Enantioselective binding of chiral 1,14-dimethyl[5]helicene-spermine ligands with B- and Z-DNA

Article abstract of DOI:10.1016/j.bmc.2013.07.022

Duplex DNA adopts a right-handed B-DNA conformation under physiological conditions. Z-DNA, meanwhile, has a left-handed helical structure and is in equilibrium with right-handed B-DNA. We recently reported that the bisnaphthyl maleimide-spermine conjugate (1) induced a B- to Z-DNA transition with high efficiency at low salt concentrations. It was also found that the bisnaphthyl ligand (1) spontaneously transformed into the corresponding [5]helicene derivative (2). Because [5]helicene 2 can potentially be chiral and because the chiral discrimination of B- and Z-DNA is also of interest, we became interested in whether enatiomerically pure [5]helicene-spermine conjugates might discriminate the chirality of B- or Z-DNA. In this study, we have demonstrated an efficient synthesis of chiral DNA-binding ligands by the conjugation of a [5]helicene unit with a spermine unit. These chiral helicene ligands exhibited recognition of B- and Z-DNA, with (P)-3 displaying preference for B-DNA and (M)-3 for Z-DNA. The characteristic features of the helicene-spermine ligands developed in this study include two points: the cationic spermine portion produces electrostatic interactions along the phosphate backbone of the minor groove, and the helicene forms complexes in an end-stacking mode. Such binding modes, together with the thermodynamic parameters, account for the mode of chiral recognition of (P)- and (M)-3 for B- and Z-DNA.

Full text of DOI:10.1016/j.bmc.2013.07.022

Bioorganic & Medicinal Chemistry 21 (2013) 6063–6068
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enantioselective binding of chiral 1,14-dimethyl[5]
helicene–spermine ligands with B- and Z-DNA q
⇑
Genichiro Tsuji, Kyoko Kawakami, 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:
Duplex DNA adopts a right-handed B-DNA conformation under physiological conditions. Z-DNA, mean-
while, has a left-handed helical structure and is in equilibrium with right-handed B-DNA. We recently
reported that the bisnaphthyl maleimide–spermine conjugate (1) induced a B- to Z-DNA transition with
high efficiency at low salt concentrations. It was also found that the bisnaphthyl ligand (1) spontaneously
transformed into the corresponding [5]helicene derivative (2). Because [5]helicene 2 can potentially be
chiral and because the chiral discrimination of B- and Z-DNA is also of interest, we became interested
in whether enatiomerically pure [5]helicene–spermine conjugates might discriminate the chirality of
B- or Z-DNA. In this study, we have demonstrated an efficient synthesis of chiral DNA-binding ligands
by the conjugation of a [5]helicene unit with a spermine unit. These chiral helicene ligands exhibited rec-
ognition of B- and Z-DNA, with (P)-3 displaying preference for B-DNA and (M)-3 for Z-DNA. The charac-
teristic features of the helicene–spermine ligands developed in this study include two points: the cationic
spermine portion produces electrostatic interactions along the phosphate backbone of the minor groove,
and the helicene forms complexes in an end-stacking mode. Such binding modes, together with the ther-
modynamic parameters, account for the mode of chiral recognition of (P)- and (M)-3 for B- and Z-DNA.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Received 11 June 2013
Revised 10 July 2013
Accepted 10 July 2013
Available online 18 July 2013
Keywords:
Chiral recognition
Helicene
DNA binding ligand
Surface plasmon resonance
Isothermal titration calorimetry
1. Introduction
pure [5]helicene–spermine conjugates might discriminate the chi-
rality of B- or Z-DNA. Here, we show that the chiral 1,14-
Duplex DNA adopts a right-handed B-DNA conformation under
physiological conditions. Z-DNA, meanwhile, has a left-handed
helical structure and is in equilibrium with right-handed B-DNA.
