Strong positive cooperativity in binding to the A3T3 repeat by Hoechst 33258 derivatives attaching the quinoline units at the end of a branched linker

Article abstract of DOI:10.1016/j.bmc.2015.05.056

Hoechst 33258 derivatives with additional interacting moieties attached at the ends of branched linkers were synthesized, and their DNA binding properties were investigated with regard to the A3T3 repeat by measuring fluorescence spectra. The binding prop

Full text of DOI:10.1016/j.bmc.2015.05.056

Bioorganic & Medicinal Chemistry 23 (2015) 4583–4590
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Strong positive cooperativity in binding to the A T repeat
3
3
by Hoechst 33258 derivatives attaching the quinoline units
at the end of a branched linker
a
b
a
a,
⇑
Hironori Koda , John Alan Brazier , Ippei Onishi , Shigeki Sasaki
a
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
School of Pharmacy, University of Reading, Chemistry Building, Whiteknights, PO Box 224, Reading RG6 6AD, UK
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Hoechst 33258 derivatives with additional interacting moieties attached at the ends of branched linkers
were synthesized, and their DNA binding properties were investigated with regard to the A3T3 repeat by
measuring fluorescence spectra. The binding property of the ligand was investigated by fluorescence
titration, and the titration data were analyzed using the McGhee–von Hippel method. Ligand 6Q with
the quinolin-6-yloxyacetyl group and Ligand IQ with isoquinolin-6-yloxyacetyl group at the ends of
the branched linkers exhibit highly positive cooperativity for the DNA having 5 A3T3 sites with 3
base-insertions between them with sequence selectivity. The strategy developed in this study may be
generally applicable for designing ligands for repetitive DNA sequences.
Received 28 April 2015
Revised 29 May 2015
Accepted 30 May 2015
Available online 16 June 2015
Keywords:
Cooperative binding
DNA repeat
Ó 2015 Elsevier Ltd. All rights reserved.
Hoechst
Fluorescence titration
McGhee–von Hippel equation
7
1
. Introduction
peptides, metal complexes,8 intercalators,9 and minor groove
1
0
binders, show cooperativity in binding with DNA; however, few
studies have considered this approach for designing molecules
for DNA repeat. Here, we describe that the Hoechst 33258
derivatives with the additional interaction units attached at the
ends of a branched linker exhibit highly positive cooperativity for
Repetitive DNA sequences are widely distributed in genomes
and have received considerable attention because of their diverse
roles in structure and biology,
1–3
4
which include telomeric repeat
and trinucleotide repeat,5 etc. Some diseases are associated with
expansion disorders of trinucleotide repeat. For example, a trinu-
cleotide (CAG) repeat in the androgen receptor gene lengthens
from 13 to 30 repeats in the normal range to 40 or more
repeats in patients with the disorder of this receptor. Disorders
of repeat expansion cause loss of function or toxicity of proteins.
Therefore, selective compounds for targeting expansion of trinu-
cleotide repeat are desired for diagnostic and therapeutic pur-
3 3
the binding with the A T repeat.
2
2
. Results and discussion
.1. Molecular design
Figure 1 outlines our approach, in which additional interaction
units are attached at the ends of a branched linker of the ligand to
promote cooperativity between the adjacent ligands. It is well
known that Hoechst 33258 produces bright fluorescence by bind-
ing to the minor groove of duplex DNA, with a marked preference
for AT-rich regions.
depending on such factors as the DNA sequence, ligand concentra-
tions, and buffer conditions.
Hoechst derivatives with the bipyridine unit were demonstrated
to form bimolecular chelate complexes with the Cu(II) in the bind-
ing with DNA. In this study, we attempted to determine suitable
interaction units at the ends of the branched linker to validate this
6
poses. However, effective methods have not yet been developed.
Triplex-forming oligonucleotides and minor groove binders are
representative compounds for binding with duplex DNA.
Nevertheless, expansion of a trinucleotide repeat exceeds a length
for recognition of these compounds. In this study, we attempted to
establish a new strategy in designing molecules for targeting
expansion of a sequence repeat. From the perspective of small
molecules, cooperativity is of great interest for expansion of a
binding site. Certain small molecular compounds, such as
1
1–14
Other binding modes are also observed,
1
0,15–17
18,19
In our previous work,
⇑
Corresponding author. Tel./fax: +81 92 642 6615.
E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki).
3 3
concept for cooperative binding with the A T repeat.
http://dx.doi.org/10.1016/j.bmc.2015.05.056
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0

4
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H. Koda et al. / Bioorg. Med. Chem. 23 (2015) 4583–4590
Figure 1. General strategy for cooperative binding to a repetitive DNA sequence.
2
.2. Chemistry
non-linear least-squares to the theoretical curve calculated by
assuming a 1:1 binding stoichiometry between Ligand 6Q and the
3 3
A T site, and the association constants were estimated to be
Figure 2 summarizes the structure of the ligands and their com-
18
6
ꢁ1
5
ꢁ1
ponents. The carboxy derivative of Hoechst 33258 (1) was used
as the key unit. The dipropionic acid derivative of ethylenediamine
linker 2 was coupled with the mono-modified derivative of dia-
mine spacers (3–6) using standard condensation reagents to give
the corresponding branched linker. After deprotection of the Boc
group, the branched linker was coupled with 1 to form Ligand X
K
s
= 6.5 ꢀ 10 M for the change at 275 nm and 3.9 ꢀ 10 M for
the change at 327 nm. Ligand 6Q was used at M concentrations
for CD measurements, thereby forming typical minor groove bind-
ing with the A site. It turned out that it was difficult to evaluate
l
3 3
T
binding cooperativity by CD measurement, therefore, we next per-
formed the fluorescence titration experiments.
(
see Supporting information for the synthetic procedures of the
components). The synthesized ligands were purified by HPLC and
quantified by H NMR using maleic acid as an internal standard.
2.4. Fluorescent titration experiments
1
The ligand is named for its terminal group and the linker; for
example, Ligand 6Q is constructed of the branched linker with 3
The binding property of the ligand was investigated by
fluorescence titration using the ligand at a concentration of
10 nM. A portion of DNA solution was added to a buffer solution
of the ligand, and the increase in fluorescence intensity was mea-
sured. Figure 4A represents examples obtained using Ligand 6Q
and DNA5(3). As control binding experiments, the use of DNA0
did not increase the fluorescence intensity significantly; thus, the
fluorescence titration data were first analyzed based on the bind-
(
R = quinolin-6-yloxyacetyl attached at the terminal). Ligand 6QS
contains the short spacer (4) at the ends of the branched linker,
and 6QSL represents the case in which both the short (4) and long
linkers (6) are conjugated. Ligand MonoQ was synthesized as the
control ligand conjugating the non-branched quinolin-6-yloxy-
3 3
acetyl linker. For the DNA substrates, the A T sequence was cho-
sen as a favorable binding site for Hoechst 33258, and duplex
DNA sequences with 60 base pairs in length were designed. DNA
3 3
ing to A T sites. The titration results were well simulated using
the non-linear curve-fitting method by assuming two binding
modes, one with high affinity and the other with low affinity
(Fig. 4A, circles: observed values, dotted line: calculated values).
