Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site

Article abstract of DOI:10.1016/j.bmc.2012.04.009

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base exci

Full text of DOI:10.1016/j.bmc.2012.04.009

Bioorganic & Medicinal Chemistry 20 (2012) 3470–3479
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and binding properties of new selective ligands
for the nucleobase opposite the AP site
Yukiko Abe a, Osamu Nakagawa a, Rie Yamaguchi a, Shigeki Sasaki a,b,
⇑
a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
b Core Research for Evolution Science and Technology (CREST), Japan Science and Technology Agency, 4-1-8 Motomachi, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA
alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/
apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also
representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and
a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and
mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnos-
tic purposes. In this study, we have developed nucleobase–polyamine conjugates as the AP site binding
ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleo-
base opposite the AP site by the Watson–Crick base pair formation and that the polyamine part should
contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate
backbone. The nucleobase conjugated with 3,30-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-
ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on
the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand
to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that
the specific stabilization is due to specific binding of the ligands to the complementary AP site. These
results have suggested that the complementary base pairs of the Watson–Crick type are formed at the
AP site.
Received 19 March 2012
Revised 3 April 2012
Accepted 4 April 2012
Available online 10 April 2012
Keywords:
AP site
DNA
Binding ligand
Surface plasmon resonance
Isothermal titration calorimetry
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
its biological impacts.10 An attractive application of the AP site-spe-
cific molecules is to block the AP site repair in order to enhance the
DNA is continuously damaged by either endogenous or exoge-
nous agents such as reactive oxygen species, DNA alkylating agents,
etc.1,2 For example, the oxidation of guanosine at the 8-position pro-
duces 8-oxoguanosine which is an important marker of oxidative
stress. The alkylation of guanosine at the N7-position or adenosine
at the N3-position by alkylating agents produces 7-methylguano-
sine3 or 3-methyladenosine,4 respectively. These damaged nucleo-
tides are removed by the base excision repair pathway to form the
apurinic/apyrimidinic site (AP sites) as the intermediate.5 AP sites
per se are DNA damages and can spontaneously occur at a rate about
10,000 sites per day in human cells.6 AP sites affect the local and glo-
bal structures of DNA and it is known that unrepaired AP sites block
DNA polymerase leading to acute toxicities7 or preferentially incor-
porate deoxyadenosine opposite the AP site to induce mutations in
the DNA polymerization.8 The AP site stimulates topoisomerase
II-mediated DNA cleavage to induce cell death.9 Thus, the AP site-
specific molecules would be useful for a better understanding of
antitumor efficacy of the alkylating anticancer agents.11,12 The AP
site is in equilibrium between the hemiacetals, the hydrate form,
and the aldehyde, which is targeted by the covalent bond with small
molecular ligands though the Schiff base formation followed by
reduction.13 Non-covalent recognition of the AP site has also been
investigated using non-natural nucleotides,14 non-nucleoside
probes,15 nucleobase derivatives,16 etc. The compounds that can
recognize the remaining nucleobase opposite the AP sites would
be useful to analyze the nature of the damage leading to the AP site.
It can be also expected that the compound specific to the cytosine
base opposite the AP site might potentiate the effect of the guano-
sine-alkylating agents. Despite a number of examples, it is difficult
to rationally design molecular probes with selectivity to a nucleo-
base opposite the AP site. Lhomme’s group developed adenine–acri-
dine conjugates as DNA cleaving agents in which the adenine base
was expected to locate in the AP site pocket and forms the comple-
mentary base pair with the thymine opposite the AP site. The strong
binding of the acridine with the duplex DNA either by stacking or in
the external binding mode was thought to bring the adenine base
into the AP site pocket for the binding with the thymine base
⇑
Corresponding author.
E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.009

Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
3471
O
O
P
O
H
O
N
H
AP site pocket
N
N
O
O
P
O
O
P
O
O
O
O
O
O
O
OH
O
P
O
O
O
P
O
O
O
O
H
N
H
H
+
+
O
N
N
N
O
O
P
O
O
P
O
N
O
N
O
O
O
H
O
N
NH Base pairing
O
OH
+
O
P
O
O
NH2
O
Charge interaction
NH2
NH3
Figure 1. Design strategy for AP sites ligands with a selectivity to the opposing nucleobase.
NH2
N
O
N
N
N
N
N
HN
H2N
4HCl
4HCl
H
N
H
N
H
N
H
N
N
NH2
NH2
1: A-ligand
2: G-ligand
NH2
N
O
O
N
N
HN
HN
4HCl
3HCl
3HCl
H
N
H
H
N
H
N
H
N
H
N
O
O
N
O
N
NH2
NH2
NH2
3: C-ligand
4: T-ligand
Figure 2. Nucleobase–polyamine conjugates.
5: U-ligand
opposite the AP site.17 They also applied the AP site-binding ligand
to enhance the in vivo toxicity of the alkylating agent (bischloroeth-
ylnitrosourea).12 Encouraged by these studies, we hypothesized
that the nucleobase might generally form Watson–Crick base pairs
if the nucleobase is properly placed in the AP site pocket. In order to
bring the nucleobase around the duplex DNA close the AP site, we
relied on the weak electrostatic interaction of the polycation with
the phosphate backbone. We now describe in detail the molecular
design, synthesis and the binding properties with the duplex DNA
containing the AP site.
(Fig. 1). The polycation unit was expected to promote access of the
nucleobase to the duplex DNA by a non-specific, electrostatic inter-
action. When the nucleobase enters the AP site pocket, as the AP
site pocket has a relatively hydrophobic nature, the complemen-
tary base pairing with the nucleobase opposite the AP site should
produce the specific binding affinity of the ligand. Thus, the objec-
tive of this study is to confirm the binding selectivity of the nucle-
obase–polyamine conjugates in a predictable manner based on the
Watson–Crick pairing. We employed 3,30-diaminodipropylamine
(norspermidine)18 as the polyamine part in this study, because a
weak DNA binding affinity of the polycation part was thought to
be preferable in order to prove that the selectivity originated due
to the nucleobase part. Biological polyamines, such as spermine,
was not chosen because of the relatively higher affinity with some
sequence preference.19–21 Thus, five nucleobase conjugates with
3,30-diaminodipropylamine were synthesized and subjected to
measuring the binding properties (Fig. 2, A-ligand (1), G-ligand
(2), C-ligand (3), T-ligand (4) and U-ligand (5)).