Since its discovery, Z-DNA has attracted attention in regards to
its biological functions,1 its mechanism of transition to B-DNA,2
and its applications in chemical devices3 or DNA nanotechnology.4
Molecules that specifically bind Z-DNA are also of great interest,
and many examples have been investigated, including binding pro-
teins such as ADAR1,5 anti-Z-DNA antibodies,6 the virus E3L pro-
tein,7 chiral metal complexes,8,9 and organic ligands.10–12 We
recently reported that the bisnaphthyl maleimide–spermine con-
jugate (1) induced a B- to Z-DNA transition with high efficiency
at low salt concentrations.13 It was also found that the bisnaphthyl
ligand (1) spontaneously transformed into the corresponding
[5]helicene derivative (2). Because [5]helicene can potentially be
chiral and because the chiral discrimination of B- and Z-DNA is also
of interest,14 we became interested in whether enatiomerically
dimethyl[5]helicene–spermine ligand (3) exhibits recognition of
B- and Z-DNA, with (P)-3 displaying preference for B-DNA and
(M)-3 for Z-DNA (Fig. 1). We speculate that recognition takes place
in the end-stacking binding mode, which is assisted by electro-
static interactions of the cationic spermine part along the phos-
phate backbone of the minor groove.
2. Results and discussion
2.1. Design and synthesis of chiral 1,14-dimethyl[5]helicene–
spermine ligands
The bisnaphthyl maleimide–spermine ligand (1) was converted
to [5]helicene ligand (2) upon standing under visible light at ambi-
ent temperature for one week. The (P)- and (M)-enantiomers of 2
were separated by chiral column chromatography; however, they
racemized quickly.15 Therefore, 1,14-dimethyl[5]helicene16 ligand
3 was designed to obtain stable enantiomers. As it turned out that
the racemic 3 was not separated by chiral column chromatogra-
phy, enantiomers were separated at an early stage of the synthesis.
The synthesis of 3 is summarized in Scheme 1. The Suzuki–Miya-
ura coupling of 8-methylnaphthalene-2-boronic acid (5)17 with
dibromo-maleimide derivative (4) produced the bisaryl substi-
tuted compound 6, which was transformed into racemic helicene
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
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 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.022

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G. Tsuji et al. / Bioorg. Med. Chem. 21 (2013) 6063–6068
Bn
N
O
O
Bn
N
B(OH)2
O
O
a
+
Br
Br
4
5
6
Bn
N
O
O
O
O
O
d
b
c
(P)-3, (M)-3
Figure 2. Changes in the CD spectra indicate a B- to Z-DNA transition. (A) In the
presence of 1 from 0 to 160 M; (B) in the presence of ( )-3 from 0 to 160 M. An
initial CD spectrum was measured using solution containing 20 B-DNA
Boc
N
l
l
NHBoc
a
lM
H2N
N
[d(CGCGCG)2] in 5 mM Na cacodylate buffer containing 100 mM NaCl at pH 7.0 and
20 °C. The arrows indicate the direction of change in the spectra effected by the
Boc
(P)-7, (M)-7
(P)-8, (M)-8
9
addition of the ligand. The last spectra was obtained in the presence of 160 lM of 1
or ( )-3.
Scheme 1. Reagents and conditions: (a) PhCH2NH3Cl, Pd(PPh3)2Cl2, CsF, H2O–
toluene, 95%; (b) Hg lamp, 500 W, I2, THF–toluene, 92%; (c) 5 N aq KOH–EtOH, then
10% HCl, 92%; (d) (1) 9, DMF–toluene; (2) 33% TFA–CH2Cl2, 15%.
Table 1
Effects of the ligands on the Tm and binding constants (Ka) with B- or Z-DNA
determined by SPR
Sper
Sper
N
+H3N
+H2N
N
Tm °C
Ka, 106 Mꢀ1b
ka
,
kd 10ꢀ3 sꢀ1
O
O
a
b
b
Ligand
DNA
D
,
O
O
103 Mꢀ1 sꢀ1
1c
Z-DNA
B-DNA
Z-DNA
B-DNA
Z-DNA
B-DNA
+18
+16
3.0
0.7
0.028
0.21
0.22
0.11
20.2
5.2
0.163
1.72
1.17
0.406
6.66
7.49
6.68
8.59
5.48
3.70
1c
(P)-3
(P)-3
(M)-3
(M)-3
+3.3
+10.1
+7.5
+4.5
1
2
Sper=
+
NH2
Sper
N
Sper
N
a
UV-melting temperature was measured using 5 lM B- or Z-DNA in 5 mM Na
cacodylate buffer containing 100 mM NaCl in the absence and the presence of 5 lM
O
O
O
O
ligand at pH 7.0.
b
Sensor chips contain 50-d(TTTTCGCGCG-TTTT-CGCGCG) as loop B-DNA and 50-
d(TTTTCGCm8GCG-TTTT-CGCm8GCG) as loop Z-DNA. Association constants (Ka)
were obtained at 25 °C using a solution of ligand in 5 mM Na cacodylate buffer
containing 100 mM NaCl at pH 7.0.