The titration data were analyzed using the McGhee–von Hippel
method, which is an established method of analyzing the coopera-
tivity in the binding of ligands with duplex DNA and is based on
substrate is named for the numbers of A
insertions between them (Chart 1). For example, DNA5(3) is con-
structed of five A sites with three base insertion containing G
and C. DNA5(1), DNA5(3) and DNA5(6) are different in the number
of inserted bases between the A sites.
3 3
T sites and the base
3 3
T
3 3
T
2
0,21
Eq. 1.
The McGhee–von Hippel analysis produces three param-
2
.3. Binding evaluation by CD
Figure 3 presents examples of the CD spectral changes produced
eters, n (the number of base pairs covered by one ligand), K (the
ꢁ1
association constant, M ), and
A large value corresponds to strong cooperativity due to higher
ratio of the closely bound complexes. The mean cluster size C on
x (the cooperativity parameter).
x
by the binding of Ligand 6Q with DNA1 and DNA5(3). The CD spectra
of DNA1 did not show significant change by the addition of Ligand
n
2
1–23
the DNA substrate can be calculated by Eq. 2.
At first, the complex concentration at each base-pair concentra-
tion was calculated from the titration data to produce the (the
concentration of the bound ligand per base pair) and /L values
(L is the free ligand concentration). The /L values were plotted
against the values (Fig. 4B). These plots were fitted by non-linear
curve-fitting method with the McGhee–von Hippel Eq. 1 to pro-
duce the three parameters, n, K, and . For the titration data
6
Q (Fig. 3A), although the complexes were formed as discussed in
the following session. In contrast, DNA5(3) indicated significant
CD changes by binding with Ligand 6Q (Fig. 3B). Isoelliptic points
observed at 256 nm and 284 nm suggest a single mode of binding
with respect to the complex structures responsible for the CD spec-
tra. The CD bands are attributable to each structural component:
bands approximately 260 nm for DNA-Hoechst binding, bands
approximately 322 nm for 6-quinolinol, and bands approximately
m
m
m
m
x
obtained with Ligand 6Q, these parameters were determined to
3
ꢁ1
4
3
70 nm for the Hoechst part. These results show that Ligand 6Q
be n = 19.3, K = 4.2 ꢀ 10 M , and
that a large
binding of Ligand 6Q to DNA5(3).
x
= 2.5 ꢀ 10 . It should be noted
binding to DNA5(3) induces DNA conformational change. The titra-
tion profiles monitored at 275 nm and 327 nm were well fitted by
x value indicates a highly positive cooperativity for the

H. Koda et al. / Bioorg. Med. Chem. 23 (2015) 4583–4590
4585
O
O
A
N
O
NH
B
N
N
N
H
N
N
H
N
OH
Ligand X
Ligand
Name
Linker for A and B
Unit R of the linker 3
Chart 1. DNA sequences used in this study.
NH
H
2
Ac
A=B: 3
A=B: 3
Acetyl
6Q
Quinolin-6-yloxyacetyl
Nac
Py
Naphthalen-2-yloxyacetyl
Pyridine-3-yloxyacetyl
IQ
Isoquinolin-6-yloxyacetyl
5-Nitroquinolin-6-yloxyacetyl
5-Bromoquinolin-6-yloxyacetyl
Quinolin-2-yloxyacetyl
6
6
2
6
QNO
2
QBr
Q
Qx
Quinoxalin-6-yloxyacetyl
6
6
6
QS
A=B: 4
QL
A=B: 5
QSL
A: 6 , B: 4
Ligand MonoQ
O
CH
3
H
N
O
N
O
HN
O
N
H
O
N
O
N
N
N
H
N
N
N
H
OH
Componetns of Ligand X
COOH
COOH
N
N
N
N
H
BocNH
COOH
N
N
H
N
1
3
2
OH
O
O
NH
R
H N
O
H N
O
2
2
N
H
4
N
O
H
N
O
O
O
H N
O
O
N
2
O
H
N
5
O
Figure 3. The spectral changes of Ligand 6Q and DNA5(3). (A) CD of DNA1 (5 lM in
H
2
N
O
O
O
A
3
T
3
) and CD with Ligand 6Q (10
lM, 2 equiv for A
3 3
T ). (B) CD Titration of DNA5(3)
O
O
N
(
2
l
M in A ) with Ligand 6Q (0–4 l
3
T
3
M).
H
6
N
Figure 2. Structure of the ligands.
DNA1 were between 2 and 1, in good agreement with the known
preference of Hoechst 33258 for the A sequence. Interestingly,
large cluster formation is indicated for the binding of the ligand
Q and DNA5(3), even at a high [A ]/[Ligand] ratio. The binding
properties of other ligands were similarly evaluated to obtain three
parameters (Table S1). The cooperative parameters obtained
with a variety of ligands are compared in Figure 5.
Hoechst 33258 exhibited little cooperativity to DNA, regardless
of the number of A T binding sites, as expected. The ligands 6Q
3 3
ꢀð2
ꢁRꢁnꢁ1
ꢀ
ꢁ
3 3
T
2
m
L
x
þ1Þð1ꢁn
m
Þþ
m
1ꢁðnþ1Þ þR
m
¼
K ꢂð1ꢁn
m
Þꢂ
ꢂ
2ð ꢁ1Þð1ꢁn
x
mÞ
2ð1ꢁn Þ
m
6
3 3
T
ð1Þ
x
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
m
ð
x
m
ꢁ 1Þ
ꢁ 1 þ R
2
C
n
¼
R ¼ ½1 ꢁ ðn þ 1Þ
m
ꢃ þ 4
xm
ð1 ꢁ n Þ
m
ðn ꢁ 1Þ
ð2Þ
exhibited the highest selective cooperativity for DNA5(3). The
The mean cluster number (C
the n and parameters and is plotted against the [A
ratio in Figure 4C. The C values of Hoechst 33258 in binding to
n
) at a
m
value was calculated using
ligands NH , Ac MonoQ did not display marked cooperativity, indi-
cating that the two quinoline parts at the end of the branched lin-
ker are essential for high cooperativity. The x cooperativity
2
x
3
3
T ]/[Ligand]
n

4
586
H. Koda et al. / Bioorg. Med. Chem. 23 (2015) 4583–4590
DNA5(6) may be shorter or longer to maintain mutual contact of
the ligand 6Q. 6QL with the longer spacer, 6QS with the short
spacer and 6QSL with both the short and long spacers did not exhi-
bit marked cooperativity. Therefore, the symmetrical linker struc-
ture with an appropriate spacer length is crucial for 6Q to exhibit
cooperativity (Fig. 5A).