2. Results and discussion
2.1. Design and synthesis of the nucleobase-3,30-
diaminodipropylamine conjugates
In our attempt to form A–T and G–C Watson–Crick base pairs in
the AP site pocket, we designed nucleobase–polyamine conjugates

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Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
a
3,3'-Diamino-
dipropylamine
HN
Ns
N
Ns
NHBoc
6
b
c
Br
N
N
NHBoc
Ns
Ns
7
1
2
: A-ligand
: G-ligand
R
d
3: C-ligand
N
Ns
N
Ns
NHBoc
4
5
: T-ligand
: U-ligand
8
: R = 9-adenine
9: R = 9-guanine
10: R = 1-cytosine
11
: R = 1-thymine
12: R = 1-uracil
Scheme 1. The synthesis of the ligands (1–5). Reagents and conditions: (a) (i)
(Boc)2O, THF, 0 °C; (ii) NsCl, Et3N, CH2Cl2, 0 °C; (b) 1,3-dibromopropane, K2CO3,
DMF, rt; (c) (i) K2CO3, DMF, 60 °C, adenine for 1; 2-amino-6-chloropurine for 2, and
then 0.5 M HCl/MeOH; 4-N-acetylcytosine for 3; 3-N-benzoylthymine for 4; 3-N-
benzoyluracil for 5, (d) (i) thiophenol, K2CO3, DMF, rt; (ii) 0.5 M HCl/MeOH, rt, or
10% aq HCl.
Figure 4. The selective stabilization effects of the ligand to the duplex ODN
containing the AP site. [NaCl]: 100 mM for A-, G-ligands and [NaCl]: 50 mM for C-,
T- and U-ligands. AP and X represent the tetrahydrofuran part and the nucleobase
opposite the AP site, respectively.
GCG-30, where AP represents the THF ring. Duplexes were formed
with the complementary sequence of 50-dCGC ATG X GTA CGC-30,
where X represents the adenine, guanine, cytosine or thymine base
opposite the AP site (X = A, G, C, T). Figure 3 illustrates the UV-
melting curves obtained using the duplex (50-dGCG TAC AP CAT
GCG-3/50-dCGC ATG X GTA CGC-30) in the absence and presence
of the A-ligand (Fig. 3A) or the G-ligand (Fig. 3B). The A-ligand
selectively increased the Tm value of the duplex having X = T
Figure 3. UV-melting curves in the absence (gray line) and presence of A-ligand
(red line) or G-ligand (green line). (A) A-ligand and thymine opposite the AP site, (B)
G-ligand and cytosine opposite the AP site. UV-melting curves were measured in
10 mM HEPES–NaOH buffer (pH 7.0), 100 mM NaCl at the scan rate of 1 °C at
260 nm in the absence and presence of the ligand. [duplex ODNs]: 4
20 M. AP and X represent the tetrahydrofuran part and the nucleobase opposite
the AP site, respectively.
lM, [ligands]:
l
The synthesis of the ligands is shown in Scheme 1. The terminal
amino group of 3,30-diaminodipropylamine was protected with the
tert-butoxycarbonyl (t-Boc) group and the remaining amino groups
were reacted with 2-nitrobenzenesulfonyl chloride (NsCl). The
terminal nosylated amino group was reacted with 1,3-dibromopro-
pane to form the triamine bromide 7 as the common intermediate.
The nucleobase was alkylated with the bromide 7 to produce the
corresponding nucleobase derivatives (8–12). Finally, all the pro-
tecting groups were removed to produce the nucleobase–polyamine
conjugates (1–5). All ligands were purified as the hydrochloride
salts.
(
D
Tm = +6.7 °C), and the duplex incorporating X = C was selectively
stabilized by the G-ligand ( Tm = +6.6 °C). The Tm value of other
duplexes were not significantly changed (Fig. S1).
D
The increase in the Tm values are displayed in the bar graphs of
Figure 4 and the data are shown in Table 1, clearly indicating the
selective stabilization of the A-ligand for the opposing T and the
G-ligand for opposing C (Fig. 4, A and B). The UV melting experi-
ments using the C-, T- and U-ligands were performed in the buffer
containing 50 mM NaCl to observe the clear effect of the ligand
(UV-melting curves are shown in Fig. S2), and the increase in the
Tm values are summarized in Figure 4 (C and D). Interestingly,
the C-ligand showed a selective stabilization for G opposite the
2.2. Stabilization effects of ligands on duplex DNAs containing
the AP site
AP site (Fig. 4C, DTm = +5.3 °C). In the case of the T-ligand, although
the selective stabilization was observed for the opposing A, some
stabilization was also observed for the opposing T and G (Fig. 4D,
The binding property of the ligand was evaluated by measuring
the increase in the melting temperature (Tm) of the duplex DNA
containing the AP site. The tetrahydrofuran ring was used as the
stable AP site analog in the sequence of 50-dGCG TAC AP CAT
DTm = +3.6 °C for A, +2.1 °C for G, and +3.0 °C for T). The formation
of the G–T and T–T pairs might contribute to the weak stabiliza-
tion. It should be noted that the U-ligand retained its selectivity
to the opposing A, but lost the stabilization effect for the opposing

Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
3473
Table 1
Melting temperature of duplexes containing the AP site in the absence (Tm(ꢀ)) and presence (Tm(+)) of each liganda
X =
Ligand
C-Ligandc
A-Ligandb
G-Ligandb
T-Ligandc
U-Ligandc
Tm (ꢀ)
Tm (+)
Tm (ꢀ)
Tm (+)
Tm (ꢀ)
Tm (+)
Tm (ꢀ)
Tm (+)
Tm (ꢀ)
Tm (+)
A
G
C
T
39.7
39.8
34.7
37.0
40.4 (+0.7)
40.8 (+1.0)
34.6 (ꢀ0.1)
43.7 (+6.7)
40.1
40.5
34.2
37.3
40.4 (+0.3)
40.9 (+0.4)
40.8 (+6.6)
38.1 (+0.8)
34.0
35.2
32.5
32.4
35.2 (+1.2)
40.5 (+5.3)
32.6 (+0.1)
32.4 ( 0.0)
35.6
35.7
32.5
32.5
39.2 (+3.6)
37.8 (+2.1)
33.8 (+1.3)
35.5 (+3.0)
35.0
35.1
32.1
33.2
39.1 (+4.1)
36.4 (+1.3)
32.1 ( 0.0)
33.9 (+0.7)
a
b
c
Melting temperature was measured as described in the footnote of Figure 3 except for the NaCl concentration in the buffer. X represents the base opposite AP.
