The effect of linker length on binding affinity of a photoswitchable molecular glue for DNA

Article abstract of DOI:10.1016/j.bmc.2009.01.053

Molecular glue for DNA is a small synthetic ligand that adheres two single-stranded DNAs to produce a double-stranded DNA. We previously devised a photoswitchable molecular glue (PMG) that uses external light stimuli to reversibly control DNA hybridizatio

Full text of DOI:10.1016/j.bmc.2009.01.053

Bioorganic & Medicinal Chemistry 17 (2009) 2536–2543
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The effect of linker length on binding affinity of a photoswitchable
molecular glue for DNA
*
Chikara Dohno, Shin-nosuke Uno, Shun Sakai, Mika Oku, Kazuhiko Nakatani
Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecular glue for DNA is a small synthetic ligand that adheres two single-stranded DNAs to produce a
double-stranded DNA. We previously devised a photoswitchable molecular glue (PMG) that uses external
light stimuli to reversibly control DNA hybridization. To optimize the structure of PMG, we synthesized a
series of PMGs and evaluated the effect of changing the methylene linker length on the binding affinity
and photoresponse. From the comprehensive Tm and CSI-TOF-MS measurements, a PMG possessing a
three-methylene linker with carbamate linkage produced maximum binding affinity and photoswitching
ability. These results indicate that a small difference in the linker can significantly affect PMG function.
These findings are useful for designing new photoswitchable DNA-binding ligands.
Received 20 December 2008
Revised 21 January 2009
Accepted 22 January 2009
Available online 30 January 2009
Keywords:
DNA
Hybridization
Mismatch binding ligand
Photoswitchable molecular glue
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
reversible control between ssDNA and dsDNA containing a GG-
mismatch by external light stimuli (Fig. 1).
The sequence-specific hybridization property of DNA is the un-
ique and indispensable basis of the essential functions of DNA.
Being able to control DNA hybridization with external stimuli
could be useful for regulating diverse biological functions and con-
structing DNA-based materials. A wide variety of chemically mod-
ified oligonucleotides have been developed to control and
modulate DNA hybridization.1–7 We demonstrated previously a
new concept of a molecular glue for DNA that controls the hybrid-
ization of two natural unmodified DNAs.8 This molecular glue is a
small synthetic ligand that adheres two single-stranded DNAs
(ssDNA) that do not hybridize spontaneously with each other.
We have developed a series of synthetic small molecules that can
bind specifically to mismatch-containing DNAs.8,9 Because the mis-
match-binding ligands (MBLs) increase the apparent thermody-
namic stability of the mismatched DNA duplex, MBLs can
function as a molecular glue for DNA. The function of the molecular
glue in the first generation was limited to the unidirectional con-
trol of DNA hybridization because MBL induces the one-way trans-
formation from ssDNA to double-stranded DNA (dsDNA).8 We
previously extended the concept to the reversible control of DNA
hybridization by external stimuli.10 To accomplish this objective,
we have devised a photoswitchable molecular glue (PMG) for
DNA, NCDA, in which a photochromic azobenzene unit is incorpo-
rated into the GG mismatch-binding ligand. NCDA permits the
The photochromic azobenzene moiety in NCDA exhibits Z/E
isomerization in response to the light stimuli, which changes the
relative position and orientation of the two naphthyridines. Z-
NCDA can bind to DNA more strongly than can E-NCDA and, there-
fore, is more efficient as a molecular glue. The photoswitching abil-
ity is based on the difference of the stabilization effect between Z-
NCDA and E-NCDA. To achieve an ideal switching of DNA hybrid-
ization, an increased binding affinity of Z-NCDA and a decreased
binding affinity of E-NCDA, or vice versa, are desirable. To under-
stand the structure–binding relationship of PMG, we herein de-
scribe our synthesis of a series of PMGs that possess common
mismatch recognition groups and photochromic azobenzene but
different linking groups (Chart 1). Investigation of the PMG func-
tion revealed a significant correlation between the linker struc-
tures and PMG function.
5'
3'
5'
3'
N
N
G
G
N
C
G
G
N
N
G
G
C
N
360 nm
C
G
G
N
C
> 400 nm
N
3'
5'
3'
5'
ssDNA
E-NCDA
dsDNA - Z-NCDA complex
* Corresponding author. Fax: +81 6 6879 8459.
Figure 1. Control of DNA hybridization by photoswitchable molecular glue for
E-mail address: nakatani@sanken.osaka-u.ac.jp (K. Nakatani).
DNA.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.053

C. Dohno et al. / Bioorg. Med. Chem. 17 (2009) 2536–2543
2537
NCDA310 was achieved via a similar reductive amination reaction
in Scheme 1. However, it was difficult to isolate NCDA2 in accepta-
ble purity because of its instability. Decomposition with concomi-
tant generation of 14, 12, and 13 suggested that NCDA2 underwent
a facile intramolecular carbamate exchange reaction, which is fa-
vored by the formation of a five-membered ring (Chart 2).12 To
overcome this difficulty, we developed another synthetic route
where the final step is N-Boc deprotection of 21 (Scheme 2). In this
synthetic scheme, NCDA2 was obtained as a hydrochloric salt. The
hydrochloric salt of NCDA2 is chemically more stable because the
protonation of the amines in NCDA2 hinders the intramolecular
carbamate exchange reaction by decreasing the nucleophilicity.
NDA is an NCDA analogue possessing amide linkages instead of
carbamate linkages (Scheme 1). The linker length is equivalent to
one methylene linker in the NCDA series. All PMGs synthesized
here have a poor solubility in water and were therefore used as hy-
drochloric salts in following experiments.
2. Results and discussion
The parent PMG, NCDA, is constituted of base-recognizing
naphthyridine moieties, a photochromic azobenzene, and a flexible
linker connecting the two naphthyridines. The dimeric naphthyri-
dine carbamate functions to recognize GG-mismatch DNA by form-
ing complementary hydrogen bonds.8,9c The azobenzene moiety
undergoes Z/E isomerization upon photoirradiation, and this
photoisomerization induces changes in the DNA-binding affinity
and stabilization ability of NCDA.11 The photoinduced structural
change of the azobenzene moiety is transmitted as a geometry
change of the two naphthyridines through the linker. Therefore,
the photoswitching ability of NCDA will be highly dependent on
the linker group. From the standpoint of the large change in geom-
etry, a short and rigid linker is favorable for maximum photos-
witching ability of NCDA. However, a long and flexible linker is
required to locate the two naphthyridines in the proper geometry,
whereas a shorter linker is preferred to minimize the entropic
costs. Thus, optimization of the linker length is required to maxi-
mize the switching ability of PMG.