Interestingly, among the structurally resembling ligands, only
IQ exhibited cooperativity with selectivity to DNA5(3) (Fig. 5B).
Ligand Nac, which has a naphthalene-2-ol unit, exhibited coopera-
tivity to some extent, albeit with no sequence selectivity. It should
be noted that decrease in cooperativity and selectivity of Ligand
Nac arose from the absence of ring nitrogen in naphthalene. The
5
-nitro and 5-bromo substitution of the quinoline ring largely
diminished cooperativity. The low cooperativity with 6Qx, 2Q, or
Py indicated the importance of the positions of the nitrogen atom
of the quinoline ring.
The quinoline ring is well known to produce molecular interac-
2
4,25
26
tions, such as
hydrogen bonding. In aqueous media, the
presumably major assembling forces, which may occur through
p
–
p
stacking,
binding with metal cations and
2
7
Figure 4. The fluorescence titration of the ligand 6Q by DNA5(3). (A) Circles
represent raw data of titration, which are well fitted to the dotted line calculated by
p–p
interactions are
using the three parameters. (B) The Scatchard plot of
K, and , which were obtained using Eq. 1, were used to calculate the dotted line.
C) The mean cluster sizes (C ) were calculated using Eq. 2. Hoechst 33258 binding
to DNA1 (closed circles and blue line) and the ligand 6Q binding to DNA5(3) (open
squares and blue line) are plotted against the [A ]/[Ligand] ratio.
m
and
m
/L. Three parameters, n,
21,28,29
dipole-dipole interactions between quinolines.
Substitution
x
pattern of the quinoline ring produces the different dipole
moments, which might be beneficial in the case of Ligand 6Q and
IQ for the high cooperativity.
(
n
3 3
T
The UV–vis absorption bands of Ligand 6Q were shifted by bind-
ing with DNA5(3); the Hoechst region shifted to longer wavelength
(372.5 to 384.5 nm) with 50% increased absorbance, the quinoline
region shifted to shorter wavelength (328.5 to 326.5 nm) with 43%
parameter of the ligand 6Q for DNA5(3) was the largest in this
study. The linker length of 6Q seems to be fitted to DNA5(3), and
the interval of a single base pair in DNA5(1) and six base pairs in
Figure 5. Comparison of the cooperativity parameter (x).

H. Koda et al. / Bioorg. Med. Chem. 23 (2015) 4583–4590
4587
Figure 6. UV change of Ligand 6Q (A) and Ligand 6QBr (B) by binding with DNA5(3).
increased absorbance (Fig. 6A). These UV changes suggest strong
tendencies of the Hoechst parts for forming J-aggregates and the
quinoline parts for H-type aggregates. In contrast, although
Ligand 6QBr showed similar wavelength shifts for both regions,
their absorbance increases were smaller; 13% for the bromoquino-
line part and 16% for the Hoechst part (Fig. 6B). Accordingly, Ligand
(2.2 equiv), DIPEA (12 equiv) and HBT (2.2 equiv) were added to a
solution of N-Boc derivative of the ethylenediamine linker 2 in
DMF (0.30 M) at room temperature under argon, and the reaction
mixture was stirred at room temperature. After the completion of
the reaction, the reaction mixture was evaporated, and the residue
was diluted with dichloromethane. The mixture was washed with
6
QBr has less ability for the formation of both J- and H-aggregates.
saturated aqueous NaHCO
was dried over anhydrous Na
3
solution and brine. The organic layer
SO , filtered and evaporated. The resi-
The difference of Ligand 6Q and 6QBr in cooperativity may be
explained based on their different ability for J- and H-aggregates
formation. It has been reported that, in addition to a representative
minor groove binding mode, Hoechst 33258 binds to DNA with
multiple stoichiometries including stacking aggregates depending
on the concentration and buffer conditions.11 Thus, it is conceiv-
able that the ligand molecules bound in the minor groove promote
interactions between the quinoline units, which in turn facilitate
stacking aggregate formation of the ligand molecules. Further
study on the complex structure is needed for detail discussion
about the effect of the terminal binding parts on the cooperativity.
2
4
due was purified by column chromatography (Kanto 60N, dichloro-
methane/methanol) to afford the corresponding N-Boc linker
derivative. Trifluoroacetic acid (20 equiv) was added to a solution
of the N-Boc linker derivative in dichloromethane, and the reaction
mixture was stirred at room temperature. After the completion of
the reaction, the reaction mixture was evaporated. The crude pro-
duct was dissolved in DMF (0.10 M) and used for the next reaction
without purification. Hoechst-COOH (1) (1.1 equiv), HBTU
(1.1 equiv), HBT (1.1 equiv) and DIPEA (12 equiv) were added to
the above solution under argon. The reaction mixture was stirred
at room temperature. After the completion of the reaction, the reac-
tion mixture was evaporated. The residue was purified by column
chromatography (Fujisilysia NH, chloroform/methanol eluent) to
afford Ligand 6Q. Analytical sample was obtained by reverse-phase
HPLC (Nacalai Tesque COSMOSIL 5C18-AR-II 10 mm ꢀ 250 mm;
3
. Conclusions
In conclusion, this study clearly demonstrated that the ligand
Q and IQ with quinoline moieties at the ends of the branched link-
6
2 3
eluents H O (A) and CH CN (B); gradient 0–20 min, 15–100% B in
A + B; flow rate 2.5 mL/min; detection 368 nm). The ligand concen-
tration was determined by the integral values of H NMR spectrum
including maleic acid as the internal standard.
ers exhibit highly positive cooperativity with sequence selectivity.
Although it is difficult to observe detailed complex structure, the
cluster formation has been supported by fluorescence titration
and UV–vis measurements. The strategy developed in this study
may be generally applicable for designing ligands for repetitive
DNA sequences. Potential application of selective ligands for the
expanded DNA repeats include intracellular imaging of DNA and
interference of transcription of the gene, which are now ongoing
in our group.