N
O
O
Ar
Ar
c
Data obtained from the literature and kinetic parameters were recalculated
1
using the reported sensorgrams.13
(P)-3
(M)-3
Figure 1. Structures of several spermine-conjugated ligand.
racemic 3 caused B- to Z-DNA transitions at low salt concentra-
tions (Fig. 2). As the CD bands in the spectra of (P)- and (M)-3 over-
lapped with those of B- and Z-DNA (Fig. 6), these transitions were
not similarly evaluated. Accordingly, the chiral recognition of B- or
Z-DNA by (P)- or (M)-3 was investigated by measuring the thermal
denaturing temperature (Tm) and binding affinity (Ka).
derivative 7 in 92 % yield as a sole product via a photocyclization
reaction in the presence of iodine.18 The [5]helicene structure is
represented by the downfield shift of the protons at 6 and 9 posi-
tion at 9.02 ppm (doublet, J = 8.6 Hz). (P)- and (M)-7 were effi-
ciently separated using a chiral column (OJ-RH) (Fig. 6A), and
each enantiomer was transformed into thecorresponding maleic
anhydride (8). Tri-Boc-protected spermine 9 reacted with 8 to form
the corresponding imide derivative, which was treated with TFA to
induce the deprotection of the Boc groups, thus producing the heli-
cene–spermine conjugate (3). The optical active 8 was returned to
7 by the similar reaction conditions without racemization, indicat-
ing that the final helicene–spermine conjugate (3) was obtained in
optically pure form. Racemic 7 was also converted to the corre-
sponding spermine conjugate ( )-3 (Scheme 1).
As 20-deoxy-8-methylguanosine (m8G) is known to stabilize
Z-DNA, a duplex containing m8G, that is, d(CGCm8GCG)2, was used
as a Z-DNA standard.19 Tm values were obtained by measuring the
UV-melting curves of B- and Z-DNA, and the values were obtained
in the presence and absence of ligand. The ligands increased the Tm
values, as summarized in Table 1. Ligand 1 increased the Tm values
of both B- and Z-DNA to a similar extent.13 Interestingly, (P)-3 in-
creased theTm value of B-DNA more than that of Z-DNA, whereas
its enantiomer (M)-3 had a greater effect on the Tm value of
Z-DNA. The selectivities of (P)-3 to B-DNA and (M)-3 to Z-DNA
were further evaluated by surface plasmon resonance (SPR)
(Fig. 3). 50-Biotin-conjugated 50-d(TTTTCGCGCG-TTTT-CGCGCG)
(loop B-DNA) and 50-d(TTTTCGCm8GCG-TTTT-CGCm8GCG) (loop
Z-DNA) were immobilized onto a sensor chip as B- and Z-DNA,
respectively.13 The thymidine tetramer (T4) at the 50-end served
as a spacer between the duplex and the sensor surface, and the
T4 in the middle allowed for the stem loop of the intramolecular
duplex formation.The B- and Z-conformations of each duplex were
confirmed by their CD spectra in solution, and loop B-DNA did not
2.2. Stabilization effects of ligands on B- and Z-DNA
The CD spectrum of B-DNA with sequence d(CGCGCG)2 showed
a negative band at approximately 255 nm and a positive band at
approximately 275 nm. This spectrum changed at high salt concen-
trations (5 M NaCl) to a CD spectrum with a positive band at
approximately 270 nm and a negative band at approximately
295 nm, indicating a B- to Z-DNA transition. It was shown that

G. Tsuji et al. / Bioorg. Med. Chem. 21 (2013) 6063–6068
6065
Figure 3. SPR sensorgrams obtained using a chip immobilized with loop DNA. (A) (M)-3 to loop B-DNA; (B) (M)-3 to loop Z-DNA. The SPR spectrum was obtained using a
ligand solution in 5 mM Na cacodylate buffer containing 100 mM NaCl at 25 °C, pH 7.0 and a flow rate of 20 lL/min. Kinetic parameters were obtained using manufacturer
supplied software, and summarized in Table 2.
