95/5)
afforded compound 11 (19 mg, 62%) as a yellow crystals. 1H NMR
(400 MHz, CDCl3) d: 7.63 (1H, s), 7.15 (4H, m), 7.06 (1H, m), 6.77
(1H, t, J = 8 Hz), 6.49 (1H, br), 6.37 (2H, m), 6.23 (1H, t, J = 6 Hz),
4.37 (1H, m), 3.94 (2H, m), 3.89 (2H, m), 3.75 (1H, d, J = 11 Hz),
3.58 (2H, m), 2.93 (2H, t, J = 8 Hz), 2.50 (2H, t, J = 8 Hz), 2.34 (1H,
m), 2.05 (1H, m), 0.90 (9H, s), 0.86 (9H, s), 0.13 (3H, s), 0.10 (3H,
s), 0.04 (6H, s). IR mmax (film): 2929, 1671, 1558, 1473. ESI-MS
(m/z): 737.5 (M+H)+.
Under Ar atmosphere, a solution of 1-pyrenyl acetic acid
(200 mg, 0.77 mmol) in 3.5 mL dry THF was added into a suspen-
sion of LiALH4 (117 mg, 3.07 mmol) in dry THF (2.5 mL), and the
mixture was stirred for 2 h at room temperature. The reaction mix-
ture was quenched by a sequential addition of methanol (2.5 mL)
and 10% aqueous H2SO4, and extracted with ether. The organic lay-
ers were dried over Na2SO4, evaporated to give a crude oil, which
was chromatographed on a silica gel column (CHCl3) to give 1-
pyrenyl-2-ethanol (160 mg, 84%). 1H NMR (400 MHz, CDCl3) d:
8.35 (1H, m), 8.16 (4H, m), 7.96 (4H, m), 3.98 (2H, t, J = 7 Hz),
3.58 (2H, t, J = 7 Hz). IR mmax (film): 3293, 2324, 1044, 841. ESI-
MS (m/z): 247.0883 (M+H)+.
4.6. OEtPyr-8-oxoG-clamp (7)
Under Ar atmosphere, N,N0-carbodiimidazole was added into a
solution of 1-pyrenylethanol (160 mg) in dry CH2Cl2 (2.5 mL), and
the mixture was stirred for 2.5 h at room temperature. Evaporation
of the solvents gave a crude product, which was crystallized from
ether to give a white powder (imidazole/1H-imidazole-1-carboxylic
acid 1-pyrenyl-2-ethanol ester = 3:1 by NMR). The above power
(18 mg, 0.028 mmol) was added into a solution of 1 (15 mg,
0.025 mmol) in dry CH3CN (1 mL), and the mixture was stirred for
24 h. The reaction mixture was quenched with aqueous NaHCO3
(10 mL) and extracted with CHCl3 (15 mL). The organic layers were
evaporated to give a crude oil, which was chromatographed on a sil-
ica gel column (AcOEt/n-hexane = 1:1to give 7 (14 mg, 65%). 1H NMR
(400 MHz, CDCl3) d: 8.34 (1H, d, J = 9 Hz), 8.08 (4H, m), 7.93 (4H, m),
7.70 (1H, s), 6.84 (1H, t, J = 9 Hz), 6.67 (1H, br), 6.42 (1H, d, J = 9 Hz),
6.32 (1H, d, J = 8 Hz), 6.21 (1H, t, J = 6 Hz), 4.49 (2H, m), 4.36 (1H, m),
3.98 (2H, m), 3.94 (2H, m), 3.76 (1H, m), 3.66 (2H, m), 3.61 (2H, m),
2.36 (1H, m), 2.05 (1H, m), 0.94 (9H, s), 0.87 (9H, s), 0.15 (3H, s), 0.13
(3H, s), 0.05 (6H, s). 13C NMR (125 MHz, CDCl3) d: 172.7, 153.1, 146.3,
143.0, 140.7, 128.3, 127.4, 126.1, 123.7, 122.8, 115.7, 108.3, 106.8,
87.6, 86.0, 70.8, 68.0, 62.3, 42.0, 38.8, 38.2, 31.7, 26.0, 25.7, 18.5,
17.9, ꢀ4.6, ꢀ4.9, ꢀ5.5, ꢀ5.5. IR mmax (film): 2951, 2323, 1696,
1473 cmꢀ1. High-resolution MS (ESI): calcd for C48H61N4O8Si2
(M+H)+: 877.4023. Found 877.4062.