To evaluate the effect on the switching property and binding af-
finity of PMG possessing different linkers, we synthesized a series
of NCDA variants with methylene linker lengths ranging from two
to four (Chart 1). NCDA with a four-methylene linker (NCDA4) was
synthesized as shown in Scheme 1. 4-Aminobutanol 5 was con-
verted to the four-methylene-linked naphthyridine derivatives 9b
via coupling its activated carbonic acid ester with 2-amino-7-
methyl-1,8-naphthyridine. Reductive amination of the naphthyri-
dine carbamate 9b with 4,40-diformyl-azobenzene furnished the
desired NCDA4 in moderate yield. Synthesis of NCDA2 and
Azobenzene is
a well-known photochromic molecule that
shows E to Z isomerization by UV light irradiation and Z to E isom-
erization by visible light irradiation.12 Photoisomerization of
NCDA4 in aqueous 10 mM Naꢀcacodylate buffer (pH 7.0) and
100 mM NaCl was monitored by UV–vis absorption spectroscopy
(Fig. 2). The spectrum before photoirradiation exhibited an absorp-
tion band at around 317 nm attributed to the overlap of the azo-
benzene
p–
p* transition and absorption of naphthyridine
chromophores. Upon exposure to 360 nm light, the band intensity
at 317 nm decreased and the absorbance at around 405 nm in-
creased concomitantly. These spectral changes are typical of the
E to Z photoisomerization of azobenzene.13 The photoisomeriza-
tion of NCDA4 was confirmed further by HPLC analysis. The azo-
benzene of NCDA4 exists exclusively in the
E form under
ambient light condition (Fig. 3a). We examined the E to Z photoiso-
merization of NCDA4 using UV light in 10 nm intervals at wave-
lengths ranging from 330 to 380 nm. The maximum Z isomer
content in the photostationary states was achieved by 360 nm irra-
diation. After 5 min irradiation at 360 nm, the photostationary
state was reached, and the fraction of Z-NCDA4 increased to about
55% (Fig. 3b). The photostationary mixture was then irradiated
with 430 nm light, resulting in conversion back to the mixture
comprising exclusively E-NCDA4 (Fig. 3c). Similar photoisomeriza-
tion behaviors were observed for NCDA2 and NCDA3, whereas
NDA showed considerably higher E to Z photoisomerization effi-
ciency (81% Z in the photostationary state).
O
N
H
N
H
N
N
N
N
N
N
H
N
H
N
N
N
N
N
1: NDA
O
O
O
n
N
H
O
N
H
N
N
H
N
H
N
O
n
2: NCDA2 (n=2)
3: NCDA3 (n=3)
4: NCDA4 (n=4)
We next examined the stabilization of mismatch DNA duplex by
the series of NCDA derivatives and NDA. The melting temperatures
Chart 1.
O
O
a
b
c
NH2
NHBoc
NHBoc
NH2
HO
HO
N
N
N
O
N
N
N
O
H
H
5
N
N
6
8
9b
O
CHO
N
N
N
N
N
N
H
H
O
N
N
N
d
+
N
H
N
H
N
N
N
N
NH2
N
N
H
OHC
1, NDA
O
O
10
11
O
N
H
O
N
H
n
O
N
N
H
N
H
N
N
H
O
NH2
N
N
O
n
n
3, NCDA3 (n = 3)
4, NCDA4 (n = 4)
9a (n=3)
9b (n=4)
Scheme 1. Reagents and conditions: (a) Boc2O, CHCl3, quant.; (b) (i) DSC, Et3N, CH3CN; (ii) 2-amino-7-methyl-1,8-naphthyridine, Et3N, CH2Cl2, 61% (two steps); (c) HCl,
AcOEt, CHCl3, 80%; (d-i) 10, NaBH3CN, AcOH, MeOH, CHCl3, 14%; (d-ii) 9a, NaBH3CN, AcOH, MeOH, CHCl3, 37%; (d-iii) 9b, NaBH3CN, AcOH, MeOH, CHCl3, 50%.

2538
C. Dohno et al. / Bioorg. Med. Chem. 17 (2009) 2536–2543
OTBDPS
N
a
b
H2N
H2N
H
N
OH
OTBDPS
N
H
N
16
15
TBDPSO
17
OTBDPS
OH
N
N
Boc
Boc
c
d
N
N
N
N
Boc
Boc
N
N
TBDPSO
HO
18
19
O
H
O
N
N
N
O
O
e
f
N
N
N
Boc
Boc
O
O
N
N
N
N
O
O
O
O
Boc
Boc
N
N
N
N
N
N
O
O
O
H
21
O
g
20
H
O
N
N
N
N
H2
O
N
N
Cl-
Cl-
O
H2
N
NCDA2
N
N
N
O
H
Scheme 2. Reagents and conditions: (a) TBDPSCl, imidazole, 43%; (b) 4,40-diformylazobenzene 11, NaBH3CN, AcOH, CH3OH, CHCl3, 45%; (c) Boc2O, CHCl3, 84%; (d) TBAF,
AcOH, THF, 93%; (e) DSC, Et3N, CH3CN, 67%; (f) 2-amino-7-methyl-1,8-naphthyridine, Et3N, CH2Cl2, 56%. (g) HCl, AcOEt, CHCl3, quant.
O
(a)
(b)
E-NCDA4
E-NCDA4
N
O
N
N
N
N
NH2
O
N
Z-NCDA4
12
14
O
H
N
O
N
N
N
H
O
N
N
(c)
O
N
13
O
0
10
20
30
40
50
60
Chart 2.
t / min
Figure 3. HPLC analysis of the photoisomerization of NCDA4. The solution of
NCDA4 (40
l
M) in 10 mM Naꢀcacodylate (pH 7.0) and 100 mM NaCl was photoir-
radiated, and analyzed by HPLC. The peak with a longer retention time is attributed
to E-NCDA4. Key: (a) Before irradiation; (b) after 360 nm irradiation for 5 min, (c)
subsequent irradiation at 430 nm for 5 min.
0.25
0.2
0.15
0.1
(Tm) of 11-mer duplexes 50-(CTAA CXG AATG)-30/50-(CATT CYG
TTAG)-30 containing a single mismatch site (X/Y) were measured
in the presence of the PMGs before and after photoisomerization
(Table 1). All synthesized photoswitchable ligands have two guan-
ine-recognizing naphthyridine groups, and, as expected, showed
an apparent selectivity for duplexes containing a GG mismatch.
