Stabilization of DNA duplex by 2-substituted adenine as a minor groove modifier

Article abstract of DOI:10.1016/j.bmcl.2011.09.104

2-(1-Naphthalenylethynyl)-2′-deoxyadenosine (^NA) was synthesized and incorporated into oligodeoxynucleotides. DNA duplexes containing newly designed 5′-^NAT-3′/3′-T^NA- 5′ base pairs are considerably stabilized than unmodifi

Full text of DOI:10.1016/j.bmcl.2011.09.104

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7021–7024
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Bioorganic & Medicinal Chemistry Letters
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Stabilization of DNA duplex by 2-substituted adenine as a minor
groove modifier
Makiko Tanaka a,b, , Ryota Kozakai a,b, Yoshio Saito a, , Isao Saito b,
⇑
⇑
⇑
a Department of Chemical Biology and Applied Chemistry, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
b NEWCAT Institute, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
a r t i c l e i n f o
a b s t r a c t
2-(1-Naphthalenylethynyl)-20-deoxyadenosine (NA) was synthesized and incorporated into oligodeoxy-
nucleotides. DNA duplexes containing newly designed 50-NAT-30/30-TNA-50 base pairs are considerably
stabilized than unmodified duplexes by stacking interaction of naphthalene rings in the narrow minor
groove as characterized by a new emission at longer wavelength and exciton coupled CD signals.
Ó 2011 Elsevier Ltd. All rights reserved.
Article history:
Received 27 July 2011
Revised 24 September 2011
Accepted 27 September 2011
Available online 1 October 2011
Keywords:
DNA
Minor groove
Modified nucleoside
p-Stacking
Ethynyl linker
DNA has attracted considerable attention as a building block of
ous 5-substituted pyrimidine bases have been used for modified
DNA.6–12,22,23 To our knowledge, the accesses to minor groove by
appending an external molecule onto the base moiety of natural
nucleoside had so far been reported only for A-form RNA.24
We report herein that the modification of both complementary
strands upon tethering naphthalene to deoxyadenosine at the 2-
position though an ethynyl linker effectively lead to a stabilization
new nanomaterials for potential nanotechnological applications
because of their unique helical structures and properties.1–5 From
a supramolecular view point, DNA can be viewed as a versatile
scaffold for the attachment of functional molecules.6–16 In the field
of DNA electron transfer chemistry, double-helical DNA, which
consists of
p
-stacked four nucleobases, is known to provide an
ideal medium of electron transfer (ET) mediated by a
p
-stack.17,18
of the DNA duplex due to the p-stacking between naphthalene
Various chemical modifications of oligodeoxynucleotides (ODNs)
have been developed for the systematic investigation of ET process
though DNA containing electron donor and acceptors at given dis-
tances.19 Some studies have also been initiated to modify DNA
using base analogs20,21 in order to enhance ET efficiency for the
development of new DNA-based nanotechnological devices. How-
ever, the introduction of external molecules onto natural nucleo-
sides (A, T, G, and C) through a linker instead of base analogs
possessing different frameworks from natural nucleobases often
destabilizes or disturbs the original DNA duplex system to a certain
extent. The introduced parts should at least be placed in DNA
major or minor groove to maintain the helical structure and the
base pairing.14 Modification at 5-position of pyrimidine bases in
the major groove has been more common than that located in
the narrower minor groove due to a synthetic reason, and numer-
moieties in the minor groove without disturbing original B-form
DNA. A series of DNA duplexes synthesized for the system was
shown in Figure 1a. 2-(1-Naphthalenylethynyl)-20-deoxyadenosine
(NA) was prepared via palladium(0) mediated Sonogashira cross
coupling (Supplementary data). After protection of amino group
with N,N-dimethylformamide diethylacetal, protected NA was con-
verted to phosphoramidite, which was used for ODN synthesis by
automated DNA/RNA synthesizer. Aliquots of DNA duplex samples
were prepared by annealing equimolar amount of desired DNA
complements. The schematic illustration of
p-stacked zipper-like
block, 50-NAT-30/30-TNA-50 base pairs in DNA duplex is shown in
Figure 1b.
The UV–visible absorption and fluorescence spectra of naphtha-
lene-modified DNA assemblies are shown in Figure 2. The signifi-
cant red shift of absorption bands of naphthalene upon tethering
to DNA through an ethynyl linker results from a strong electron
coupling between deoxyadenosine and naphthalene.25 For du-
plexes I-c and -d, the absorption bands of naphthalene moieties
in the range from 300 to 380 nm showed significant change of
the ratio between the two maxima with a spectral shift compared
⇑
Corresponding authors. Tel.: +81 24 956 8923 (M.T.); tel.: +81 24 956 8809
(Y.S.); tel.: +81 24 956 8911; fax: +81 24 956 8924 (I.S.).
E-mail addresses: makikot@ao.ce.nihon-u.ac.jp (M. Tanaka), saitoy@chem.ce.
nihon-u.ac.jp (Y. Saito), saito@chem.ce.nihon-u.ac.jp (I. Saito).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.104