Figure 4. Unsubtracted calorimetric binding heats and binding isotherm obtained from the titration of (P)-3. (A) Binding to B-DNA. (B) Binding to Z-DNA. Data were obtained
using a solution of 10
lM B-DNA or Z-DNA in 5 mM Na cacodylate, 100 mM NaCl at pH 7.0 and 25.0 °C.
Table 2
change to the corresponding Z-conformation at the ligand concen-
trations used in these measurements. Examples of SPR sensor-
grams are shown in Figure 3. The binding parameters are
summarized in Table 1. (P)-3 Showed more than 7 times higher
affinity for B-DNA than Z-DNA. In contrast, (M)-3 displayed 2 times
higher affinity for Z-DNA. These data are in good agreement with
the preferences observed by UV-melting experiments. The kinetic
parameters suggest that a larger ka value contributes to a selectiv-
ity of (P)-3 to B-DNA and (M)-3 to Z-DNA. As it seemed to us that-
detailed interpretation of the binding mechanism was difficult
based on the SPR data, we next investigated binding by isothermal
calorimetry (ITC) measurements.
A solution of the ligand (P)- or (M)-3 was added to a solution of
d(CGCGCG)2 as B-DNA or d(CGCm8GCG)2 as Z-DNA, and binding
isotherms were obtained. Figure 4 illustrates examples obtained
from the titration of (P)-3. The obtained Ka values and thermody-
namic parameters are summarized in Table 2. An analysis of the
binding heats obtained from the titration of (P)-3 to B-DNA indi-
cated a DNA:ligand = 2:1 ratio of complexation with an association
constant of Ka = 3.7ꢁ 108 Mꢀ1, indicating high-affinity binding
Thermodynamic parameters obtained by ITC using (P)- and (M)-3 against B- and
Z-DNAa
Complex to B- or
Z-DNA
Ka,
Nb
D
H
D
G
-TDS
(kJ/mol)
107 Mꢀ1
(kJ/mol)
(kJ/mol)
1 versus Zc
0.31
0.62
0.67
36.7
0.17
0.083
4.1
3.5
1.7
2.0
1.8
4.0
ꢀ32.6
ꢀ21.8
ꢀ46.6
ꢀ36.5
ꢀ47.7
ꢀ23.6
ꢀ37.0
ꢀ33.1
ꢀ38.9
ꢀ48.9
ꢀ35.6
ꢀ33.8
ꢀ4.5
ꢀ11.3
7.7
1 versus Bc
(P)-3 versus Z
(P)-3 versus B
(M)-3 versus Z
(M)-3 versus B
ꢀ12.4
12.1
ꢀ10.2
a
b
c
Thermodynamic parameters were obtained as shown in Figure 4.
The binding stoichiometry is defined as molar ratio [ligand]/[duplex].
Data are taken from the literature.13 The
G and ꢀT S values were miscalcu-
D
D
lated in the literature and are corrected in this table.
between (P)-3 and B-DNA (Fig. 4A). This affinity is 50 times greater
than the complexation between (P)-3 and Z-DNA (Fig. 4B), again
confirming the preference of (P)-3 for B-DNA. A preference of
(M)-3 for Z-DNA was also observed by ITC measurements with
two times higher affinity than binding to B-DNA (Table 2).

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G. Tsuji et al. / Bioorg. Med. Chem. 21 (2013) 6063–6068
(B)
(A)
T4 Loop
O
N
O
O
N
O
O
N
O
T4 spacer
SPR chip
(D)
(C)
O
spermine
N
spermine
O
N
O
O
3
(M)-
3
(P)-
O
O
O
O
P
Figure 6. (A) The HPLC Chart of (M)-7 and (P)-7 separated by OJ-RH. Column
DAICEL OJ-RH column (4.6 mm
min, 35 °C. (A) Faster peak at 8.3 min and a peak at 9.8 min correspond to (M)-7 and
(P)-7, respectively; (B) CD Spectra of (P)-3 (green line) and (M)-3 (blue line). Spectra
were obtained using a 10 lM ligand solution in 5 mM Na Cacodylate buffer
containing 100 mM NaCl at pH 7.0 and 20 °C.