Regardless of the linker length, the Tm values of the GG-mismatch
duplex were higher after photoirradiation at 360 nm (Tm2) than be-
fore irradiation (Tm1), showing that PMGs in the Z configuration
have a higher stabilization ability than the E isomer. This is proba-
bly because, for inducing the appropriate spatial arrangements of
the two naphthyridine groups, the folded Z azobenzene linkage
0.05
0
200
250
300
350
400
450
500
wavelength / nm
Figure 2. UV–vis spectra of NCDA4 (6
lM) in 10 mM Naꢀcacodylate (pH 7.0) and
100 mM NaCl (black line). The sample was irradiated with 360 nm light for 10 min
(red solid line). Subsequent irradiation at 430 nm for 5 min resulted in a recovery of
the original spectrum (blue dotted line).

C. Dohno et al. / Bioorg. Med. Chem. 17 (2009) 2536–2543
2539
Table 1
ing affinity and produced a less effective photoswitchable molecu-
lar glue. An ideal photoswitchable molecular glue will bind
strongly in the Z configuration (high Tm2 value) while keeping
the affinity weak in the E configuration (low Tm1 value). Z-NCDA3
provides a significantly higher binding affinity to the GG-mismatch
DNA (Tm2 = 48.0 °C) than the other ligands, and this can overwhelm
Melting temperature (Tm/°C) of DNA duplexes in the presence of a variety of PMGs
before and after photoirradiation
a
X–Y
Tm0
+NCDA2, dark
+NCDA2, h
m
+NCDA3, dark
+NCDA3, hm
b
c
d
e
b
c
d
e
Tm1
D
Tm1
Tm2
D
Tm2
Tm1
D
Tm1
Tm2
D
Tm2
15.2
n.d.f
G–G
A–A
A–C
C–C
G–A
G–T
T–C
T–T
A–T
G–C
25.6
17.8
16.1
18.2
25.7
28.3
18.6
25.1
34.3
40.3
28.9
17.6
18.5
24.5
27.1
30.9
21.1
22.3
33.7
40.8
3.4
42.7
17.9
19.4
24.6
31.6
32.6
21.2
24.1
33.5
40.9
13.8
0.3
0.9
0.1
4.5
1.7
0.1
1.8
32.7
n.d.f
23.3
25.7
32.0
32.0
27.2
28.6
34.5
38.6
7.1
48.0
21.3
20.4
22.7
33.4
31.5
23.8
27.8
35.2
40.3
ꢁ0.2
n.df
7.2
the relatively strong affinity of E-NCDA3 (
methylene unit insertion significantly decreased the Tm2 value by
9.7 °C and Tm2 value by 7.2 °C (Tm2 = 38.3 °C and Tm2 = 8.0 °C
for NCDA4). NCDA2 showed a photoresponse ( Tm2 = 13.8 °C)
comparable to NCDA3, but further shortening of the linker abol-
ished the photoswitching ability ( Tm2 = 1.3 °C for NDA). The
DTm1 = 7.1 °C). A single
2.4
ꢁ2.9
6.3
1.4
2.6
2.5
ꢁ2.8
ꢁ0.6
0.5
7.4
6.3
3.7
8.6
ꢁ3.0
1.4
D
D
D
ꢁ0.5
ꢁ3.4
–0.8
0.7
3.5
D
ꢁ0.2
ꢁ0.2
exceptionally small Tm difference for NDA before and after photoir-
radiation is attributed to the considerably strong binding of E-NDA
0.1
ꢁ1.7
1.7
to GG-mismatch DNA (DTm1 = 11.7 °C). The effective binding of E-
X–Y
+NCDA4, dark
+NCDA4, h
m
+NDA, dark
+NDA, h
m
NDA raises the possibility of rational design of a new PMG possess-
ing an opposite photoresponse, that is, higher binding affinity in
the E configuration than in the Z configuration. The Tm data for
NDA differed markedly from the NCDA series, suggesting a differ-
ent binding mode. The maximum photoresponse was observed for
NCDA3 possessing three methylene units. These results clearly
demonstrate that the linker structure and length affect the photo-
response significantly.
b
c
d
e
b
c
d
e
Tm1
D
Tm1
Tm2
D
Tm2
Tm1
D
Tm1
Tm2
D
Tm2
G–G
A–A
A–C
C–C
G–A
G–T
T–C
T–T
A–T
G–C
30.3
n.d.f
n.d.f
25.8
26.0
30.0
24.4
28.3
36.2
41.7
4.7
n.d.f
n.d.f
7.6
0.3
1.7
5.8
3.2
1.9
1.4
38.3
n.d.f
n.d.f
25.3
25.4
30.3
24.0
27.1
36.1
41.7
8.0
37.3
20.8
18.3
23.1
30.0
24.2
22.0
n.d.f
31.8
40.1
11.7
2.9
2.2
4.9
4.3
38.6
21.0
17.9
22.5
30.1
25.4
22.2
n.d.f
30.2
40.2
1.3
0.2
ꢁ0.4
ꢁ0.6
0.1
n.d.f
n.d.f
ꢁ0.5
ꢁ0.6
0.2
ꢁ4.1
1.2
0.2
ꢁ0.4
ꢁ1.2
ꢁ0.1
0.0
3.4
n.d.f
n.d.f
To examine the binding stoichiometry of PMGs, cold-spray ion-
ization time-of-flight mass spectrometry (CSI-TOF-MS)15 was used
to measure the ligand–DNA complex before and after photoirradi-
ation. The 11-mer duplex DNA 50-(CTAA CGG AATG)-30/50-(CATT
CGG TTAG)-30 was used in these experiments because we previ-
ously found that dimeric naphthyridine derivatives bind selec-
tively to the CGG/CGG sites with 2:1 stoichiometry or two
ligands for a single CGG–CGG site (Fig. 1).8,9 In the presence of
3 equiv of E-NCDA derivatives, the 5ꢁ ions of the 1:1 complex
(m/z of 1481.29, 1487.05, and 1492.54 for NCDA2, NCDA3, and
NCDA4, respectively) and, to a lesser extent, the 2:1 complex (m/
z of 1621.17, 1632.55, and 1643.43 for NCDA2, NCDA3, and NCDA4,
respectively) were observed (Fig. 4a–d). After photoirradiation at
360 nm for 5 min, the ion intensity corresponding to the 2:1 com-
plex increased markedly for all tested ligands (Fig. 4e–h); in partic-
ular, Z-NCDA3 produced the 2:1 complex almost exclusively
(Fig. 4f). The higher ion intensities compared with those before
irradiation suggest that the Z-NCDA derivatives formed a more sta-
ble complex with the CGG–CGG sequence than did the E-NCDA,
and these results agreed with the results of the Tm experiments.