7022
M. Tanaka et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7021–7024
Figure 1. (a) Sequences of the naphthalene-modified ODNs synthesized in this study. (b) Schematic illustration of the base pairs, 50-NAT-30/30-TNA-50 in DNA duplex.
1.0
a
b
0.8
0.6
0.4
0.2
0
600
350
400
450
500
550
250
300
350
400
450
Wavelength, nm
Wavelength, nm
Figure 2. (a) Absorption spectra of ODN I-a (green line), I-b (black line), I-c (red line), and I-d (blue line); 2.0 lM duplex, 50 mM phosphate buffer, pH 7.0, 100 mM NaCl. (b)
Fluorescence spectra of ODN I-b (black line), I-c (red line), and I-d (blue line); 2.5
lM duplex, 50 mM phosphate buffer, pH 7.0, 100 mM NaCl. Excitation wavelength is
335 nm.
to the duplex I-b. This spectral change suggests the presence of
p–p interaction between two naphthalene moieties on the differ-
Table 1
ent strands. Fluorescence spectra of ODN I-b–d are shown in Figure
2b. The doubly modified duplex I-c exhibits an excimer-like
intense fluorescence that is 31 nm red-shifted with respect to the
singly modified duplex I-b. Duplex I-d showed a similar fluores-
cence maximum (kflꢀmax = 425 nm) as duplex I-c with a higher
intensity.26
The melting temperatures (Tm) of DNA assemblies obtained by
monitoring the characteristic DNA duplex absorption at 260 nm
are given in Table 1 together with their wavelength of fluorescence
maxima. The doubly modified duplex I-c was found to be more
Melting temperatures (Tm
) and wavelength of fluorescence maxima for DNA
duplexesa
Duplex
Tm (°C)
D
Tm (°C)
kflꢀmax (nm)
I-a
I-b
I-c
I-d
II-a
II-c
45.3
42.6
50.8
47.6
46.6
39.3
—
—
ꢁ2.7
+5.5
+2.3
—
394
425
425
—
ꢁ7.3
—
a
Experimental conditions: [DNA duplex] = 2.0
lM (for Tm mesuaurements), or
stable (
DTm = +5.5 °C) than unmodified duplex I-a. The Tm value
2.5 lM (for fluorescence spectra mesurements) in 50 mM sodium phosphate, pH
7.0, 100 mM NaCl.
of duplex I-d possessing four NA in total was also higher than that

M. Tanaka et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7021–7024
7023
of the unmodified duplex (
D
Tm = +2.3 °C), although the Tm value
between naphthalenes in the narrow minor groove and indicates
was lower than that of duplex I-c. On the other hand, duplex II-c,
which has two modifications in the same strand was less stable
right-handed and
p-stacked arrangement of naphthalene
moieties.27
(
D
Tm = ꢁ7.3 °C) than the corresponding unmodified duplex. The
The calculated structures (MacroModel, Amber⁄) of naphtha-
lene-modified duplexes are shown in Figure 4. Naphthalene moie-
ties of one strand and the other strand are shown in blue and
green, respectively. A single naphthalene moiety was located in
the minor groove along DNA duplex in duplex I-b (Fig. 4a). It
should be noted that the single modification in the minor groove
increase in Tm values of DNA duplexes containing naphthalene-
modified deoxyadenosine in both strands (I-c and -d) suggests that
p-stacking between naphthalene moieties in the minor groove en-
hanced duplex stabilization.
The circular dichroism (CD) spectra of duplexes at the wave-
length shorter than 300 nm indicated that the overall B-form
DNA double strand structures were retained (Fig. 3). On the other
hand, positive Cotton effects of I-c and -d in the region of
300–400 nm can be attributed to the strong exciton coupling
does not destabilize the DNA duplex to
a large extent
(D
Tm = ꢁ2.7 °C) as compared to the modification of 5-position of
pyrimidine base by 1-ethynylnaphthalene in the major groove.28
The second naphthalene moiety on the other strand of the duplex
I-c can form p–p stacking with the first one like a piece of zipper
(Fig. 4b), although the minor groove of duplex II-c does not seem
2
to provide enough space for two consecutive naphthalene moieties
in the same strand (Fig. 4d). The second p-stacked zipper-like block
1
by introduction of four NA can be fitted in the minor groove of DNA
(Fig. 4c). These results are consistent with the assumption that the
0
p-stacking between multiple naphthalenes in the narrow minor
groove of DNA results in an effective enhancement of duplex sta-
bility. The present study clearly indicated that introduction of
50-NAT-30/30-TNA-50 base pairs in duplex induced a remarkably
large thermal stability as characterized by an excimer-like new
emission at 425 nm.
In summary, we have devised a system for enhancing the stabil-
ity of DNA duplex using naphthalene-modified adenine–thymine
base pairs. Applications of chemically modified DNA have recently
attracted much attention for designing nanomaterials. Since sub-
stitutional functional groups may be easily introduced on naphtha-
lene moiety of the deoxyadenosine, such AT base pairs with
-1
-2
-3
250
300
350
400
Wavelength, nm
Figure 3. CD spectra of ODN I-a (green line), I-b (black line), I-c (red line), and I-d
(blue line); 2.5 M duplex, 50 mM phosphate buffer, pH 7.0, 100 mM NaCl.
l
Figure 4. Structures of (a) I-b, (b) I-c, (c) I-d, and (d) II-c calculated by MacroModel, Amber⁄.

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M. Tanaka et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7021–7024
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Acknowledgments
We are grateful for financial support from a Grant-in-Aid for
Scientific research of MEXT, Japanese Government. M. T. thanks
the Fumi Yamamura Memorial Foundation for Female Natural
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D
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