1
4.1.3. Ligand 6Q
An orange solid. IR (cmꢁ1) 3285.7, 3091.4, 1671.4, 1200.1,
1
1130.1; H NMR (400 MHz, CD OD) d (ppm) 8.78 (dd, J = 4.8,
3
1.5 Hz, 2H), 8.52 (d, J = 8.2 Hz, 2H), 8.48 (d, J = 0.9 Hz, 1H), 8.33
(
dd, J = 8.6, 1.5 Hz, 1H), 8.00 (d, J = 8.9 Hz, 2H), 7.98 (d, J = 9.2 Hz,
4
4
4
. Experimental
2H), 7.81 (d, J = 8.9 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.66 (dd,
J = 8.5, 4.9 Hz, 2H), 7.61 (dd, J = 9.1, 2.7 Hz, 2H), 7.37 (d,
J = 2.7 Hz, 2H), 7.34 (d, J = 2.4 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H),
.1. Chemical synthesis
4
.63 (s, 4H), 4.03 (s, 2H), 3.63–3.58 (m, 8H), 3.53–3.48 (m, 12H),
.1.1. General
3.46–3.40 (m, 10H), 3.34–3.32 (m, 4H), 3.16 (t, J = 5.3 Hz, 4H),
1H NMR (400 MHz) and 13C NMR (100, 125, 150 MHz) spectra
13
3
3.01 (s, 3H), 2.80 (t, J = 6.3 Hz, 4H); C NMR (125 MHz, CD OD) d
were recorded on a Varian UNITY-400 or INOVA-500. Infrared (IR)
spectra were obtained using a SHIMADZU FTIR-8400 spectrometer.
High resolution mass spectra analyses were recorded on an Applied
Biosystems Mariner System 5299 spectrometer using bradykinin,
neurotensin, and angiotensin as an internal standard. See
Supplementary data for the synthesis of the components (1–6).
(ppm) 172.1, 169.9, 169.2, 164.4, 159.0, 157.0, 153.2, 152.4,
148.2, 144.7, 144.5, 138.1, 137.4, 135.2, 134.3, 131.8, 131.2,
128.0, 126.0, 124.9, 123.2, 122.4, 117.9, 116.4, 115.3, 114.7,
113.6, 108.8, 104.8, 71.2, 70.3, 70.2, 68.5, 54.8, 52.1, 43.6, 40.3,
40.0, 36.5, 29.7; HRESI-MS (m/z) calcd for
68 80 14 12
C H N O
2
+
([M+2H] ) 643.3113, found 643.3123.
4
.1.2. Synthesis of Ligand 6Q as a general procedure for the
4.1.4. Ligand NH2
The title compound was prepared as a yellow solid. IR (cmꢁ1)
synthesis of the ligand
Quinolin-6-yloxyacetyl derivative of 1,2-bis(2-aminoethoxy)-
ethane (3, R = Quinolin-6-yloxyacetyl) (2ꢄ3 equiv), HBTU
1
3432.4, 1675.0, 1202.4, 1133.6; H NMR (400 MHz, CD3OD) d
(ppm) 8.50 (s, 1H), 8.26 (d, J = 7.3 Hz, 1H), 8.03 (d, J = 8.9 Hz, 2H),

4
588
H. Koda et al. / Bioorg. Med. Chem. 23 (2015) 4583–4590
7
1
.79 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 2.1 Hz, 1H), 7.39 (d, J = 2.4 Hz,
H), 7.02 (d, J = 8.5 Hz, 2H), 4.03 (t, J = 5.6 Hz, 2H), 3.63 (t,
J = 8.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 2.1 Hz, 1H),
7.57 (dd, J = 9.2, 2.4 Hz, 2H), 7.44 (d, J = 2.1 Hz, 2H), 7.33 (d,
J = 2.1 Hz, 1H), 6.99 (d, J = 8.5 Hz, 2H), 4.73 (s, 4H), 4.04 (s, 2H),
3.66–3.58 (m, 10H), 3.53–3.49 (m, 12H), 3.45–3.40 (m, 10H),
3.23–3.20 (m, 2H), 3.17 (t, J = 5.5 Hz, 4H), 3.02 (s, 3H), 2.83 (t,
J = 5.6 Hz, 2H), 3.60–3.56 (m, 12H), 3.52–3.50 (m, 6H), 3.38 (t,
J = 5.6 Hz, 4H), 3.22 (t, J = 5.6 Hz, 6H), 3.08 (t, J = 5.2 Hz, 4H), 3.01
(
(
s, 3H), 2.82 (t, J = 6.4 Hz, 4H); 13C NMR (150 MHz, CD3OD) d
ppm) 172.3, 169.5, 163.0, 151.1, 147.9, 138.7, 130.5, 119.1,
1
3
3
J = 6.3 Hz, 4H); C NMR (125 MHz, CD OD) d (ppm) 172.2, 169.4,
1
4
C
17.5, 117.2, 116.0, 105.0, 71.3, 71.2, 70.4, 67.9, 54.9, 54.4, 52.1,
165.4, 164.4, 148.2, 146.6, 142.9, 133.6, 132.7, 131.2, 126.6,
126.0, 125.2, 124.9, 124.6, 122.4, 117.9, 116.3, 115.3, 113.6,
107.9, 71.2, 70.3, 70.2, 68.5, 54.7, 52.1, 43.6, 40.3, 40.0, 36.5,
5.2, 43.6, 40.6, 40.2, 36.5, 29.8; HRESI-MS (m/z) calcd for
2
+
46
66 12 8
H N O ([M+2H] ) 458.2636, found 458.2672.
68 80 14
C H N O )
12 ([M+2H]2+
2
9.7; HRESI-MS (m/z) calcd for
4
.1.5. Ligand Ac
643.3113, found 643.3158.
The title compound was obtained as an orange solid. IR (cmꢁ1)
1
3
286.0, 3089.9, 2876.3, 1669.7, 1196.7, 1134.4; H NMR (400 MHz,
4.1.9. Ligand Py
CD
3
OD) d (ppm) 8.56 (s, 1H), 8.38 (d, J = 8.2 Hz, 1H), 8.04 (d,
Ligand Py was obtained as an orange solid. IR (cmꢁ1) 3275.3,
1
J = 8.9 Hz, 2H), 7.85 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 2.4 Hz, 1H),
1672.8, 1200.1, 1131.0; H NMR (400 MHz, CD3OD) d (ppm) 8.57
7
3
.40 (d, J = 2.1 Hz, 1H), 7.07 (d, J = 8.9 Hz, 2H), 4.04 (s, 2H), 3.63–
.58 (m, 8H), 3.50–3.41 (m, 16H), 3.30–3.27 (m, 10H), 3.12 (t,
(s, 1H), 8.40 (dd, J = 8.7, 1.5 Hz, 1H), 8.35 (d, J = 2.8 Hz, 2H), 8.24
(dd, J = 4.9, 1.2 Hz, 2H), 8.05 (d, J = 8.9 Hz, 2H), 7.86 (d, J = 8.2 Hz,
1H), 7.74 (d, J = 2.4 Hz, 1H), 7.61 (ddd, J = 8.6, 2.7, 1.2 Hz, 2H),
7.51 (dd, J = 8.7, 4.9 Hz, 2H), 7.39 (d, J = 2.4 Hz, 1H), 7.08 (d,
J = 8.9 Hz, 2H), 4.62 (s, 4H), 4.04 (t, J = 5.2 Hz, 2H), 3.65–3.58 (m,
8H), 3.51–3.46 (m, 12H), 3.44–3.39 (m, 10H), 3.21 (t, J = 7.3 Hz,
2H), 3.20 (t, J = 7.3 Hz, 2H), 3.14 (t, J = 5.2 Hz, 4H), 3.02 (s, 3H),
J = 5.2 Hz, 4H), 3.02 (s, 3H), 2.80 (t, J = 5.5 Hz, 4H), 1.90 (s, 6H);
1
3
3
C NMR (125 MHz, CD OD) d (ppm) 173.4, 172.2, 169.3, 164.4,
1
1
5
53.2, 152.6, 148.2, 138.2, 135.3, 134.4, 131.2, 127.3, 126.1,
22.5, 117.9, 116.4, 115.2, 114.8, 113.6, 104.9, 71.2, 70.5, 70.2,
4.8, 54.6, 52.1, 43.6, 43.6, 40.3, 40.2, 36.5, 29.6, 22.6; HRMS
+
13
(m/z) calcd for C50
70
H N
12
O
10 ([M+H] ) 999.5411, found 999.5402.