With increasing amounts of NCDA from 1 to 3 equiv, the ions cor-
responding to the 2:1 complex increased, but complexes with
higher binding stoichiometries could not be detected under these
conditions. The preferential formation of the 2:1 complex is typical
ꢁ2.5
ꢁ1.5
ꢁ0.2
0.1
The synthetic ligands were designed to bind selectively to GG-mismatch DNA, and
the values for GG-mismatch were emphasized by the bold characters.
Tm values of DNA duplexes in the absence of PMG. Thermal melting curves were
measured for duplex DNA, d(CTA ACX GAA TG)/d(CAT TCY GTT AG) (4.54 lM) in
10 mM sodium cacodylate buffer (pH 7.0) containing 0.1 M NaCl. Tm values (°C)
were calculated by median method.
a
b
Tm values in the presence of PMG (18.2
l
M).
Tm1 is calculated as the difference between Tm0 and Tm1
Tm values in the presence of PMG after photoirradiation at 360 nm for 5 min.
Tm2 is calculated as the difference between Tm1 and Tm2
Tm value could not be determined due to the unclear transition.
c
d
e
f
D
.
D
.
gives preferable binding compared with the extended E azoben-
zene linkage.10 The binding of the E isomer is less selective to the
GG mismatch than the binding of the Z isomer. The intercalative
interaction of the planar E azobenzene may contribute to this less
selective binding.14
The
DTm2 value was calculated as the difference in Tm from be-
fore to after photoirradiation, and was used as an index of the
photoswitchable function of the molecular glues. Among the four
ligands tested, NCDA3 possessing the three-methylene linker
exhibited the highest
(NCDA4) and the shorter linkers (NDA, NCDA2) reduced the bind-
DTm2 value (15.2 °C). Both the longer linker
(a)
15000
(b)
(c)
(d)
[dsDNA]4-
[ssDNA]2-
10000
5000
[dsDNA]5-
[1:1]5-
[1:1]5-
[1:1]5-
[1:2]5-
[1:2]5-
[1:2]5-
[1:2]5-
[1:1]5-
[1:1]5-
0
(e)
(f)
(g)
(h)
15000
[1:2]5-
10000
5000
[1:2]5-
[1:2]5-
[1:1]5-
[1:1]5-
[1:1]5-
0
1200
1500
1800
1200
1500
1800
1200
1500
1800
1200
1500
1800
mass / m/z
mass / m/z
mass / m/z
mass / m/z
Figure 4. Cold-spray ionization time-of-flight mass spectra of DNA duplex in the presence of various PMGs. Samples contain 20
aqueous methanol and ammonium acetate (100 mM). The sample was cooled at ꢁ10 °C during the injection with flow rate 10
photoirradiation; (e)–(h) after photoirradiation at 360 nm for 5 min; (a) and (e) NCDA2; (b) and (f) NCDA3; (c) and (g) NCDA4; (d) and (h) NDA.
l
M duplex DNA and 60
l
M PMG in 50%
a
l
L/minꢁ1
. Key: (a)–(d) Before

2540
C. Dohno et al. / Bioorg. Med. Chem. 17 (2009) 2536–2543
form the above-mentioned 2:1 complex. The different binding
mode may have caused the distinct binding properties observed
in the Tm experiments (Table 1). The linker length is one methylene
unit shorter in NDA than in NCDA2 and has an amide linkage in-
stead of a carbamate linkage. These changes in the linker have a
significant effect on the DNA binding and the photoswitching prop-
erties. The optimal linker length is determined by a delicate bal-
ance of the enthalpic gain and the entropic loss upon ligand
binding.
3. Conclusion
We synthesized and examined a series of the photoswitchable
DNA-binding ligands to evaluate the effect of the differing methy-
lene linker length on the binding affinity and photoresponse. This
study reveals that the linker length has a significant effect on the
binding affinity and the photoswitching ability. Therefore, deter-
mination of an optimal linker, which locates the base-recognizing
moiety in the appropriate positions and minimize steric hindrance
and entropic loss upon binding to the target DNA, is a crucial step.
From the comprehensive Tm and CSI-TOF-MS measurements, both
maximum binding affinity and photoswitching ability were ob-
served for the three-methylene linker with carbamate linkage,
NCTA3. The significantly higher binding affinity of Z-NCDA3 than
E-NCDA3 contributes the remarkable photoresponse. A difference
of only one methylene unit in the linker length has a profound
influence on PMG function. In the case of NDA in particular, the
two-methylene linker with amide linkage diminished the effective
photoswitching. These findings provide useful guidance for design-
ing new PMGs possessing different binding selectivity and
photoresponse.
Figure 5. Molecular modeling of the complex between NCDA derivatives and the
duplex DNA 50-(CTAA CGG AATG)-30/50-(CATT CGG TTAG)-30. The model structures
were optimized by use of the AMBER* force field in water with MacroModel
Version9.1. (a) Schematic illustration of the complex. (b) NCDA2; (c) NCDA3; (d)
NCDA4. The ligands are represented in red, while their linker groups are
represented in magenta.
4. Experimental
4.1. General
for MBLs we have synthesized previously.8–10 Figure 5 shows the
predicted structure of the 2:1 complex of the Z-NCDA derivatives
and the CGG/CGG containing 11-mer duplex DNA. These structures
were obtained by energy minimization of the model structures
constructed manually from the NMR-structure we previously
determined9b using MacroModel 9.1 with an AMBER* force field
in water. As increasing the linker length, the azobenzene moieties
were more extruded from the major groove. Solvent accessible sur-
faces of the NCDA derivatives in the complex are calculated as 648,
665, and 772 Å2 for NCDA2, NCDA3, and NCDA4, respectively, in
the energy minimized structures. The hydrophobic azobenzene
and the linking methylene groups of NCDA4 in the complex are
more exposed to solvent water (Fig. 5d). Thus, the long flexible
methylene linker of NCDA4 is unfavorable with regard to entropic
costs that originate from the loss of degrees of freedom and the
exposure of the hydrophobic groups to solvent water. The struc-
ture of the complex with NCDA2 and NCDA3 are similar in terms
of the solvent accessible surfaces. The modestly weak binding of
NCDA2 may come from the existence of the remarkably stable con-
formations of the free ligand, which were obtained from conforma-
tional search calculations. The conformational change of the
ligands upon the complex formation was estimated from the dif-
ference between the global minimum conformation of the free li-
gand and the conformation extracted from the ligand in the
minimized structure of the complex. The conformational change
of NCDA2 in the complex formation was energetically less favor-
able than that of the NCDA3 by 6.4 kJ/mol.