The buffer contained 100 mM NaCl.
The buffer contained 50 mM NaCl.
Figure 5. The SPR sensorgrams obtained with A-ligand using a sensor chip immobilizing the hairpin duplex. (A) X = T, (B) X = A, (C) X = C and D) X = G. Experiments were
performed in 10 mM HEPES–NaOH buffer (pH 7.0) and 100 mM NaCl at 25 °C and the concentration of the A-ligand was from 0.05 to 2.00 lM at the flow rate of 20 ll/min.
Table 2
T, resulting in an improvement of the selectivity to the opposing A
Equilibrium association constants (Ka, 106 Mꢀ1) of the ligands obtained by the SPR
(Fig. 4E,
DTm = +4.0 °C). In the control experiments, these ligands
measurementsa
did not show any stabilization effect to the full match duplexes
lacking the AP site (Table S1). Also 3,30-diaminodipropylamine,
adenine, and cytosine, thymine, or uracil alone did not produce
any significant stabilization to the duplex ODN containing the AP
site. Guanine was not tested due to its insolubility. These results
clearly suggested that the selective stabilization was not exhibited
by the polycation, but by the selective interaction of the nucleo-
base with the nucleobase opposite the AP site pocket.
Ligand
Ka (ꢁ 106 Mꢀ1
)
X = A
X = G
X = C
X = T
A-Ligand
G-Ligand
C-Ligand
0.057
0.016
0.002
0.222
0.045
0.087
0.047
0.039
0.043
0.001
0.010
6.300
0.002
0.063
0.022
3.900
0.080
0.001
0.053
0.027
<0.001
T-Ligandb
U-Ligandb
norspermidine
c
c
c
—
—
—
The increase in the Tm values of the duplex ODN containing the
AP site is produced due to the stabilization effect of the ligand not
only by the hydrogen bonds and the electrostatic interaction, but
also by the stacking interaction of the nucleobase part inserted in
the AP site pocket. Therefore, it was assumed that the pyrimidine
a
Equilibrium association constants were obtained from the SPR response by an
affinity analysis by steady-state. The measurement was done as described in the
footnote of Figure 5.
b
50 mM NaCl was used.
Not measured.
c

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Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
Figure 6. The ITC binding isotherm of the ligand to the selective duplex ODN containing the AP site. Each ligand was added to a solution of the duplex ODN (10 l
M, 50-
dGCGTAC AP CATGCG-30/ 50-dCGCATG X GTACGC-30; AP = THF, X = A, G, C, T) in 10 mM HEPES–NaOH buffer (pH 7.0) containing 100 mM NaCl at 25 °C. (A) A-ligand to X = T,
(B) G-ligand to X = C, (C) C-ligand to X = G, D) T-ligand to X = A.
ligands (C-, T- and U-ligand) would exhibit a lower stacking inter-
action than the purine ligands (A- and G-ligand) and would pro-
duce the lower stabilization effect for the duplex containing the
AP site. Thus, the effect of pyrimidine ligands on the Tm values
were measured under the lower salt conditions (50 mM NaCl) than
those with the purine ligands (100 mM NaCl). Stronger charge
interactions were expected to compensate the weaker stacking
interactions. The stabilization effect of the T- or U-ligand was

Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
3475
Table 3
The thermodynamic parameters of the binding of the ligand with its selective duplexa
Ligand
X
Nb
D
H (kcal/mol)
D
S (kcal/kmol)
ꢀTD
S (kcal/mol)
DG (kcal/mol)
A-Ligand
G-Ligand
C-Ligand
T-Ligand
U-Ligand
T
0.94
0.87
1.02
0.97
1.04
ꢀ18.17
ꢀ26.30
ꢀ7.38
ꢀ9.56
ꢀ3.08
ꢀ32.60
ꢀ59.80
ꢀ1.07
ꢀ7.53
13.50
9.72
17.83
0.32
2.25
ꢀ4.03
ꢀ8.45
ꢀ8.47
ꢀ7.06
ꢀ7.32
ꢀ7.11
C
G
A
A
a
The parameters were obtained from the ITC binding isotherm shown in Figures 6 and S6.
Binding stoichiometry.
b
lower than that of the C-ligand probably because the A-T or A-U
base pair is weaker than the G–C base pair. The CD spectrum of
the duplex DNA containing the AP site either in the absence or
the presence of the ligand showed the typical B-DNA conformation
(Fig. S3), suggesting that the duplex DNA conformation was not af-
fected by the binding of the ligand in the AP site pocket.
2.3. Evaluation of the binding affinity of the ligands to the
duplex DNA by SPR
We measured the surface plasmon resonance (SPR) to evaluate
the binding affinity of the ligand to the duplex DNA containing the
AP site. The hairpin duplex DNA having the TTTT loop structure and
the AP site (50-dTTTT CGC ATG X GTA CGC TTTT GCG TAC AP CAT
GCG-30, X = A, G, C, T) labeled with the biotin unit at the 50 termi-
nus was immobilized onto a streptavidin surface of the sensor chip.
The A-ligand produced a sensorgram showing the strong binding
to the duplex having the thymidine opposite the AP site (Fig. 5A),
but a much lower response was observed for the duplexes having
A, G, or C opposite the AP site (Fig. 5B–D).