O
O
P
O
P
O
O
u
ꢁ 150 mm), solvent CH3CN, flow rate of 0.500 mL/
O
O
O
H
N
O
H
O
H
O
O
N
H
H
N
N
O
H
O
N
N
N
H
H
O
H
N
O
O
N
O
N
H
N
N
N
O
O
N
H
H
O
N
P
O
H
N
O
O
H
anti dG
Z-DNA
syn dG
B-DNA
This notion is partly supported by the fact that more than 100-fold
smaller Ka values were obtained by SPR because end-stacking
interactions should be hampered by steric hindrance of a T4 loop
on one side. Also, the T4 spacer most likely causes an additional
steric hindrance (Fig. 5B). In such an end-stacking complex, the po-
sitive helix face of (P)-3 seems to interact with dG preferably in an
anti-conformation, as schematically shown in Figure 5C. X-ray
analysis of a crystalline complex between d(CGCGCG)2 with a poly-
amine has shown that polyamines bind in the minor groove of the
Z-form of d(CGCGCG)2.21 The minor groove of Z-DNA is narrower
than that of B-DNA. The binding of the cationic spermine portion
of (P)-3 in the minor groove would produce higher electrostatic
interactions for a favorable enthalpy effect. Meanwhile, the limited
degrees of freedom of the spermine portion can cause a loss of en-
tropy. Thus, the low affinity of (P)-3 to Z-DNA is rationalized in
terms of favorable enthalpy and unfavorable entropy. The binding
of (M)-3 to Z-DNA may be considered based on the formation of a
similar complex as follows: a negative helical face of (M)-3 is pref-
erable for end-stacking with dG in the syn conformation of Z-DNA,
as illustrated in Figure 5D. On the contrary, enthalpic contributions
during the binding of (M)-3 to B-DNA decreased largely
Figure 5. Schematic illustrations of speculative ligand binding. (A) End-stacking
mode of the binding with B-DNA; (B) T4 loop and T4 spacer inhibit end-stacking
binding; (C) Space-filling model of (P)-3 and its schematic binding mode to a dG in
anti conformation; (D) Space filling model of (M)-3 and its speculative binding to a
dG in syn conformation at the end of the Z-DNA. The spermine moiety has been
omitted from the space-filling model for clarity.
Examination of the thermodynamic parameters indicates that the
binding of (P)-3 and B-DNA is an enthalpically and entropically
favorable process. In contrast, the binding of (P)-3 with Z-DNA is
enthalpically favorable, but it is associated with an unfavorable
change in entropy (ꢀT
DS = 7.7 kJ/mol) and results in a low binding
affinity. For (M)-3, although binding with B-DNA is also enthalp-
ically and entropically favorable, the enthalpy contribution is rela-
tively small (
is associated with an unfavorable entropy term (ꢀT
mol), but a large enthalpic contribution (
H = ꢀ47.7 kJ/mol) com-
D
H = ꢀ23.6 kJ/mol). The binding of (M)-3 with Z-DNA
D
S = 12.1 kJ/
D
pensates to produce the observed selectivity for Z-DNA. The Ka val-
ues obtained by ITC were larger than those obtained by SPR. Based
on the NMR measurements obtained in the previous study,13 the
binding of 1 with duplex DNA contains a stacking mode at the ends
of the duplex. The importance of an end stacking interaction for
Z-DNA has also been suggested in other study.20 The stoichiometry
of the binding of (P)-3 with B- or Z-DNA was estimated to be
ligand:DNA = 2:1, suggesting that (P)-3 binds the duplex in an
end-stacking mode, as illustrated in Figure 5A.
(
D
H = ꢀ23.6 kJ/mol), most likely due to fewer end-stacking inter-
actions between a negative helical face of (M)-3 and the dC–dG
base pair at the end of B-DNA, thus resulting in the selectivity of
(M)-3 for Z-DNA.
3. Conclusion
We speculate that an end-stacking binding mode such as shown
in Figure 5A is assisted by electrostatic interactions of the cationic
spermine part along the phosphate backbone of the minor groove.