Reagents and solvents were purchased from standard suppliers
and used without further purification. Reactions were monitored
with TLC plates precoated with Merck Silica Gel 60 F254. Spots were
visualized with UV light or ninhydrin. Wakogel C-200 was used for
silica gel flash chromatography. 1H NMR spectra were measured
with JEOL JNM-LA400 and LA600. The chemical shifts are ex-
pressed in ppm relative to residual solvent as an internal standard.
ESI mass spectra were recorded on a JEOL AccuTOF JMS-T100N
mass spectrometer. FAB mass spectra were recorded on a JEOL
JMS-M600H.
4.1.1. N-(7-Methyl-1,8-naphthyridin-2-yl)carbamic acid 4-(tert-
butoxycarbonylamino)butyl ester (8)
To a mixture of N-(tert-butoxycarbonyl)-4-amino-1-butanol 6
(2.00 g, 10.7 mmol) and N,N-disuccinimidyl carbonate (DSC)
(4.06 g, 15.9 mmol) in acetonitrile (30 mL) was added triethyl-
amine (4.42 mL, 31.7 mmol) and the reaction mixture was stirred
at ambient temperature for 3 h. Solvent was removed in vacuo
and the residue was diluted with saturated aqueous NaHCO3 solu-
tion and extracted with ethyl acetate. The organic phase was
washed with brine, dried over anhydrous MgSO4, filtered, and con-
centrated in vacuo. The crude product was purified by column
chromatography on silica gel (CHCl3/methanol = 100:1) to give
crude succinimidyl carbonic ester 7 (3.36 g). To a solution of 7
(2.00 g, 6.05 mmol) in anhydrous dichloromethane (50 mL) was
successively added 2-amino-7-methyl-1,8-naphthyridine (1.06 g,
6.66 mmol) and triethylamine (1.27 mL, 9.08 mmol) and the reac-
tion mixture was stirred overnight at ambient temperature. The
reaction mixture was diluted with saturated aqueous NaHCO3
In contrast to the preferential formation of the 2:1 complex by
NCDA derivatives, NDA prefers to form a 1:1 complex even in the Z
configuration (Fig. 4h). The binding mode of NDA differs from that
of the NCDA derivatives, probably because the linker is too short to

C. Dohno et al. / Bioorg. Med. Chem. 17 (2009) 2536–2543
2541
solution and extracted with CHCl3. The organic phase was washed
with brine, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The crude product was purified by column chromatogra-
phy on silica gel (ethyl acetate/n-hexane = 1:1) to give 8 (1.44 g,
3.85 mmol, 61% 2 steps from 6) as a pale yellow solid: 1H NMR
(400 MHz, CDCl3) d = 1.42 (s, 9H), 1.59 (m, 2H), 1.74 (m, 2H),
2.75 (s, 3H), 3.16 (m, 2H), 4.22 (t, 2H, J = 6.6 Hz), 4.55 (br, 1H),
7.26 (d, 1H, J = 8.3 Hz), 7.72 (br, 1H), 7.99 (d, 1H, J = 8.3 Hz), 8.11
(d, 1H, J = 8.8 Hz), 8.25 (d, 1H, J = 8.8 Hz); 13C NMR (150 MHz,
CDCl3) d = 25.44, 25.98, 26.41, 28.26, 39.96, 65.25, 79.00, 112.49,
117.79, 121.14, 136.23, 138.81, 153.11, 153.31, 154.53, 155.85,
163.00; ESI-MS, m/e 397 ([M+Na]+); HRMS (FAB): calcd for
C19H27O4N4 ([M+H]+) 375.2032, found 375.2042.
over anhydrous MgSO4. The solvent was evaporated in vacuo and
the crude residue was purified by column chromatography on sil-
ica gel (CHCl3/methanol = 25:1) to give 16 (1.30 g, 4.34 mmol, 25%)
as a pale-yellow oil: 1H NMR (400 MHz, DMSO) d 0.98 (s, 9H), 266
(t, 2H, J = 6.08 Hz), 3.58 (t, 2H, J = 4.88 Hz), 7.43 (m, 6H), 7.61 (m,
4H); 13C NMR (100 MHz, CDCl3) d 18.9, 26.5, 43.9, 65.8, 127.3,
129.3, 133.3, 135.2; HRMS (ESI) calcd for C18H27NOSi [(M+H)+]
300.1783, found 300.1779.
4.1.5. N,N0-Bis[2-(tert-butyldiphenylsilyloxy)ethyl]-4,40-
aminomethylazobenzene (17)
To a solution of 16 (398 mg, 1.33 mmol) in CHCl3/metha-
nol = 2:1 (12 ml) was added 4,40-diformylazobenzene 11 (113 mg,
0.475 mmol) and glacial acetic acid (pH 5). The reaction mixture
was stirred at ambient temperature for 1 h under argon. To the
reaction mixture was added sodium cyanotrihydroborate
(NaBH3CN) (83.5 mg, 1.32 mmol). The reaction mixture was stirred
at room temperature for 2 h. The solvent was concentrated in va-
cuo. The residue was diluted with CHCl3, washed with saturated
aqueous NaHCO3, H2O and brine, and dried over anhydrous MgSO4.
The solvent was concentrated in vacuo and the crude residue was
purified by column chromatography on silica gel (from CHCl3 100%
to CHCl3/methanol = 50:1) to give 17 (306 mg, 0.38 mmol, 80%) as
an orange solid: 1H NMR (400 MHz, CDCl3) d 1.04 (s, 18H), 2.78 (t,
4H, J = 5.1 Hz), 3.81 (t, 4H, J = 5.1 Hz), 3.88 (s, 4H), 7.34–7.46 (16H),
7.61–7.66 (8H), 7.87 (d, 4H, J = 8.3 Hz); 13C NMR (150 MHz, CDCl3)
d 19.3, 27.0, 50.9, 53.2, 63.1, 123.0, 127.8, 128.8, 129.8, 133.6,
135.6, 143.5, 151.9; HRMS (ESI) calcd for C50H61N4O2Si2 [(M+H)+]
805.4333, found 805.4326.