The polyamine part alone did not produce a significant SPR re-
sponse, clearly indicating that the selective binding is obtained by
the conjugation of the polyamine with the nucleobase part. The
selective affinity of the A-ligand to the duplex containing the AP
site was well correlated with the increase in the Tm value. Accord-
ingly, the selective interaction of the A-ligand with the duplex DNA
containing the AP site has been proved to be responsible for the
selective increase of the Tm value. Similarly, the G-, C-, and T-li-
gands showed the strongest binding to the duplex having the
opposing C, G and A, respectively (Fig. S4). Since the T-ligand did
not produce a sufficient response for the affinity analysis in the
buffer containing 100 mM sodium chloride, the equilibrium associ-
ation constants were obtained in the presence of 50 mM sodium
chloride. The selective combination; that is A-ligand to T, G-ligand
to C, C ligand to G, and T-ligand to A, produced the equilibrium
association constants ranging from 0.22 to 6.3 ꢁ 106 Mꢀ1, which
were 5ꢂ390-fold higher than those for the non-selective combina-
tions (Table 2). In contrast to the UV-melting experiments, the T-li-
gand exhibited a clear selectivity to the opposing A. In the case of
the U-ligand, the A-selectivity observed in the UV-melting experi-
ment was not clear for the SPR measurement.
Figure 7. Comparison of thermodynamic parameters summarized in Table 3.
a clear binding isotherm was not obtained for the combination of
the A-ligand and the non-selective duplex having the A/AP, G/AP
or C/AP site. The single-base loss reduces the free energy of the du-
plex to the extent of 8–11 kcal/mol and the enthalpy of the duplex
to the extent of 29–46 kcal/mol in these sequences.22 It has been
shown that the ligand can compensate for the duplex instability
caused by the loss of the single base by binding with the remaining
nucleobase in the AP site pocket (the free energy was 7–8 kcal/mol
and the enthalpy was 3–33 kcal/mol).
The thermodynamic parameters for the G-ligand binding to
X = C were similar to those of the A-ligand in terms of the 1:1 bind-
ing stoichiometry, the high enthalpy gain and the entropy loss
(Fig. 7). The binding enthalpy must be due to the interactions by
hydrogen bonds between the purine base of the ligand and the
pyrimidine base opposite the AP site, stacking of the purine base
within the flanking cytosine bases, and charge interactions between
the polycation part and the phosphate backbone. MD calculations
on the duplex having the C/AP or T/AP sites have suggested that
the AP site is flexible and adopts a range of conformations.23 The
CD spectra showed a slight change in the B-form conformation of
the duplex DNA by binding of the ligand to the AP site (Fig. S3).
Therefore, the entropy loss associated with the binding might re-
flect the decreased flexibility of the local conformation neighboring
the AP site.
2.4. Thermodynamic parameters of the binding of the ligand for
the duplex containing the AP site
Interestingly, the binding parameters of the C- and T-ligands
were similar to each other and showed a remarkable contrast to
those of the A- and G-ligands in that almost no entropy loss is asso-
ciated with the binding. In the MD calculation on the G/AP site, the
The thermodynamic parameters of the binding of the ligand
with the duplex containing the AP site were obtained by isothermal
titration calorimetry (ITC). The ligand solution was added to a solu-
tion of the duplex and the thermal change was observed. The ITC
binding isotherms of the ligands are summarized in Figure 6 and
S6, and the obtained parameters are summarized in Table 3. The
A-ligand binding to the duplex having the T/AP site was analyzed
to produce the 1:1 binding stoichiometry (N = 0.94, Table 3) with
a high enthalpy gain and entropy loss (Fig. 7). On the other hand,
a
-anomer of the 20-deoxyribose part adopts an intrahelical confor-
mation, and the guanine is stacked between the flanking bases. The
slight entropy loss associated with the binding of the pyrimidine
ligands may imply that the conformational change around the AP
site is not induced by the binding of the pyrimidine ligand. In the
UV-melting experiment, the U-ligand showed a better selectivity
to the opposing A than the T-ligand (Fig. 4D vs E). However, the

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Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
binding affinity obtained by SPR and also by ITC indicated that the
T-ligand has a higher affinity to the adenine opposite the AP site.
The thermodynamic parameters of these ligands revealed a sharp
contrast, namely, the relatively high enthalpy gain and low entropy
loss were observed for the T-ligand, whereas lower enthalpy gain
and entropy gain for the U-ligand (Fig. 7). The higher enthalpy gain
for the T-ligand may reflect the effects of the methyl group on the
hydrogen bonding, stacking interaction, and the hydrophobic
interaction.24 Such interactions also increase the binding affinity
for the mismatch base pairs, resulting in low selectivity in the
UV-melting experiment (Table 1). The entropy gain observed for
the U-ligand binding to the opposing A may be beneficial for keep-
ing the complex during the thermal denaturation in UV-melting
experiment. This may account for the fact that a better selectivity
to the opposing A was obtained by the U-ligand (Fig. 4E).
chromatography (silica gel, CH2Cl2/MeOH: 28% aqueous NH3 =
40:8:1to10:4:1) 40:8:1–10:4:1)to give1-N-Bocderivativeasa pale
yellow oil (8.5 g, 36.7 mmol, 91%).
To a solution of the above 1-N-Boc derivative (8.7 g, 37.8 mmol)
in anhydrous CH2Cl2 (20 ml) were added anhydrous Et3N (26.3 ml,
189 mmol) and a solution of 2-nitrobenzenesulfonyl chloride
(17.6 g, 79.4 mmol) in anhydrous CH2Cl2 (40 ml) over a period of
15 min under an argon atmosphere at 0 °C, and the mixture was
stirred for 45 min. The reaction mixture was poured into CH2Cl2
(150 ml), washed with 1 M aqueous KHSO4 (200 ml), saturated
aqueous NaHCO3 (200 ml), and brine (150 ml). The organic extracts
were dried over anhydrous Na2SO4, filtered and evaporated to af-
ford a crude product (20.9 g), which was purified by column chro-
matography (silica gel, CH2Cl2 to 5% MeOH) to yield 6 as a green
foam (17.3 g, 28.7 mmol, 76%). 1H NMR (400 MHz, CDCl3) d ppm:
8.10–8.08 (1H, m), 7.99–7.97 (1H, m), 7.85–7.83 (1H, m), 7.75–
7.67 (4H, m), 7.62–7.60 (1H, m), 5.63 (1H, t, J = 6 Hz), 4.74 (1H,
br s), 3.36 (2H, t, J = 7 Hz), 3.31 (2H, t, J = 7 Hz), 3.16–3.09 (4H,
m), 1.83 (1H, quint., J = 7 Hz), 1.70 (1H, quint., J = 7 Hz), 1.41 (9H,
s). ESI-MS m/z: 602.2 [M+H]+. IR cmꢀ1: 1694, 1542, 1367, 1344,
1164.