4.9. PrPh-8-oxoG-clamp (12)
Under Ar atmosphere DCC (9 mg, 0.044 mmol), DMAP (2 mg,
0.019 mmol) and 1 (19 mg, 0.032 mmol) were added to a solution
of 4-phenyl-n-butyric acid (6 mg, 0.038 mmol) in anhydrous
dichloromethane (2 mL) at room temperature and the mixture
was stirred for 1 h. Evaporation of the solvents gave a crude prod-
uct, which was purified by silica gel column chromatography
(CHCl3/MeOH = 95:5) to give compound 12 (22 mg, 82%) as a yel-
low crystals. 1H NMR (400 MHz, CDCl3) d: 7.62 (1H, s), 7.21 (2H,
m), 7.12 (3H, m), 6.77 (1H, t, J = 8 Hz), 6.57 (1H, br), 6.44 (1H, d,
J = 8 Hz), 6.34 (1H, d, J = 8 Hz), 6.23 (1H, t, J = 6 Hz), 4.37 (1H, m),
4.03 (2H, m), 3.92 (1H, d, J = 11 Hz), 3.88 (1H, m), 3.76 (1H, d,
J = 11 Hz), 3.62 (2H, m), 2.60 (2H, t, J = 8 Hz), 2.33 (1H, m), 2.21
(2H, t, J = 8 Hz), 2.03 (1H, m), 1.94 (2H, m), 0.94 (9H, s), 0.80 (9H,
s), 0.14 (3H, s), 0.12 (3H, s), 0.05 (6H, s). 13C NMR (125 MHz, CDCl3)
d: 173.3, 153.1, 146.4, 143.1, 141.6, 128.4, 128.3, 127.3, 125.9, 123.
9, 122.8, 115.6, 108.4, 106.8, 87.6, 86.0, 70.8, 68.1, 62.3, 45.0, 38.8,
35.9, 35.2, 27.1, 26.1, 25.7, 18.5, 18.0, ꢀ4.6, ꢀ4.9, ꢀ5.5, ꢀ5.5. IR
mmax (film): 2952, 1671, 1558, 1473, 1256. ESI-MS (m/z): 751.5
(M+H)+.
4.10. NHEt-8-oxoG-clamp (13)
4.7. OMs-8-oxoG-clamp (8)
Under Ar atmosphere, N,N0-carbodiimidazole was added into a
solution of ethylamine (70% in water) (500 lL) in dry CH2Cl2
(10 mL), and the mixture was stirred for 2.5 h at room tempera-
Under Ar atmosphere, N,N0-carbodiimidazole was added into a
solution of 2,4,6-trimethyl phenol (270 mg) in dry CH2Cl2 (5 mL),
ture. Evaporation of the solvents gave a crude product, which

3998
Z. Li et al. / Bioorg. Med. Chem. 18 (2010) 3992–3998
was crystallized from n-hexane to give a white powder (imidazole/
1H-imidazole-1-carboxylic acid N-ethyl ester = 3:1 by NMR). The
above power (16 mg, 0.036 mmol) was added into a solution of 1
(15 mg, 0.025 mmol) in dry CH3CN (0.5 mL), and the mixture was
stirred for 24 h. The reaction mixture was quenched with aqueous
NaHCO3 (10 mL) and extracted with CHCl3 (10 mL). The organic
layers were evaporated to give a crude oil, which was chromato-
graphed on a silica gel column (CHCl3/MeOH = 50:1 to give 13
(11 mg, 65%). 1H NMR (400 MHz, CDCl3) d: 7.61 (1H, s), 6.7 (1H,
t, J = 8 Hz), 6.46 (1H, d, J = 8 Hz), 6.33 (1H, d, J = 8 Hz), 6.21 (1H, t,
J = 6 Hz), 5.72 (1H, br), 4.91 (1H, br), 4.36 (1H, m), 4.04 (2H, m),
3.91 (1H, m), 3.87 (1H, m), 3.75 (1H, m), 3.57 (1H, m), 3.20 (2H,
m), 2.33 (1H, m), 2.05 (1H, m), 1.11 (3H, t, J = 7 Hz), 0.94 (9H, s),
0.86 (9H, s), 0.14 (3H, s), 0.12 (3H, s), 0.05 (6H, s). 13C NMR
(125 MHz, CDCl3) d: 158.8, 153.2, 152.9, 146.8, 143.0, 127.6,
122.7, 115.4, 108.4, 108.2, 107.4, 87.7, 86.0, 70.8, 69.3, 62.3, 41.9,
39.4, 35.4, 25.7, 25.0, 18.5, 17.9, 15.5, ꢀ4.6, ꢀ4. 9, ꢀ5.5, ꢀ5.5. IR
mmax (film): 3318, 1673, 1350, 1169 cmꢀ1. High-resolution MS
(ESI): calcd for C32H54N5O7Si2 (M+H)+: 676.3556. Found 676.3602.