4.1.2. N-(7-Methyl-1,8-naphthyridin-2-yl)carbamic acid 4-
aminobutyl ester (9b)
To a solution of 8 (500 mg, 1.34 mmol) in CHCl3 (10 mL) was
added ethyl acetate (1.67 mL) containing 4 M HCl and the reaction
mixture was stirred at ambient temperature for 1 h. To the reaction
mixture was added additional ethyl acetate containing 4 M HCl
(1.00 mL). After 30 min stirring, solvent was removed in vacuo
and the residue was washed with CHCl3, diluted with saturated
aqueous NaHCO3 solution and extracted with CHCl3. The organic
phase was washed with brine, dried over anhydrous MgSO4, fil-
tered, and concentrated in vacuo to give 9b (292 mg, 1.07 mmol,
80%) as a pale yellow solid: 1H NMR (400 MHz, CDCl3) d = 1.52
(m, 2H), 1.72 (m, 2H), 2.69 (s, 3H), 2.72 (t, 2H, J = 7.1 Hz), 4.18 (t,
2H, J = 6.6 Hz), 7.20 (d, 1H, J = 8.0 Hz), 7.93 (d, 1H, J = 8.0 Hz),
8.05 (d, 1H, J = 8.8 Hz), 8.19 (d, 1H, J = 8.8 Hz); 13C NMR
(150 MHz, CDCl3) d = 25.59, 26.22, 29.69, 41.62, 65.66, 112.67,
117.94, 121.28, 136.40, 138.91, 153.32, 153.50, 154.69, 163.15;
ESI-MS, m/e 297 ([M+Na]+), 571 ([2M+Na]+); HRMS (FAB): calcd
for C14H19O2N4 ([M+H]+) 275.1508, found 275.1508.
4.1.6. N,N0-Bis(tert-butoxycarbonyl)-N,N0-bis[2-(tert-
butyldiphenylsilyloxy)ethyl]-4,40-aminomethylazobenzene (18)
To a solution of 17 (276 mg, 0.34 mmol) in CHCl3 (10 mL) was
added di-tert-butyl dicarbonate (224 mg, 1.03 mmol). The reaction
mixture was stirred at ambient temperature for 3 h under argon
and concentrated in vacuo. The residue was extracted with ethyl
acetate. The organic phase was washed with saturated aqueous
NaHCO3, H2O and brine, and dried over anhydrous MgSO4. The or-
ganic phase was concentrated in vacuo and the crude residue was
purified by column chromatography on silica gel (CHCl3/metha-
nol = 200:1) to give 18 (345 mg, 0.343 mmol, quantitative yield)
as an orange oil. The NMR spectra showed the rotameric mixture
due to high rotation barriers caused by the tertiary amide bond
of t-Boc protective group: 1H NMR (400 MHz, CDCl3) (rotameric
mixture) d 1.04 (s, 18H), 1.41 (s, 18H), 3.32–3.44 (4H), 3.71–3.80
(4H), 4.57–4.61 (4H), 7.32–7.43 (16H), 7.60–7.64 (8H), 7.84 (d,
4H, J = 8.0 Hz); 13C NMR (150 MHz, CDCl3) (rotameric mixture) d
19.1, 26.8, 28.3, 48.5, 48.8, 50.8, 51.6, 62.4, 62.5, 79.8, 85.1,
122.9, 127.7, 128.2, 129.7, 133.3, 135.5, 141.7, 142.1, 151.8,
155.5, 155.8; HRMS (ESI) calcd for C60H76N4O6Si2Na [(M+Na)+]
1027.5201, found 1027.5172.
4.1.3. N-(7-Methyl-1,8-naphthyridin-2-yl)carbamic acid [1,2-
diazenediylbis (4,1-phenylenemethyleneimino-4,1-butanediyl)]
ester (4, NCDA4)
To a mixture of 9b (80 mg, 0.292 mmol) and 4,40-formylazoben-
zene 11 (31.6 mg, 0.13 mmol) in CHCl3 (5 mL) and methanol
(2.5 mL) was added acetic acid (19.0 mL, 0.33 mmol) and the reac-
tion mixture was stirred at ambient temperature for 1 h. Sodium
cyanotrihydroborate (NaBH3CN) (16.7 mg, 0.27 mmol) was added
to the reaction mixture. After 2.5 h stirring, solvent was removed
in vacuo and the residue was diluted with saturated aqueous NaH-
CO3 solution and extracted with CHCl3. The organic phase was
washed with brine, dried over anhydrous MgSO4, filtered, and con-
centrated in vacuo. The crude product was purified by column
chromatography on silica gel (CHCl3/methanol = 20:1) to give 4
(49.5 mg, 0.066 mmol, 50%) as an orange solid: 1H NMR
(400 MHz, CDCl3) d = 1.60 (m, 4H), 1.73 (m, 4H), 2.65–2.68 (10H),
3.83 (s, 4H), 4.18 (t, 4H, J = 6.5 Hz), 7.14 (d, 2H, J = 8.3 Hz), 7.40
(d, 4H, J = 8.1 Hz), 7.63 (br, 2H), 7.80 (d, 4H, J = 8.1 Hz), 7.92 (d,
2H, J = 8.3 Hz), 8.04 (d, 2H, J = 8.8 Hz), 8.19 (d, 2H, J = 8.8 Hz); 13C
NMR (150 MHz, CDCl3) d = 25.95, 26.70, 26.98, 49.17, 53.92,
66.05, 112.93, 118.30, 121.64, 123.24, 129.05, 136.73, 139.29,
143.77, 152.11, 153.55, 153.69, 155.04, 163.54; ESI-MS, m/e 777
([M+Na]+); HRMS (FAB): calcd for C42H47O4N10 ([M+H]+)
755.3782, found 755.3772.