3. Conclusion
In this study, we hypothesized the selective formation of Wat-
son–Crick base pairs in the AP site using the nucleobase–polyca-
tion conjugates (1–5). Measurements of the UV-melting showed
a high selectivity of A-, G-, C-, and U-ligands for the complemen-
tary nucleobases except T-ligand. SPR measurements showed the
larger association constants between base-selective conjugates
and their complementary nucleobases including C-ligand. And
ITC measurements clearly showed that the 1:1 binding stoichiom-
etry between conjugates and their complementary bases. Binding
of A- and G-ligands was favored by a high enthalpy gain and entro-
py loss, suggesting the strong stacking of purine bases. The entropy
loss was ascribed to the conformational flexibility from the CD
measurements. C- and T-ligands showed almost no entropy loss
for the base pairing, and U-ligand showed a lower enthalpy gain
compared to T-ligand. In conclusion, this study has clearly demon-
strated that the Watson–Crick base pair is generally formed in the
AP site pocket using the nucleobase–polyamine conjugates. The
new AP site selective ligands may find utility as recognition mole-
cules for the AP site as well as the lead compounds for interference
of the AP site associated biological reactions such as polymeriza-
tion and repair systems.
4.3. 1-N-Boc-7-N-(3-bromopropyl)-4-N,7-N-dinosyl-4-
azeheptane-1,7-diamine (7)
To a solution of 5 (14.6 g, 24.3 mmol) in anhydrous DMF (40 ml)
were added K2CO3 (16.8 g, 22 mmol) and 1,3-dibromopropane
(12.4 ml, 122 mmol) under an argon atmosphere at 0 °C, and the
mixture was stirred for 8.5 h at room temperature. The reaction
mixture was poured into water (200 ml) and extracted with ether
(200 ml ꢁ 2). The organic layers were washed with brine (200 ml),
dried over anhydrous Na2SO4, filtered and evaporated to afford a
crude product (20.9 g), which was purified by column chromatog-
raphy (silica gel, CH2Cl2 to 1% MeOH) to yield 7 as a clear yellow
viscous oil (12.6 g, 17.4 mmol, 72%). 1H NMR (400 MHz, CDCl3) d
ppm: 8.03–7.97 (2H, m), 7.71–7.66 (4H, m), 7.62–7.59 (2H, m),
3.40 (2H, t, J = 7 Hz), 3.36–3.25 (8H, m), 3.13–3.08 (2H, m), 2.06
(2H, quint., J = 7 Hz), 1.86 (2H, quint., J = 7 Hz), 1.71 (2H, quint.,
J = 7 Hz), 1.42 (9H, s). ESI-MS m/z: 722.5, 724.5 [M+H]+. IR cmꢀ1
:
1705, 1543, 1368, 1346, 1161.
4. Experimental section
4.1. Method
4.4. A-Ligand (1)
A
suspension of adenine (393 mg, 2.90 mmol), 7 (1.05 g,
1.45 mmol) and K2CO3 (804 mg, 5.81 mmol) in anhydrous DMF
(3 ml) was heated under an argon atmosphere at 60 °C for 5.5 days,
and then quenched by the addition of water. The reaction mixture
was extracted with CHCl3, the organic extracts were washed with
brine, dried over anhydrous Na2SO4, filtered and evaporated to af-
ford crude products (1.58 g), which was purified by column chro-
matography (silica gel, CHCl3/MeOH = 20:1) to yield the coupling
product 8 as a pale yellow foam (676 mg, 0.87 mmol, 60 %). 1H
NMR (400 MHz, CDCl3) d ppm: 8.29 (1H, s), 7.93 (1H, s), 7.96–
7.88 (2H, m), 7.70–7.58 (6H, m), 4.25 (2H, t, J = 7 Hz), 3.33 (2H, t,
J = 7 Hz), 3.30–3.22 (6H, m), 3.09 (2H, q, J = 6 Hz), 2.19 (2H, quint.,
J = 7 Hz), 1.85–1.77 (2H, m), 1.67 (2H, quint., J = 7 Hz),1.41 (9H, s).
ESI (MS) m/z: 777.4 [M+H]+. IR cmꢀ1: 1704, 1543, 1367, 1347,
1162.
Melting points were determined using a Stuart Scientific MELT-
ING POINT APPARATUS SMP 3. 1H and 31P NMR spectra were re-
corded at 400 MHz on a Varian 400 UNITY using CDCl3, CD3OD,
CD3CN or D2O as a solvent. 13C NMR spectra were recorded at
125 MHz on a Varian INOVA 500 using D2O as a solvent. Chemical
shifts are reported in ppm, in d units. ESI-MS spectra were recorded
on an Applied Biosystems Marinar System 5299 API-TOF instru-
ment. IR spectra were recorded on a PerkinElmer Spectrum One
FT-IR spectrometer.