2.81 (t, J = 6.3 Hz, 4H); C NMR (125 MHz, CD3OD) d (ppm)
1
72.2, 169.7, 169.3, 164.4, 157.0, 153.3, 152.7, 148.2, 140.0,
4
2
.1.6. Ligand Nac
138.3, 135.5, 131.2, 128.0, 127.4, 126.0, 122.5, 117.9, 116.4,
115.3, 115.1, 113.6, 104.9, 103.9, 71.2, 70.3, 70.2, 68.6, 54.8, 54.6,
52.1, 43.6, 40.3, 40.0, 36.5, 29.6; HRESI-MS (m/z) calcd for
Ligand Nac was obtained as a yellow solid. IR (cmꢁ1) 3070.1,
1
866.9, 1667.6, 1200.2, 1183.3, 1128.7;
H NMR (400 MHz,
2
+
CD
3
OD) d (ppm) 8.43 (d, J = 0.9 Hz, 1H), 8.31 (dd, J = 8.5, 1.5 Hz,
60 76 14
C H N O12 ([M+2H] ) 593.2956, found 593.2968.
1
7
3
H), 7.98 (dd, J = 7.8, 2.1 Hz, 2H), 7.76 (d, J = 8.6 Hz, 1H), 7.72–
.70 (m, 3H), 7.68 (s, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.37–7.32 (m,
H), 7.29–7.24 (m, 2H), 7.13 (dd, J = 8.9, 2.4 Hz, 2H), 7.10 (d,
4.1.10. Ligand 6QBr
Ligand 6QBr was obtained as an orange solid. IR (cmꢁ1) 3274.6,
1
J = 2.4 Hz, 2H), 7.05 (dd, J = 7.8, 2.1 Hz, 2H), 4.53 (s, 4H), 3.90 (t,
J = 5.2 Hz, 2H), 3.56 (t, J = 5.2 Hz, 2H), 3.52-3.44 (m, 16H), 3.41–
3095.7, 1669.4, 1198.8, 1133.0; H NMR (400 MHz, CD
3
OD) d
(ppm) 8.73 (dd, J = 4.4, 1.4 Hz, 2H), 8.57 (d, J = 8.9 Hz, 2H), 8.43
(s, 1H), 8.32 (dd, J = 8.7, 1.4 Hz, 1H), 7.99 (d, J = 8.9 Hz, 2H), 7.94
(d, J = 9.2 Hz, 2H), 7.78 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 2.1 Hz, 1H),
7.58 (dd, J = 8.7, 4.4 Hz, 2H), 7.54 (d, J = 9.2 Hz, 2H), 7.30 (d,
J = 2.1 Hz, 1H), 7.06 (d, J = 8.9 Hz, 2H), 4.68 (s, 4H), 4.01 (t,
J = 5.5 Hz, 2H), 3.64–3.58 (m, 8H), 3.56–3.52 (m, 10H), 3.48–3.42
(m, 10H), 3.38–3.34 (m, 6H), 3.19 (t, J = 5.5 Hz, 4H), 3.02 (s, 3H),
3
(
(
.35 (m, 12H), 3.27 (t, J = 5.2 Hz, 4H), 3.13 (t, J = 5.2 Hz, 4H), 2.98
s, 3H), 2.75 (t, J = 6.1 Hz, 4H); 13C NMR (125 MHz, CD
OD) d
ppm) 172.0, 171.0, 169.3, 164.2, 156.7, 153.0, 152.7, 148.0,
3
1
1
1
4
38.2, 135.7, 135.3, 134.5, 131.1, 130.7, 130.6, 130.6, 128.6,
27.9, 127.5, 125.8, 125.1, 122.3, 119.3, 117.8, 116.3, 115.0,
13.3, 108.4, 108.3, 71.2, 71.2, 70.4, 70.2, 68.1, 54.7, 52.0, 43.6,
1
3
0.3, 39.9, 36.5, 29.7; HRESI-MS (m/z) calcd for C70
82
H N
12
O
12
2.82 (t, J = 6.1 Hz, 4H); C NMR (125 MHz, CD
172.2, 170.0, 169.2, 164.5, 154.2, 153.1, 152.5, 149.4, 148.1,
43.9, 138.1, 137.4, 135.1, 134.3, 131.2, 129.8, 129.5, 126.0,
3
OD) d (ppm)
2
+
(
[M+2H] ) 642.3160, found 642.3174.
1
4
.1.7. Ligand 2Q
Ligand 2Q was obtained as a yellow solid. IR (cmꢁ1) 3275.3,
088.0, 1647.2, 1199.9, 1129.1; 1H NMR (400 MHz, CD
OD) d
124.0, 122.3, 120.2, 117.9, 116.3, 115.2, 114.7, 113.4, 109.3,
104.8, 71.3, 70.4, 70.3, 69.7, 54.8, 52.3, 43.6, 40.3, 40.0, 36.7, 30.7,
2+
3
3
68 78 14 2
29.7; HRESI-MS (m/z) calcd for C H N O12Br ([M+2H] )
(
ppm) 8.46 (s, 1H), 8.31 (dd, J = 8.6, 1.5 Hz, 1H), 7.99 (d,
721.2218, found 721.2261.