4.1.7. N,N0-Bis(tert-butoxycarbonyl)-N,N0-bis(2-hydroxyethyl)-
4,40-aminomethylazobenzene (19)
To a solution of 18 (368 mg, 0.37 mmol) in THF (7.5 mL) was
added TBAF (1 M in THF) (1.6 mL, 5.6 mmol) and acetic acid (50 lL,
0.87 mmol) and the mixture was stirred at ambient temperature
for 12 h. To the reaction mixture was added additional TBAF
(0.2 mL, 0.70 mmol) at ambient temperature. Then the solvent was
concentrated in vacuo and the crude product was extracted with
ethylacetate. Theorganicphasewaswashedwithsaturatedaqueous
NaHCO3, H2O and brine, and dried over anhydrous MgSO4. The sol-
vent was concentrated in vacuo and the crude residue was purified
by column chromatography on silica gel (CHCl3/methanol = 30:1)
to give 19 (190 mg, 0.359 mmol, 98%) as an orange oil. The NMR
spectra showed the rotameric mixture due to high rotation barriers
4.1.4. 2-(tert-Butyldiphenylsilyloxy)ethylmine (16)
To a solution of 2-aminoethanol 15 (1.06 g, 17.3 mmol) in dry
DMF (20 ml) was added t-butylchlorodiphenyl silane (4.58 g,
17.3 mmol) and imidazole (1.18 g, 17.4 mmol). The reaction mix-
ture was stirred at ambient temperature for 3 h. Then the solvent
was concentrated in vacuo. The residue was diluted with CHCl3,
washed with saturated aqueous NaHCO3, H2O and brine, and dried

2542
C. Dohno et al. / Bioorg. Med. Chem. 17 (2009) 2536–2543
caused by the tertiary amide bond of t-Boc protective group: 1H NMR
(400 MHz, CDCl3) (rotameric mixture) d 1.44 (s, 18H), 3.43 (4H),
3.58–3.72 (4H), 4.39–4.53 (4H), 7.36 (d, 4H, J = 7.56 Hz), 7.86 (d,
4H, J = 8.32); 13C NMR (150 MHz, CDCl3) (rotameric mixture) d
28.3, 49.1, 50.1, 50.9, 51.9, 61.3, 62.3, 80.3, 123.1, 127.6, 128.3,
141.4, 151.9, 157.3; HRMS (ESI) calcd for C28H40N4O6Na [(M+Na)+]
551.2845, found 551.2846.
ent temperature for 1 h. Then to the reaction mixture was added
sodium cyanotrihydroborate (7 mg, 0.11 mmol) and the reaction
mixture was stirred at ambient temperature for 6 h. To the reaction
mixture was added additional sodium cyanotrihydroborate (7 mg,
0.11 mmol) and the reaction mixture was stirred at ambient tem-
perature 18 h. Then the reaction mixture was extracted from
CHCl3, washed with brine, and dried over anhydrous MgSO4. The
solvent was concentrated in vacuo and the crude residue was puri-
fied by column chromatography on silica gel (CHCl3/MeOH = 5:1),
GPC (MeOH) and preparative TLC to give 1 (8.6 mg, 0.013 mmol,
14%). The hydrochloric salt of 1 was used for 1H and 13C NMR mea-
surements: 1H NMR (400 MHz, D2O) d 2.68 (s, 6H), 2.93 (t, 4H,
J = 6.0), 3.44 (t, 4H, J = 6.0), 4.34 (s, 4H), 7.50 (d, 2H, J = 8.5), 7.57
(s, 8H), 8.20 (d, 2H, J = 9.0), 8.41 (d, 2H, J = 9.4), 8.61 (d, 2H,
J = 8.3); 13C NMR (150 MHz, D2O) d 20.8, 33.2, 43.1, 51.1, 117.9,
120.6, 123.1, 124.0, 131.8, 135.0, 141.3, 146.6, 147.5, 152.6,
156.4, 160.4, 172.0; HRMS (ESI) calcd for C38H39N10O2 [(M+H)+]
667.3257, found 667.3253.
4.1.8. N-(7-Methyl-1,8-naphthyridin-2-yl)carbamic acid [1,2-
diazenediylbis [(4,1-phenylenemethylene)(tert-
butoxycarbonyl)amino-2,1-ethanediyl]] ester (21)
To a solution of 19 (37 mg, 0.07 mmol) in CH3CN (2.6 mL) was
added N,N0-dissuccinimidyl carbonate (DSC) (62 mg, 0.24 mmol)
and triethylamine (48 lL, 0.33 mmol) and the reaction mixture
was stirred at ambient temperature under argon for 3 h. To the
reaction mixture was added additional DSC (18 mg, 0.081 mmol)
diluted with CH3CN (0.5 mL). After 2 h stirring, the solvent was
concentrated in vacuo and the crude product was extracted with
ethyl acetate. The organic layer was washed with saturated aque-
ous NaHCO3 solution, H2O and brine, and dried over anhydrous
MgSO4. The solvent was concentrated in vacuo and the crude res-
idue was purified by column chromatography on silica gel (CHCl3/
methanol = 200:1) to give crude N-hydroxysuccinimidyl carobonic
ester 20 (45.2 mg). To a solution of 20 (44 mg, 0.054 mmol) in
CH2Cl2 (2 mL) was added 2-amino-7-methyl-1,8-naphthyridine
4.2. Measurements of UV–vis spectra
NCDA (6 lM) derivatives or NDA (6 lM) was dissolved in a so-
dium cacodylate buffer (10 mM, pH 7.0) containing NaCl
(100 mM). UV–vis absorption spectra was recorded on BECKMAN
COULTER DUÒ800 spectrometer using 1 cm path length cell at
ambient temperature before and after photoirradiation at 360 nm
for indicated time period.
12 (30 mg, 0.018 mmol) and triethylamine (34 lL, 0.23 mmol)
and the reaction mixture was stirred at ambient temperature for
1 day. Then the solvent was concentrated in vacuo and the crude
product was extracted with ethyl acetate. The organic layer was
washed with saturated aqueous NaHCO3, H2O and brine, and dried
over anhydrous MgSO4. The solvent was concentrated in vacuo and
the crude residue was purified by column chromatography on sil-
ica gel (from CHCl3 100% to CHCl3/methanol = 100:1) to give 21
(34 mg, 0.037 mmol, 56% 2 steps) as an orange solid. The NMR
spectra showed the rotameric mixture due to high rotation barriers
caused by the tertiary amide bond of t-Boc protective group: 1H
NMR (400 MHz, CDCl3) (rotameric mixture) d 1.34ꢁ1.50 (18H),
2.72 (s, 6H), 3.50–3.61 (4H), 4.28–4.35 (4H), 4.55–4.59 (4H),
7.20–7.22 (2H), 7.32 (4H), 7.75 (4H), 7.93 (4H), 7.98 (2H), 8.07
(2H), 8.18–8.20 (2H); 13C NMR (150 MHz, CDCl3) (rotameric mix-
ture) d 25.5, 28.3, 46.1, 46.4, 50.7, 51.7, 63.5, 63.8, 80.5, 112.4,
117.9, 121.3, 127.5, 128.2, 136.3, 139.0, 141.0, 141.3, 151.7,
152.6, 153.0, 154.5, 155.7, 163.2; HRMS (ESI) calcd for
C48H54N10O8Na [(M+Na)+] 921.4023, found 921.4021.