deoxynucleoside analog and then analyzed by curve-fitting meth-
od based on 1:1 complexation as described previously.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Re-
search (S) from the Japan Society for the Promotion of Science
(JSPS) and CREST from the Japan Science and Technology Agency.
Z.L. is grateful for the Research Fellowship from Kobayashi Interna-
tional Scholarship Foundation.
References and notes
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Under Ar atmosphere benzaldehyde (6 lL, 0.06 mmol) and
MgSO4 (30 mg) were added to a solution of compound 1 (30 mg,
0.05 mmol) in anhydrous dichloromethane (2 mL) at room temper-
ature and the mixture was refluxed for 15 h. The reaction mixture
was filtered to remove the magnesium sulfate and the solvent was
concentrated under reduced pressure gave a crude product. With-
out farther purified, To a stirred solution of the above crude prod-
uct (37 mg) in methanol (2 mL) was added 10 mg (0.16 mmol) of
sodium cyanoborohydride. The reaction mixture was stirred at
room temperature for 1 h. The reaction was quenched by addition
of water. The mixture was extracted with CHCl3. The organic phase
was dried over Na2SO4 and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(CHCl3/MeOH = 95:5) afforded compound 14 (21 mg, 57%) as a yel-
low crystals. 1H NMR (400 MHz, CDCl3) d: 7.60 (1H, s), 7.33 (4H, m),
7.23 (1H, m), 6.73 (1H, t, J = 8 Hz), 6.46 (1H, d, J = 8 Hz), 6.35 (1H, d,
J = 8 Hz), 6.23 (1H, t, J = 6 Hz), 4.37 (1H, m), 4.08 (2H, m), 3.92 (3H,
m), 3.87 (1H, m), 3.75 (1H, d, J = 11 Hz), 3.01 (2H, m), 2.36 (1H, m),
2.05 (1H, m), 0.94 (9H, s), 0.86 (9H, s), 0.14 (3H, s), 0.12 (3H, s), 0.05
(6H, s). 13C NMR (125 MHz, CDCl3) d: 154.1, 153.5, 146.2, 143.0,
128.5, 127.5, 127.4, 123.2, 122.6, 116.6, 108.6, 108.0, 87.5, 85.9,
70.7, 68.8, 62.3, 53.3, 47.5, 42.0, 26.0, 25.7, 18.5, 18.0, ꢀ4.6, ꢀ4.9,
ꢀ5.5, ꢀ5.3. IR mmax (film): 2928, 1671, 1558, 1473. ESI-MS (m/z):
695.6 (M+H)+.
4.12. Fluorescence quenching experiments
A stock solution of the 30-O-, 50-O-diTBS protected 20-deoxynu-
cleoside (0–10
lM final concentration) was added to a solution
of the 8-oxoGclamp analog (1
l
M) in CHCl3 containing 10 mM
ETA and 2.7 mM AcOH at 25 °C. Fluorescence spectra were re-
corded with excitation at 365 nm. The fluorescence intensity at
450 nm was plotted against the concentration of the added 20-
13. Cubberley, M. S.; Iverson, B. L. J. Am. Chem. Soc. 2001, 123, 7560.