J = 8.9 Hz, 2H), 7.82 (d, J = 9.8 Hz, 2H), 7.79 (d, J = 8.5 Hz, 1H),
7
2
.69 (d, J = 2.1 Hz, 1H), 7.60 (d, J = 7.9 Hz, 2H), 7.52 (t, J = 7.9 Hz,
H), 7.32 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 8.9 Hz, 2H), 7.23 (t,
4.1.11. Ligand 6QNO
2
Ligand 6QNO
2
was obtained as a yellow solid. IR (cmꢁ1) 3303.4,
1
J = 7.6 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 6.61 (d, J = 9.5 Hz, 2H),
.99 (s, 4H), 4.01 (t, J = 5.5 Hz, 2H), 3.64–3.54 (m, 10H), 3.49–3.39
m, 10H), 3.36–3.34 (m, 6H), 3.28–3.27 (m, 2H), 3.11 (t,
3088.3, 1667.6, 1185.6, 1131.4; H NMR (400 MHz, CD
3
OD) d
4
(
(ppm) 8.77 (dd, J = 4.3, 1.5 Hz, 2H), 8.43 (s, 1H), 8.30 (dd, J = 8.6,
1.5 Hz, 1H), 8.10 (d, J = 9.5 Hz, 2H), 8.08 (d, J = 8.6 Hz, 2H), 7.99
(d, J = 8.9 Hz, 2H), 7.77 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 2.1 Hz, 1H),
7.62 (d, J = 9.5 Hz, 2H), 7.55 (dd, J = 8.9, 4.3 Hz, 2H), 7.32 (d,
J = 2.4 Hz, 1H), 7.07 (d, J = 8.9 Hz, 2H), 4.73 (s, 4H), 4.02 (t,
J = 5.8 Hz, 2H), 3.66–3.58 (m, 8H), 3.53–3.49 (m, 10H), 3.48–3.45
(m, 6H), 3.39 (t, J = 5.3 Hz, 4H), 3.36–3.32 (m, 6H), 3.17 (t,
J = 5.3 Hz, 4H), 3.02 (s, 3H), 2.82 (t, J = 5.8 Hz, 4H); 13C NMR
13
J = 5.5 Hz, 4H), 2.97 (s, 3H), 2.78 (t, J = 6.1 Hz, 4H); C NMR
125 MHz, CD OD) d (ppm) 179.3, 172.1, 169.8, 169.4, 164.2,
63.8, 153.5, 153.0, 147.9, 142.0, 142.0, 140.7, 138.4, 132.2,
(
3
1
1
1
4
30.9, 130.2, 127.2, 125.4, 123.9, 122.3, 121.3, 117.7, 116.1,
15.5, 113.6, 98.9, 71.3, 71.2, 70.4, 70.2, 54.8, 52.1, 46.2, 43.6,
80 14 12
0.5, 40.3, 36.6, 29.7; HRESI-MS (m/z) calcd for C68H N O
2
+
(
[M+2H] ) 643.3113, found 643.3080.
3
(125 MHz, CD OD) d (ppm) 172.1, 169.4, 169.2, 164.4, 153.1,
1
52.5, 151.0, 149.2, 148.1, 143.4, 138.2, 136.0, 135.1, 134.7,
4
.1.8. Ligand IQ
Ligand IQ was obtained as an orange solid. IR (cmꢁ1) 3283.7,
079.1, 1671.4, 1200.9, 1133.9; 1H NMR (400 MHz, CD
OD) d
134.3, 131.2, 131.0, 127.3, 125.9, 125.1, 122.4, 122.3, 119.3,
117.9, 116.3, 115.1, 114.8, 113.4, 104.8, 71.3, 71.2, 70.3, 70.3,
69.6, 54.8, 54.6, 52.2, 43.6, 40.3, 40.1, 36.6, 30.7, 29.7; HRESI-MS
3
3
2
+
(
ppm) 9.36 (s, 2H), 8.43 (s, 1H), 8.35 (d, J = 6.4 Hz, 2H), 8.25 (d,
(m/z) calcd for
C H
68 78
N
16
O16 ([M+2H]
)
688.2964, found
J = 9.5 Hz, 2H), 8.22 (s, 1H), 8.07 (d, J = 6.4 Hz, 2H), 7.98 (d,
688.2930.

H. Koda et al. / Bioorg. Med. Chem. 23 (2015) 4583–4590
4589
4
.1.12. Ligand 6Qx
70.1, 68.4, 68.2, 54.9, 54.8, 52.3, 52.1, 43.6, 40.3, 40.1, 39.7, 36.8,
Ligand 6Qx was obtained as a yellow solid. IR (cmꢁ1) 3271.9,
29.9, 29.6, 24.1; HRESI-MS (m/z) calcd for
68 80 14 12
C H N O
077.9, 1668.1, 1199.9, 1128.0; 1H NMR (400 MHz, CD
([M+2H] ) 643.3113, found 643.3098.
2+
3
3
OD) d
(
ppm) 8.70 (d, J = 1.8 Hz, 2H), 8.63 (d, J = 1.8 Hz, 2H), 8.38 (s, 1H),
8
2
.22 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 9.5 Hz,
H), 7.73 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 7.48 (dd,
4.1.16. Ligand MonoQ
IR (cm 1) 3247.0, 3073.3, 1669.2, 1186.4, 1131.1; 1H NMR
ꢁ
J = 9.2, 2.7 Hz, 2H), 7.29 (d, J = 2.1 Hz, 1H), 7.26 (d, J = 2.7 Hz, 2H),
3
(400 MHz, CD OD) d (ppm) 8.89 (dd, J = 5.2, 1.2 Hz, 1H), 8.77
7
3
.01 (d, J = 8.9 Hz, 2H), 4.62 (s, 4H), 4.03 (t, J = 5.5 Hz, 2H), 3.63–
.58 (m, 10H), 3.52–3.46 (m, 12H), 3.42-3.39 (m, 10H), 3.33–3.32
(d, J = 8.5 Hz, 1H), 8.53 (s, 1H), 8.38 (dd, J = 8.9, 1.5 Hz, 1H), 8.08
(d, J = 9.5 Hz, 1H), 8.04 (d, J = 9.2 Hz, 2H), 7.88 (d, J = 8.6 Hz, 1H),
7.83 (dd, J = 8.2, 5.2 Hz, 1H), 7.76 (d, J = 2.1 Hz, 1H), 7.74 (dd,
J = 10.1, 2.7 Hz, 1H), 7.49 (d, J = 2.7 Hz, 1H), 7.38 (d, J = 2.1 Hz,
1H), 7.08 (d, J = 8.9 Hz, 2H), 4.67 (s, 2H), 4.03 (s, 2H), 3.92 (s, 2H),
3.72–3.50 (m, 10H), 3.48–3.44 (m, 2H), 3.41 (t, J = 5.5 Hz, 4H),
(
m, 2H), 3.16 (t, J = 5.5 Hz, 4H), 3.02 (s, 3H), 2.82 (t, J = 6.3 Hz,
1
3
4
1
1
6
3
H); C NMR (125 MHz, CD OD) d (ppm) 172.1, 170.2, 169.5,
63.1, 160.1, 154.4, 147.8, 146.5, 145.2, 144.2, 140.2, 131.3,
30.5, 124.4, 117.5, 116.0, 115.3, 109.0, 71.2, 71.2, 70.4, 70.2,
8.4, 54.9, 54.7, 52.2, 43.6, 40.3, 40.0, 36.6, 29.8; HRESI-MS (m/z)
3.36 (t, J = 5.5 Hz, 4H), 3.21 (t, J = 5.5 Hz, 2H), 3.10 (s, 3H), 3.01 (s,
12 ([M+2H]2 ) 644.3065, found 644.3081.