4.3. HPLC analysis
A solution (total volume 100
derivatives or NDA (40 M) in
l
L) containing NCDA (80
lM)
l
a sodium cacodylate buffer
(10 mM, pH 7.0) containing NaCl (100 mM) was irradiated using
an ASAHI SPECTRA LAX-102 equipped with 360 nm band path filter
at a distance of 10 cm. After photoirradiation for 5 min, the sample
solution was analyzed by reverse-phase HPLC on a GILSON system
(805 MANOMETRIC MODULE, 811C DYNAMIC MIXER, 305 PUMP,
306 PUMP, 118 UV–vis DETECTOR, Nacalai tesque COSMOSIL
packed column (4.6 ꢂ 150 mm)) detected at 260 nm; elution with
a solvent mixture of 0.1 M triethylammonium acetate (pH 7.0), 5–
25% acetonitrile/20 min, and 25–85%/20–60 min at a flow rate
1.0 mL/min.
4.4. Measurements of melting temperature (Tm) of DNA
duplexes
4.1.9. Hydrochloride salt of N-(7-methyl-1,8-naphthyridin-2-
yl)carbamic acid [1,2-diazenediylbis(4,1-
The sample solution were prepared by mixing DNA duplex
phenylenemethyleneimino-2,1-ethanediyl)] ester (2, NCDA2)
To a solution of 21 (32 mg, 0.036 mmol) in CHCl3 (6 mL) was
added ethyl acetate containing 4 M HCl (6 mL) and the reaction
mixture was stirred at room temperature for 2.5 h. The solvent
was concentrated in vacuo and freeze-dried to give 2 (quantitative
yield) as an orange solid: 1H NMR (600 MHz, D2O) d 2.73 (s, 9H),
3.53 (br, 4H), 4.38 (br, 4H), 4.40 (s, 4H), 7.31 (d, 2H, J = 8.4), 7.36
(d, 4H, J = 8.1), 7.57 (d, 4H, J = 8.4), 7.88 (d, 2H, J = 8.8), 8.27 (d,
2H, J = 8.7), 8.35 (d, 2H, J = 8.4); 13C NMR (150 MHz, D2O) d 20.6,
45.5, 50.8, 61.9, 116.6, 119.8, 122.4, 123.9, 132.4, 134.6, 140.9,
146.3, 147.2, 151.8, 153.1, 156.6, 159.5; HRMS (ESI) calcd for
C38H38N10O4Na [(M+Na)+] 721.2975, found 721.2977.
(4.5 lM) and 18.2 lM NCDA or NDA in 10 mM sodium cacodylate
buffer (pH 7.0), and 100 mM NaCl. Thermal denaturation profiles
were recorded on a SHIMADZU UV-2550 UV–vis spectrometer
equipped with a SHIMADZU TMSPC-8 temperature controller.
The absorbance of the samples were monitored at 260 nm from
2 °C to 80 °C with heating rate of 1 °C/min. The measurements
were carried out before and after photoirradiation (360 nm,
5 min). The Tm values were determined as the temperature cross-
ing the melting curve and the median of two straight lines drawn
for the single and duplex region in the melting curve.
4.5. CSI-TOF-MS measurements of ligand/DNA complex
4.1.10. N-(7-Methyl-1,8-naphthyridin-2-yl)-3,30-[1,2-
diazenediylbis(4,1-phenylenemethyleneimino)]dipropanamide
(1, NDA)
Samples were prepared by mixing DNA duplex (20
ACG GAA TG)/d(CAT TCG GTT AG)) and NCDA or NDA (0–60
in 50% methanol in water containing 100 mM ammonium acetate.
Mass spectra were obtained with a JEOL AccuTOF JMS-T100 N mass
spectrometer in the negative ion mode (orifice 1 voltage = ꢁ60 V).
Spray temperature was fixed at ꢁ10 °C with a sample flow rate of
l
M, d(CTA
M)
l
To a solution of 10 (41 mg, 0.18 mmol) and 4,40-formylazoben-
zene 11 (21 mg, 0.090 mmol) in CHCl3/CH3OH = 2:1 (9 ml) was
added acetic acid (pH 5). The reaction mixture was stirred at ambi-

C. Dohno et al. / Bioorg. Med. Chem. 17 (2009) 2536–2543
2543
2. (a) Asanuma, H.; Ito, T.; Yoshida, T.; Liang, X.; Komiyama, M. Angew. Chem., Int.
Ed. 1999, 38, 2393; (b) Asanuma, H.; Liang, X.; Yoshida, T.; Komiyama, M.
ChemBioChem. 2001, 2, 39.
10 lL/min. Nitrogen gas was used as a desolvation gas as well as a
nebulizer.
3. Lewis, F. D.; Liu, X. J. Am. Chem. Soc. 1999, 121, 11928.
4. Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S. G.; Jacobson, J. M.
Nature 2002, 415, 152.
4.6. Calculation of structures
5. (a) Horsey, I.; Krishnan-Ghosh, Y.; Balasubramanian, S. Chem. Commun. 2002,
1950; (b) Bianké, H.; Häner, R. ChemBioChem. 2004, 5, 1063; (c) Göritz, M.;
Krämer, R. J. Am. Chem. Soc. 2005, 127, 18016.
Molecular modeling simulation was carried out with MacroMod-
el 9.1 with an AMBER* force field. Initial structures were constructed
manually from the NMR-structure we determined previously.9b En-
ergy minimization was done for the initial structures of the complex
with Generalized-Born/Surface-Area (GB/SA) solvation treatment of
water. Solvent accessible surface area (ASA) was calculated with a
probe molecule of radius of 1.4 Å (water) over van der Waals surface.
Solvent accessible surfaces of the ligands in the complex were ob-
tained from the calculation of ASAL ꢁ (ASAD + ASAL ꢁ ASADL)/2,
where the solvent accessible surface area of the entire complex,
the DNA duplex, and ligand extracted from the complex are repre-
sented by ASADL, ASAD, and ASAL, respectively. For conformational
search, the Monte Carlo Multiple Minimum (MCMM) search proto-
col was used to generate an initial family of 2000 conformers of
the each ligands. Each conformer was energy-minimized using the
6. (a) Höbartner, C.; Mittendorfer, H.; Breuker, K.; Micura, R. Angew. Chem., Int. Ed.
2004, 43, 3922; (b) Höbartner, C.; Silverman, S. K. Angew. Chem., Int. Ed. 2005,
44, 7305; (c) Ghosn, B.; Haselton, F. R.; Gee, K. R.; Monroe, W. T. Photochem.
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*
AMBER force field in water.
Acknowledgments
This work was supported by Grant in Aid for Scientific Research
(S) (18105006) for K.N. and Grant in Aid for Young Scientists (B)
(18710187) for CD from the Japan Society for the Promotion of
Science.
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References and notes
1. Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900.