4.2. 1-N-Boc-4-N,7-N-dinosyl-4-azeheptane-1,7-diamine (6)
A solution of di-tert-butyl dicarbonate (9.2 ml, 40.0 mmol) in
anhydrous THF (120 ml) was added dropwise over a period of 2.5 h
to a solution of 3,30-diaminodipropylamine (24.3 ml, 172 mmol) in
anhydrous THF (180 ml) under an argon atmosphere at 0 °C. This
reaction mixture was stirred for 1 day at 0 °C, and then THF was
evaporated. The residue was poured into water (100 ml) and the
mixture was extracted with CH2Cl2 (100 ml ꢁ 7). The organic
extracts were dried over anhydrous Na2SO4, filtered and evaporated
to afford a crude product (10.7 g), which was purified by column
To a solution of the above coupling product 8 (671 mg, 0.86
mmol) in anhydrous DMF (1.5 ml) were added thiophenol (195 ll,
1.90 mmol) and K2CO3 (448 mg, 3.45 mmol) under an argon atmo-
sphere at room temperature. The reaction mixture was stirred for
13 h at room temperature and then quenched by the addition of
methanol. This mixture was filtered and the filtrate was evaporated
to afford a crude product, which was purified by column chromatog-
raphy (silica gel, CHCl3/MeOH/aq NH3 = 20:8:1) to yield the

Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
3477
partially purified products (416 mg). This product was dissolved in
0.5 M HCl/MeOH (26 ml) and the reaction mixture was stirred for
43 h at room temperature, and then the precipitates were collected
on a filter, and washed with MeOH to produce 1 as white solids
(317 mg). The filtrates were evaporated to give a crude product,
which was extracted with water. The water extracts were washed
with CHCl3 and freeze-dried to give 1 as white solids (67 mg). The
obtained 1 was purified by precipitation from water/EtOH (4 ml/
40 ml) at ꢀ78 °C. The purified 1 was collected by centrifuged
(1500 rpm, 10 min), and dried under vacuum to give 1 as white sol-
ids (266 mg, 68% after re-precipitation twice). mp 270–271 °C. 1H
NMR (400 MHz, D2O) d ppm: 8.36 (1H, s), 8.28 (1H, s), 4.38 (2H, t,
J = 7 Hz), 3.13–3.02 (10H, m), 2.28 (2H, quint., J = 7 Hz), 2.10–2.00
(4H, m). 13C NMR (125 MHz, D2O) d ppm: 152.9, 151.2, 147.7,
147.1, 120.8, 47.5, 47.3, 47.2, 44.0, 39.2, 28.7, 26.3, 25.3. HR-ESI-
6 days, and then quenched by the addition of water. The reaction
mixture was extracted with CHCl3, the organic extracts were
washed with brine, dried over anhydrous Na2SO4, filtered and
evaporated to afford the crude product (2.22 g), which was purified
by column chromatography (silica gel, CHCl3:MeOH = 20:1) to
yield the coupling product 10 as a pale yellow foam (806 mg,
1.01 mmol, 56 %). 1H NMR (400 MHz, CDCl3) d ppm: 7.97–7.93
(2H, m), 7.72–7.66 (4H, m), 7.62–7.56 (2H, m), 7.40 (1H, d,
J = 7 Hz), 3.95–3.86 (2H, m), 3.36 (2H, t, J = 7 Hz), 3.32–3.23 (6H,
m), 3.16–3.08 (2H, m), 2.07 (2H, quint., J = 7 Hz), 1.88 (2H, quint.,
J = 7 Hz), 1.42 (9H, s). ESI (MS) m/z: 794.9 [M+H]+. IR cmꢀ1: 1708,
1544, 1370, 1347, 1162.
To a solution of the above coupling product 10 (800 mg,
1.01 mmol) in anhydrous DMF (1.5 ml) were added thiophenol
(227 ll, 2.21 mmol) and K2CO3 (695 mg, 5.03 mmol) under an ar-
MS m/z: 307.2353 [M+H]+ (307.2366 calcd for C14H27N8). IR cmꢀ1
:
gon atmosphere at room temperature. The reaction mixture was
stirred for 3.5 h at room temperature and then quenched by the
addition of methanol. This mixture was filtered and the filtrate
was evaporated to afford a crude product (1.27 g), which was puri-
fied by column chromatography (silica gel, CHCl3/MeOH/aqueous
NH3 = 20:8:1–10:4:1) to yield the purified product (399 mg). This
product was dissolved in 0.5 M HCl/MeOH (29 ml) and the reaction
mixture was stirred for 2 days at room temperature, and then the
solvent was removed by Liebig condenser to produce 3 as white
solids (362 mg). The obtained 3 was purified by precipitation from
water/EtOH (1.5 ml/40 ml) at ꢀ78 °C. The purified 3 was collected
by centrifuged (1500 rpm, 10 min), and dried under vacuum to
give 3 as white solids (291 mg, 67% after re-precipitation twice).
Mp 248–249°C. 1H NMR (400 MHz, D2O) d ppm: 7.77 (1H, d,
J = 8 Hz), 6.12 (1H, d, J = 8 Hz), 3.91 (2H, t, J = 7 Hz), 3.16–3.04
(10H, m), 2.13–2.01 (6H, m). 13C NMR (125 MHz, D2O) d ppm:
162.7, 152.6, 151.8, 97.6, 49.4, 47.4, 47.3, 47.2, 47.2, 39.2, 27.6,
26.3, 25.2. HR-ESI-MS m/z: 283.2240 [M+H]+ (C13H27N6O requires
283.2266). IR cmꢀ1: 2959, 2752, 1707, 1682.
2954, 2756, 1699, 1595, 1416.
4.5. G-Ligand (2)
A suspension of 2-amino-6-chloropurine (354 mg, 2.09 mmol),
7 (1.00 g, 1.39 mmol) and K2CO3 (770 mg, 5.57 mmol) in anhy-
drous DMF (1.5 ml) was heated under an argon atmosphere at
60 °C for 4.5 days, and then quenched by the addition of water.
The reaction mixture was extracted with CHCl3, the organic ex-
tracts were washed with brine, dried over anhydrous Na2SO4, fil-
tered and evaporated to afford the crude product (1.40 g), which
was purified by column chromatography (silica gel, CHCl3 to
CHCl3/MeOH = 40:1) to yield the coupling product as a pale yellow
foam (536 mg, 0.66 mmol, 47 %). This coupling product (532 mg,
0.66 mmol) was dissolved in 0.5 M HCl/MeOH (19.7 ml) and stirred
at 60 °C for 4 days. The solvent was removed by Liebig condenser
to yield Boc-deprotected product 9 (511 mg, quant.). 1H NMR
(400 MHz, CD3OD) d ppm: 9.00 (1H, s), 8.02–7.96 (2H, m), 7.85–
7.72 (6H, m), 4.29 (2H, t, J = 7 Hz), 3.45 (2H, t, J = 8 Hz), 3.41 (2H,
t, J = 7 Hz), 3.37–3.31 (4H, m), 2.95 (2H, t, J = 8 Hz), 2.24 (2H, quint.,
J = 7 Hz), 1.96 (2H, quint., J = 7 Hz),1.89 (2H, quint., J = 7 Hz). ESI
(MS) m/z: 692.9 [M+H]+. IR cmꢀ1: 1706, 1633, 1604, 1541, 1371,
1343, 1160.