In this study, we have demonstrated an efficient synthesis of
chiral DNA-binding ligands by the conjugation of a [5]helicene unit

G. Tsuji et al. / Bioorg. Med. Chem. 21 (2013) 6063–6068
6067
with a spermine unit. These chiral helicene ligands exhibited rec-
ognition of B- and Z-DNA, with (P)-3 displaying preference for
B-DNA and (M)-3 for Z-DNA. Although several studies have
claimed chiral recognition of B- and Z-DNA, the modes of this
chiral recognition remain poorly understood. The characteristic
features of the helicene–spermine ligands developed in this study
include two points: the cationic spermine portion produces elec-
trostatic interactions along the phosphate backbone of the minor
groove, and the helicene forms complexes in an end-stacking
mode. Such binding modes, together with the thermodynamic
parameters, account for the mode of chiral recognition of
(P)- and (M)-3 for B- and Z-DNA. The results of this study may
provide useful knowledge for designing small molecular ligands
for the recognition of B- or Z-DNA.
ESI–HRMS (m/z) calcd for C33H26NO2 468.1958 [M+H]+, found
468.1977.
4.3. (P)-7 and (M)-7
A
solution of 6 (10 mg, 0.021 mmol) and iodine (6 mg,
0.024 mmol) in tolunene-THF was stirred under irradiation of
high-pressure mercury lamp (500 W) for 6 h. The reaction mixture
was quenched with saturated aqueous Na2S2O3 (20 mL), and ex-
tracted with AcOEt (20 mL ꢁ 2). The organic layers were washed
with water, brine, and dried over Na2SO4, then evaporated. The res-
idue was chromatographed (silica gel, n-hexane:toluene = 10:1) to
give 7 as yellow solids (9 mg, 92%).
4.4. Optical resolution of (P)-7 and (M)-7
4. Experimental section
A solution of racemic 7 was separated by HPLC equipped with a
1H NMR spectra were recorded 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 instrument. IR spectra were recorded on a
PerkinElmer Spectrum One FT-IR spectrometer.
chiral column (DAICEL OJ-RH 4.6 mm
u
ꢁ 150 mm) using CH3CN as
an eluent at a flow rate of 0.500 mL/min and at 35 °C (Fig. 6A).
IR(cmꢀ1) 1754, 1700, 1389, 1345, 826. ½a 2D5
ꢂ
(CHCl3) +356.7
(M)-7, ꢀ398.3 (P)-7. 1H NMR(400 MHz, CDCl3) d (ppm) 9.02 (2H,
d, J = 8.6 Hz), 8.04 (2H, d, J = 8.6 Hz), 7.87 (2H, d, J = 7.9 Hz),
7.56–7.52 (4H, m), 7.32 (2H, t, J = 7.3 Hz), 7.27 (1H, d, J = 1.2 Hz),
7.10 (2H, d, J = 7.3 Hz), 4.98 (2H, s), 0.86 (6H, s). 13C NMR(125 MHz,
CDCl3) d (ppm) 169.4, 136.7, 135.4, 133.7, 131.7, 130.6, 130.5,
129.7, 128.7, 128.5, 127.7, 127.6, 126.0, 125.7, 124.9, 120.6, 41.5,
22.1. ESI–HRMS (m/z) calcd for C33H23NO2 C33H24NO2 466.1802
[M+H]+, found 466.1840.
4.1. 1-Methylnaphthalene-7-boronic acid (5)
A
solution of n-butyl lithium in hexane (1.54 M, 1.9 mL,
2.94 mmol) was added into a solution of 7-bromo-1-methylnaph-
thalene (0.59 g, 2.67 mmol) in dry THF (10 mL) at ꢀ78 °C, and
the mixture was stirred at the same temperature for 1 h. A solution
of 2,4,6-trimethoxyboroxin (0.46 mL, 5.34 mmol) in dry THF
(10 mL) was dropped into the above solution, and the mixture
was stirred for 30 min at the same temperature. The mixture was
warmed to 0 °C, stirred for 1 h at the same temperature, and at
room temperature for additional 2 h. The mixture was quenched
by the addition of 10% aqueous HCl (20 mL), and extracted with
ether (20 mL ꢁ 2). The organic layers were washed with brine,
dried over Na2SO4, evaporated. The residue was chromatographed
(silica gel, n-hexane only to n-hexane:AcOEt = 3:1) to give 5 as col-
orless solids (384 mg, 77%). 1H NMR (400 MHz,CDCl3) d (ppm) 8.28
(1H, s), 7.87 (1H, dd, J = 7.6, 1.8 Hz), 7.82 (1H, d, J = 7.6 Hz), 7.38–
7.31 (2H, m), 2.78 (1H, s), 2.64 (3H, s). 13C NMR(125 MHz, CD3OD) d
(ppm) 131.9, 131.2, 130.9, 130,5, 128.4, 127.6, 127.3, 127.2, 127.1,
19.4. ESI–HRMS (m/z) calcd for C11H10BO2 185.0781 [M+H]ꢀ, found
185.0762.