+
13
calcd for C66
H
78
N
16
O
3
3H), 2.78 (t, J = 6.4 Hz, 2H); C NMR (125 MHz, CD OD) d (ppm)
1
72.1, 169.8, 169.2, 164.4, 158.9, 152.8, 152.2, 148.2, 145.0,
4
.1.13. Ligand 6QL
144.2, 137.8, 137.0, 135.9, 134.9, 133.9, 131.8, 131.2, 128.2,
127.0, 125.8, 124.6, 123.3, 122.3, 117.9, 116.3, 115.2, 114.4,
113.3, 108.8, 104.7, 98.9, 71.2, 70.3, 70.2, 68.4, 57.9, 54.7, 54.5,
43.6, 43.6, 41.3, 40.3, 40.0, 36.3, 29.9; HRESI-MS (m/z) calcd for
IR (cm 1) 3294.5, 3078.2, 1666.9, 1184.6, 1129.8; 1H NMR
ꢁ
(
400 MHz, CD OD) d (ppm) 8.83 (dd, J = 4.9, 1.5 Hz, 2H), 8.61 (d,
3
J = 8.2 Hz, 2H), 8.51 (s, 1H), 8.36 (dd, J = 8.6, 1.5 Hz, 1H), 8.03 (d,
J = 3.1 Hz, 2H), 8.01 (d, J = 2.4 Hz, 2H), 7.83 (d, J = 8.5 Hz, 1H),
2
+
49 57 11 7
C H N O ([M+2H] ) 456.7294, found 456.7298.
7
2
.73 (d, J = 5.2 Hz, 1H), 7.71 (d, J = 5.2 Hz, 2H), 7.67 (dd, J = 9.5,
.8 Hz, 2H), 7.44 (d, J = 2.8 Hz, 2H), 7.35 (d, J = 2.1 Hz, 1H), 7.06
4
.2. CD titration experiment using Ligand 6Q
(
3
(
2
1
1
1
1
4
d, J = 8.9 Hz, 2H), 5.76 (s, 4H), 4.04 (s, 2H), 3.92 (s, 4H), 3.64–
.59 (m, 8H), 3.57–3.52 (m, 16H), 3.52–3.48 (m, 8H), 3.48–3.43
CD spectrum was measured using a sample solution containing
lM DNA1 (5.0 lM in A T concentration), 3.0 mM HEPES and
3 3
m, 10H), 3.38–3.35 (m, 8H), 3.16 (t, J = 6.4 Hz, 4H), 3.02 (s, 3H),
5
.0
.0 mM NaCl in at pH 7.0 and 25 °C. In the titration experiment,
M (0–
). The observed spectral changes are sum-
marized in Figure 3A. Similarly, CD spectrum was measured using
a sample solution containing 2.0 M DNA5(3) (10.0 M in A
concentration), 3.0 mM HEPES and 3.0 mM NaCl at pH 7.0 and
5 °C. In the titration experiment, the concentration of Ligand 6Q
was varied from 0 to 20 M (0–2 equiv in regard to A ). The
.82 (t, J = 6.4 Hz, 4H); 13C NMR (150 MHz, CD3OD) d (ppm)
72.7, 172.2, 170.0, 169.2, 164.4, 162.6, 158.9, 153.2, 152.5,
48.2, 144.7, 144.4, 138.1, 137.8, 135.3, 134.5, 131.8, 131.2,
27.8, 126.0, 125.2, 123.3, 122.4, 117.9, 116.3, 115.3, 113.6,
08.8, 104.8, 71.8, 71.3, 70.4, 70.4, 70.2, 68.5, 54.8, 54.6, 52.1,
3
the concentration of Ligand 6Q was varied from 0 to 10
equiv in regard to A
l
2
3 3
T
l
l
3 3
T
3.6, 40.3, 40.1, 39.9, 39.8, 36.5, 30.7, 29.6; HRESI-MS (m/z) calcd
18 ([M+2H]2 ) 788.3852, found 788.3831.
+
for C80
102
H N
16
O
2
l
3 3
T
4
.1.14. Ligand 6QS
observed spectral changes are summarized in Figure 3B. The titra-
tion data observed at 275 nm and those at 370 nm were fitted with
the non-linear least-square method using Eq. 3, where
IR (cm 1) 3282.4, 3094.7, 1667.9, 1185.8, 1128.4; 1H NMR
ꢁ
(
400 MHz, CD3OD) d (ppm) 8.65 (dd, J = 6.7, 1.5 Hz, 2H), 8.36 (s,
D
Fmax and
2
7
1
7
2
1
H), 8.34 (s, 1H), 8.27 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 8.9 Hz, 2H),
.88 (d, J = 9.5 Hz, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.66 (d, J = 2.4 Hz,
H), 7.53 (dd, J = 9.3, 5.2 Hz, 2H), 7.50 (dd, J = 9.7, 2.7 Hz, 2H),
.30 (d, J = 2.1 Hz, 1H), 7.21 (d, J = 2.4 Hz, 2H), 7.01 (d, J = 8.9 Hz,
H), 4.51 (s, 4H), 4.02 (s, 2H), 3.64–3.56 (m, 10H), 3.26–3.16 (m,
2H), 3.02 (s, 3H), 2.81 (s, 4H); 13C NMR (150 MHz, CD3OD) d
ppm) 172.8, 170.0, 169.2, 164.3, 162.7, 158.5, 152.9, 152.3, 148.1,
44.7, 143.8, 137.9, 135.1, 134.3, 131.6, 131.1, 127.4, 125.8, 125.3,
23.1, 122.2, 117.8, 116.1, 115.2, 114.7, 113.3, 108.5, 104.7, 68.2,
30
K
d
were optimized.