4.7. T-Ligand (4)
A suspension of N3-benzoylthymine (495 mg, 2.15 mmol), 7
(1.04 g, 1.43 mmol) and K2CO3 (792 mg, 5.73 mmol) in anhydrous
DMF (1.5 ml) was heated under an argon atmosphere at 60 °C for
6.5 days, and then quenched by the addition of water. The reaction
mixture was extracted with CHCl3, the organic extracts were
washed with brine, dried over anhydrous Na2SO4, filtered and
evaporated to afford crude products (1.55 g), which was purified
by column chromatography (silica gel, CHCl3/MeOH = 60:1 to
20:1) to yield the coupling product 11 as a pale yellow foam
(748 mg, 0.97 mmol, 68 %). 1H NMR (400 MHz, CDCl3) d ppm:
7.97–7.95 (2H, m), 7.73–7.66 (4H, m), 7.62–7.59 (2H, m), 7.04
(1H, br s), 3.72 (2H, t, J = 7 Hz), 3.36 (2H, t, J = 7.0 Hz), 3.32–3.24
(6H, m), 3.11 (2H, q, J = 6 Hz), 1.97 (4H, quint., J = 7 Hz), 1.90 (3H,
s), 1.71 (2H, quint., J = 7 Hz), 1.44 (9H, s). ESI (MS) m/z: 768.1
[M+H]+. IR cmꢀ1: 1684, 1543, 1368, 1347, 1162.
Toa solutionoftheaboveproduct9 (504 mg, 0.66 mmol)inanhy-
drous DMF (1.5 ml) were added thiophenol (148 ll, 1.44 mmol) and
K2CO3 (362 mg, 2.62 mmol) under an argon atmosphere at room
temperature. The reaction mixture was stirred for 14 h at room tem-
perature and then quenched by the addition of water. The solvent
was evaporated to afford the crude product, which was purified by
column chromatography (silica gel, CHCl3/MeOH/aqueous NH3 =
10:5:4) to yield the Ns-deprotected product (517 mg, quant.). This
productwasdissolved in0.5 MHCl/MeOH(19.7 ml)and thereaction
mixture was stirred for 1 day at room temperature, and then the sol-
vent was removed by Liebig condenser to produce 2 as white solids
(347 mg). The obtained 2 was purified by precipitation from water/
EtOH (2 ml/40 ml) at ꢀ78 °C. The purified 2 was collected by centri-
fuged(1500 rpm, 10 min), anddriedundervacuumtogive 2 aswhite
solids (289 mg, 94% after re-precipitation three times). Mp 210–
213 °C. 1H NMR (400 MHz, D2O) d ppm: 8.08 (1H, s), 4.19 (2H, t,
J = 7 Hz), 3.17–3.05 (10H, m), 2.23 (2H, quint., J = 7 Hz), 2.14–2.03
(4H, m). 13C NMR (125 MHz, D2O) d ppm: 160.2, 157.0, 153.8,
141.9, 115.9, 47.4, 47.4, 47.3, 47.2, 43.9, 39.3, 28.5, 26.4, 25.3. HR-
ESI-MS m/z: 323.2302 [M+H]+ (C14H27N8O requires 323.2319). IR
cmꢀ1: 2959, 2756, 1716, 1644, 1607, 1137, 1108.
To a solution of the above coupling product 11 (738 mg,
0.96 mmol) in anhydrous DMF (1.5 ml) were added thiophenol
(217 ll, 2.11 mmol) and K2CO3 (664 mg, 4.80 mmol) under an ar-
gon atmosphere at room temperature. The reaction mixture was
stirred for 11.5 h at room temperature and then quenched by the
addition of methanol. This mixture was filtered and the filtrate
was evaporated to afford a crude product (1.71 g), which was puri-
fied by column chromatography (silica gel, CHCl3/MeOH = 2:1 to
CHCl3:MeOH:aqueous NH3 = 20:8:1) to yield the purified product
(376 mg). This product was dissolved in 0.5 M HCl/MeOH (28 ml)
and the reaction mixture was stirred for 1 day at room tempera-
ture, and then the solvent was removed by Liebig condenser to
produce 4 as white solids (361 mg). The obtained 4 was purified
by reprecipitation from water/EtOH (3 ml/40 ml) at ꢀ78 °C. The
4.6. C-Ligand (3)
A suspension of N4-acetylcytosine (636 mg, 4.15 mmol), 7
(1.30 g, 1.80 mmol) and K2CO3 (588 mg, 4.25 mmol) in anhydrous
DMF (2.5 ml) was heated under an argon atmosphere at 60 °C for

3478
Y. Abe et al. / Bioorg. Med. Chem. 20 (2012) 3470–3479
purified 4 was collected by centrifuged (1500 rpm, 10 min), and
dried under vacuum to give 4 as white solids (276 mg, 70% after
precipitation twice). Mp 243–244 °C. 1H NMR (400 MHz, D2O) d
ppm: 7.42 (1H, s), 3.81 (2H, t, J = 7 Hz), 3.14–3.03 (10H, m), 2.10–
2.00 (6H, m), 1.82 (3H, s). 13C NMR (125 MHz, D2O) d ppm:
169.5, 155.1, 145.2, 113.9, 48.0, 47.4, 47.3, 47.2, 47.1, 39.2, 27.8,
26.3, 25.2, 13.9. HR-ESI-MS m/z: 298.2237 [M+H]+ (C14H28N5O2 re-
quires 298.2266). IR cmꢀ1: 2952, 2756, 1699, 1664.
at 80 °C and then gradually cooled down. Obtained melting curves
were analyzed by non-linear fitting and each melting temperature
was determined.
D
Tm = Tm(+)ꢀTm(ꢀ).