4.5. Synthesis of (P)-3
A solution of (P)-7 (0.135 g, 0.290 mmol) in ethanol-5 M aque-
ous KOH (10ꢀ5 mL) was stirred at room temperature for 2 h, then
the mixture was acidified with 10% aqueous HCl (20 mL). The pre-
cipitates were collected and washed with water, then dried under
vaccum to give (P)-8 as orange solids (9 mg, 83%). This material
was used for the next reaction without purification. A mixture of
9 (7.3 mg, 0.145 mmol) and (P)-8 (6 mg, 0.159 mmol) in toluene–
DMF (3–1 mL) was heated at 80 °C for 16 h. The solvents were
evaporated and the residue was chromatographed (silica gel, CH2-
Cl2 to CH2Cl2–MeOH = 200:1) to give yellow solids (3.2 mg, 26%).
This material was treated with 33% TFA in CH2Cl2 (10 mL) at 0 °C
for 3 h, and the solvents were evaporated. The crude material
was purified by HPLC (nacalai tesque COSMOSIL 5C18-AR-II,
4.6 ꢁ 250 mm, solvents 0.05% TFA in water:0.05% TFA in CH3-
CN = 60:40) to give (P)-3 as yellow solids (2 mg, 15%). (M)-3 and
( )-3 were obtained similarly from (M)-7 and ( )-7, respectively
(Fig. 6B). Concentration of a stock solution of the ligand was deter-
mined by NMR integration value using an internal standard.
4.2. Compound 6
IR(cmꢀ1), 1676, 1202, 1130, 828, 723, ½a 2D5
ꢂ
(MeOH) +240.3(M),
1-Benzyl-3,4-dibromopyrrole-2,5-dione(4) (0.15 g, 0.435 mmol),
5 (0.24 g, 1.3 mmol), dichlorobis-(triphenylphosphine)palladium(II)
(16 mg, 0.022 mmol) and benzyltriethylammonium chloride (5 mg,
0.022 mmol) were dissolved in degassed toluene (4 mL), followed
by the addition of CsF (0.265 g, 1.74 mmol) in water (3 mL). The mix-
ture was stirred at 80 °C for 2 h. The mixture was quenched by the
addition of saturated aqueous NH4Cl (20 mL) and extracted with
AcOEt (20 mLꢁ 2). The combined organic layers were dried over Na2-
SO4, evaporated. The residue was chromatographed (silica gel, n-hex-
ane:AcOEt = 10:1) to give 6 as pale yellow solids (198 mg, 97%). 1H
NMR(400 MHz, CDCl3) d (ppm) 8.38 (2H, s), 7.67(2H, d, J = 8.6 Hz),
7.63(2H, d, J = 7.9 Hz), 7.52(2H, d, J = 7.3 Hz), 7.39(4H, t, J = 7.9 Hz),
7.36(2H, d, J = 7.3 Hz), 7.35(3H, d, J = 7.3 Hz), 7.21–7.13 (2H, m),
4.88(2H, s), 2.57(6H, s). 13C NMR(125 MHz, CDCl3), d (ppm) 1170.7,
136.6, 136.0, 135.5, 132.3, 128.8, 128.8, 128.8, 128.7, 128.7, 128.6,
127.9, 127.3, 127.2, 42.1, 19.2. IR (cmꢀ1) 1627, 1601, 1208, 823.
ꢀ240.6(P), 1H NMR(400 MHz, CDCl3) d (ppm) 8.99 (2H, d, J
= 8.8 Hz), 8.15 (2H, d, J = 8.8 Hz), 7.97 (2H, d, J = 1.6 Hz), 7.62
(2H, t, J = 7.6 Hz), 7.16 (2H, d, J = 6.7 Hz), 3.96 (2H, t, J = 6.1 Hz),
3.19 (2H, br), 3.13ꢀ3.01 (8H, br), 2.19 (2H, br), 2.08 (2H, br), 1.80
(4H, br), 0.87 (6H, s). ESI–HRMS(m/z) calcd for
C36H41N4O2
561.3224 [M+H]+ found 5631.3230.