0
0
@
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
11
2
0
D
DNA
F
max
L
2
0
A
2
L ꢁ 2L
0
B
@
AA
D
F ¼
þ
1 ꢁ
þ 1
ð3Þ
2
0
A
A ¼DNA
B ¼DNA
0
þ K
ꢁ K
d
d
(
0
1
1
5
D
F ¼
D
F
H
þ
D
F
L
ð4Þ
ð5Þ
5.1, 54.8, 52.2, 43.6, 40.3, 39.7, 36.9, 30.7, 29.9; HRESI-MS (m/z)
+
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffiffi !!
calcd for C60
H
64
N
14
O
8
([M+2H]2 ) 555.2589, found 555.2576.
H
2
0
D
F
L
2
0
A
H
L ꢁ 2L
0 H
B
max
D
F
H
¼
þ
2
1 ꢁ
þ 1
2
DNA
0
A
4
.1.15. Ligand 6QSL
H
IR (cm 1) 3272.2, 3091.0, 1669.4, 1199.5, 1185.4, 1129.4; 1
ꢁ
H
H
A
B
H
H
¼DNA
¼DNA
0
þ Kd
NMR (400 MHz, CD OD) d (ppm) 8.74 (dd, J = 4.7, 1.5 Hz, 1H),
3
H
ꢁ Kd
8
1
7
1
7
.63 (dd, J = 4.7, 1.5 Hz, 1H), 8.43 (s, 1H), 8.41 (s, 1H), 8.30 (s,
H), 8.26 (dd, J = 8.5, 1.5 Hz, 1H), 7.95 (dd, J = 8.8, 2.8 Hz, 2H),
.87 (d, J = 9.2 Hz, 1H), 7.76 (d, J = 8.9 Hz, 1H), 7.66 (d, J = 2.4 Hz,
H), 7.58 (dd, J = 8.5, 4.4 Hz, 1H), 7.56 (dd, J = 9.5, 2.6 Hz, 1H),
.49 (dd, J = 7.6, 3.8 Hz, 1H), 7.46 (dd, J = 9.6, 2.7 Hz, 1H), 7.31 (d,
0
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi!!
L
2
0
D
F
L
2
0
A
L
L ꢁ 2L
0 L
B
max
D
F
L
¼
þ
2
1 ꢁ
þ 1
ð6Þ
2
DNA
0
A
L
L
J = 2.4 Hz, 2H), 7.18 (d, J = 2.8 Hz, 1H), 7.02 (d, J = 8.9 Hz, 2H),
4
3
A
L
L
¼DNA
¼DNA
0
þ K
d
.61 (s, 2H), 4.50 (s, 2H), 4.02 (s, 2H), 3.62–3.52 (m, 20H), 3.48–
L
B
0
ꢁ Kd
.47 (m, 4H), 3.46–3.37 (m, 6H), 3.25 (t, J = 5.8 Hz, 2H), 3.23–3.18
1
3
(
m, 4H), 3.09 (s, 2H), 3.01 (s, 3H), 2.80 (t, J = 5.8 Hz, 4H);
C
4
.3. Fluorescence titration using Ligand
Q as a general
NMR (125 MHz, CD3OD) d (ppm) 172.7, 172.2, 170.3, 170.2,
procedure
1
1
1
1
69.3, 163.9, 162.7, 158.1, 157.9, 153.4, 152.8, 148.0, 146.6,
41.3, 140.9, 138.2, 136.0, 135.3, 131.3, 131.2, 130.9, 127.5,
27.1, 126.0, 125.8, 125.5, 123.1, 123.0, 122.3, 117.7, 116.1,
15.3, 113.6, 108.5, 108.3, 104.9, 71.5, 71.5, 71.4, 71.2, 71.1, 70.3,
A sample solution containing 10 nM Ligand, 3.0 mM HEPES and
.0 mM NaCl was stirred for 4 h at pH 7.0 and room temperature in
3

4
590
H. Koda et al. / Bioorg. Med. Chem. 23 (2015) 4583–4590
a quartz cell. The fluorescence spectrum was measured with exci-
tation wavelength at 363 nm at 25 °C. In the titration experiment,
an aliquot of a DNA solution was added into the sample solution,
and whole was stirred for 1 h before measuring fluorescence spec-
(Fig. 6A). The UV spectra of Ligand 6QBr showed similar shifts with
smaller changes in intensity (Fig. 6B).
Acknowledgments
trum. The concentration of DNA was varied from 0 to 0.8
M in regard to A concentrations). The observed
were plotted against the A concentration. The observed titration
l
M (0 to
4
l
T
3 3
D
F values
We are grateful for support provided by a Grant-in-Aid for
Scientific Research (S) (Grant number 21229002) and Restart
Postdoctoral Fellowship to JB (Grant number 18 06231) from the
Japan Society for the Promotion of Science (JSPS).
3 3
T
data were well fitted to the theoretical values using Eqs. 4–6 by
predicting an appropriate binding stoichiometry. The values of
H
H
L
L
K
d
,
D
F
max, K
d
, and
D
F
max were calculated by the non-linear curve-fit-
max value was obtained
ting method using KaleidaGraph 4.0 J. The
by summing
DF
Supplementary data
H
L
D
F
max and
D
F
max values.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.05.056.
4
data
.4. McGhee–von Hippel analysis of the fluorescence titration
References and notes
Based on the predicted
DFmax value obtained as above, the com-
plex concentration was calculated at each DNA concentration. The
complex concentration was divided by the base pair concentration
1. Mirkin, S. M. Curr. Opin. Struc. Biol. 2006, 16, 351.
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3
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to give the
culated to give the
produce the Scatchard plot, which is shown in Figure 4B. The
ues were used to simulate /L values by Eq. 1 using three parame-
ters, n, K and . The sum of the square values of difference
between the observed and the calculated /L values were opti-
mized by Solver installed in Microsoft Excel. The obtained n, K
and parameters for Ligand 6Q were used to calculate the ratio
m
value. Also, the free ligand concentration (L) was cal-
/L value. The and /L values were plotted to
val-
2010, 7, 1.
m
m
m
4. Brooks, T. A.; Kendrick, S.; Hurley, L. FEBS J. 2010, 277, 3459.
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1
1
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1
1
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.5. UV spectra measurements
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A sample solution (200
ature before UV measurement. Final concentrations of sample
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M DNA5(3) in 3.0 mM HEPES, 3.0 mM NaCl, pH 7.0, at 25 °C.
The UV spectral changes of Ligand 6Q are summarized in
Figure 6A. The Hoechst region Ligand 6Q shifted by binding with
DNA5(3) to longer wavelength (372.5–384.5 nm) with 50%
increase in the intensity, the quinoline region shifted to shorter
wavelength (328.5–326.5 nm) with 43% increase in the intensity
lL) was stirred for 7 h at room temper-
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2
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2
2
2
30. Wilcox, C. S.; Cowart, M. D. Tetrahedron Lett. 1986, 27, 5563.