4.11. Evaluation of binding affinities of ligands and hairpin loop
duplex ODNs by SPR
All samples were measured on a BIACORE3000. The immobiliza-
tion of hairpin loop ODNs (Biothin-50-d TTTT CGC ATG X GTA CGC
TTTT GCG TAC AP CAT GCG-3’, X = A, G, C, T) were performed in
HBS–N buffer (10 mM HEPES buffer (pH 7.4), 150 mM NaCl) with
4.8. U-Ligand (5)
A
suspension of N3-benzoyluracil (448 mg, 2.07 mmol),
7
flow rate of 10 ll/min at 25 °C. The sensorgrams for the interaction
(1.02 g, 1.41 mmol) and K2CO3 (763 mg, 5.52 mmol) in anhydrous
DMF (5 ml) was heated under an argon atmosphere at 80 °C for
4 days, and then quenched by the addition of water. The reaction
mixture was extracted with CHCl3, the organic extracts were
washed with brine, dried over anhydrous Na2SO4, filtered and
evaporated to afford crude products (1.75 g), which was purified
by column chromatography (silica gel, CHCl3/MeOH = 100:1 to
40:1) to yield the coupling product 12 as a pale yellow foam
(769 mg, 1.02 mmol, 72 %). 1H NMR (400 MHz, CD3CN) d ppm:
7.91–7.88 (2H, m), 7.79–7.70 (6H, m), 7.29 (1H, d, J = 8 Hz), 5.53
(1H, d, J = 8 Hz), 3.64 (2H, t, J = 7 Hz), 3.31–3.22 (8H, m), 2.80
(2H, q, J = 6 Hz), 1.85 (4H, m), 1.67–1.60 (2H, m), 1.39 (9H, s). ESI
(MS) m/z: 754.1 [M+H]+. IR cmꢀ1: 1682, 1543, 1369, 1346, 1162.
To a solution of the above coupling product 12 (240 mg,
of each ligand with 50-biotin-labeled ODNs were collected in
10 mM HEPES–NaOH buffer (pH 7.0), 100 mM NaCl for A-, G-li-
gands, 50 mM NaCl for C-, T-, or U-ligands at 25 °C. Each ligand
concentrations were from 0.05 to 2.00
lM and ligand solutions
were injected 100 l at a flow rate of 20
l
l
l/min over a period of
300 sec and were followed by 120 s dissociation time. Obtained
data were fitted with a one-site model to calculate an equilibrium
binding constant.
4.12. Thermodynamics of ligands against complementary
duplex ODNs containing the AP site by ITC
All samples were measured on a MicroCal VP-ITC microcalorim-
eter. 10
150 M ligand solutions 19 times, 10
first injection. The first injection volume was 2
l
M duplex ODN solutions (1.43 ml) were titrated with
l per injection except for
l and this data
0.32 mmol) in anhydrous DMF (700
l
l) were added thiophenol
l
l
(70 l, 0.70 mmol) and K2CO3 (177 mg, 1.28 mmol) under an argon
l
l
atmosphere at room temperature. The reaction mixture was stirred
for 4 h at room temperature and then quenched by the addition of
methanol. This mixture was filtered and the filtrate was evaporated
to afford a crude product (523 mg), which was purified by column
chromatography (silica gel, CHCl3/MeOH/aqueous NH3 = 40:8:1)
to yield the purified products (128 mg). This product (140 mg)
was dissolved in 0.5 M HCl/MeOH (1 ml) and the reaction mixture
was stirred for 3 days at room temperature, and then the solvent
was removed by Liebig condenser to produce 5 as white solids
(72.7 mg, 58%). Mp 260–261° C. 1H NMR (400 MHz, D2O) d ppm:
7.59 (1H, d, J = 8 Hz), 5.79 (1H, d, J = 8 Hz), 3.85 (2H, t, J = 7 Hz),
3.16–3.04 (10H, m), 2.12–2.01 (6H, m). 13C NMR (125 MHz, D2O) d
ppm: 169.3, 155.1, 149.5, 104.6, 48.3, 47.4, 47.3, 47.2, 47.1, 39.1,
27.7, 26.3, 25.2. HR-ESI-MS m/z: 284.2077 [M+H]+ (284.2081 calcd
for C13H26N5O2). IR cmꢀ1: 3405, 1544, 1368, 1164.
was excluded from analysis. Obtained data were fitted with a
one-site model to calculate the thermodynamic parameters.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Re-
search (S) from the Japan Society for Promotion Science (JSPS)
and CREST from the Japan Science and Technology Agency. Y.A. is
grateful for the financial support by the Sasagawa Scientific Re-
search Grant from the Japan Science Society.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.009.
4.9. Synthesis of ODN containing the AP site analogue and
complementary ODN
References and notes
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All oligonucleotides were synthesized using solid phase phosp-
horoamidite chemistry on a Applied Biosystems 394 DNA/RNA
synthesizer or a NIHON TECHNO SERVICE H-Series synthesizer.
The cleaved oligonucleotides were purified by HPLC on a Waters
X-Bridge C18 column using a linear gradient between the 0.1 M
TEAA buffer and CH3CN. The MALDI-TOF/MS was measured on a
BRUKER DALTONICS microflex-KS spectrometer and these data
were summarized in Supplementary data. Oligonucleotide concen-
trations were determined spectrophotometrically by measuring
the absorbance at 260 nm.
4.10. Evaluation of the stability of duplex ODNs containing the
AP site by thermal denaturation studies
10. Dahlmann, H. A.; Vaidyanathan, V. G.; Sturla, S. J. Biochemistry 2009, 48,
9347.
11. Liu, L.; Yan, L.; Donze, J. R.; Gerson, S. L. Mol. Cancer Ther. 2007, 1061.
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14. Reco, N. J.; Tor, Y. J. Am. Chem. Soc. 2005, 127, 10784.
All duplexes melting curves were measured on a UV BECKMAN
COULTER DU800 in the absence and presence of 20
lM ligand, at a
concentration of 4 M duplex in 10 mM HEPES–NaOH buffer (pH
l
7.0) at a heat rate of 1 °C/min (from 20 to 80 °C) and a scan rate
of 1 °C/min. As a pretreatment, all samples are heated for 5 min
15. Oshimoto, K.; Nishizawa, S.; Minagawa, M.; Teramae, N. J. Am. Chem. Soc. 2003,
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