4.6. DNA oligomers
DNA oligomers were synthesized by the automated DNA syn-
thesizer using the conventional amidite chemistry. The DMTr-pro-
tected DNA was cleaved from the resin with NH4OH solution and
purified by HPLC equipped with a ODS column using 0.1 M
TEAAꢀCH3CN linear gradient system. The DMTr protecting group
of the purified ODN was cleaved in 10% aqueous AcOH, and the
mixture was washed with ether. The purity of the ODN was

6068
G. Tsuji et al. / Bioorg. Med. Chem. 21 (2013) 6063–6068
confirmed by MALDI-TOF MS and UV measurements. The concen-
tration of the stock solution was estimated by the UV absorbance
at 260 nm, which was further confirmed to be less than 5% error
by HPLC analysis of the component nucleosides after enzymatic
hydrolysis using BAP and VPDE.
ate buffer containing 100 mM NaCl at pH 7.0 and 25.0 °C. The first
injection was 2 L and then 10 L each of the ligand stock was
l
l
added to the ODN solution. The data obtained by the first injection
was omitted from the analysis. The ODN concentrations are re-
ported as molar concentrations of double strands. All samples were
degassed prior to use by application of vacuum with simultaneous
stirring on a magnetic stir plate. A NanoITC SV (TA Instruments)
was used for the calorimetric measurements.
4.7. CD measurement
CD spectra were recorded by a Jasco-J720 spectrometer using a
200
A solution of d(CGCGCG)2 B-DNA (20
Z-DNA (20 M) in the buffer (5 mM Na cacodylate containing
100 mM NaCl at pH 7) was used for the CD measurement. The
ODN solution was titrated with the ligand stock solution (1
l
L 2 mm quartz glass cell at the constant temperature at 20 °C.
Acknowledgements
l
M) or d(CGCm8GCG)2
l
This work was supported by a Grant-in-Aid for Scientific Re-
search (S) from the Japan Society for Promotion Science (JSPS).
lL
each). All CD data were blank corrected, and reported as the aver-
age of quadruplicate scans.
References and notes
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4.8. Measurement UV melting temperature
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U.S.A. 1981, 78, 3546.
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8. Wu, Z.; Tian, T.; Yu, J.; Weng, X.; Liu, Y.; Zhou, X. Angew. Chem., Int. Ed. 2011, 50,
11962.
In the UV melting experiments, a solution of Z-DNA (5 lM in
Na cacodylate buffer 5 mM containing 100 mM NaCl, pH 7) was
used. The temperature was raised or lowered at the rate of 1 °C/
min between 10 and 60 °C.
4.9. Surface plasmon resonance (SPR) measurements
9. Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem. Rev. 1999, 2777.
10. Qu, X.; Trent, J. O.; Fokt, I.; Priebe, W.; Chaires, J. B. Proc. Natl. Acad. Sci. U.S.A.
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50-Biotin-conjugated
d(TTTTCGCGCG-TTTT-CGCGCG)
or
d(TTTTCGC m8GCG-TTTT-CGC m8GCG) 10–500 nM in 10 mM
HEPES and 0.15 M NaCl at pH 7.4 was immobilized on a sensor chip
SA at 20 °C. One flow channel was always left as a blank for refer-
ence. The SPR measurement was performed at 25 °C using the
5 mM Na cacodylate buffer containing 100 mM NaCl at pH 7.0 as
the running buffer. Binding was measured at 20 lL/min for 120 s
and dissociation for 150 s. A Biacore 3000 (GE Healthcare) was
used for the SPR measurements.
18. Harish, R. Talele; Monik, J. Gohil; Ashutosh, V. Bedekar Bull. Chem. Soc. Jpn.
2009, 82, 1182.
4.10. Isothermal titration calorimetry (ITC)
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T.; Grzeskowiak, K. Biochem. Biophy. Res. Commun. 2008, 368, 382.
The ligand solution (1 mM 3c) was prepared by dissolution in
the final dialysate obtained from the ODN dialysis, and was titrated
into 10.0 lM d(CGCGCG)2 or d(CGCm8GCG)2 in 5 mM Na cacodyl-