Large stabilization of a DNA duplex by the deoxyadenosine derivatives tethering an aromatic hydrocarbon group

Article abstract of DOI:10.1021/ja034465b

Novel deoxyadenosine derivatives tethering a phenyl or naphthyl group by means of an amido linker have been synthesized, and these derivatives, stacking on the 5′ end of a DNA duplex, provide free energy contributions equal to or greater than that of the

Full text of DOI:10.1021/ja034465b

Published on Web 06/14/2003
Large Stabilization of a DNA Duplex by the Deoxyadenosine Derivatives
Tethering an Aromatic Hydrocarbon Group
Shu-ichi Nakano,† Yuuki Uotani,‡ Shoji Nakashima,†,| Yosuke Anno,§ Masayuki Fujii,§ and
Naoki Sugimoto*,†,‡
High Technology Research Center and Department of Chemistry, Faculty of Science and Engineering,
Konan UniVersity, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan, and Department of Chemistry,
Kyushu School of Engineering, Kinki UniVersity, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555, Japan
Received February 3, 2003; E-mail: sugimoto@konan-u.ac.jp
Scheme 1
Complementary nucleotide strands can self-assemble in water
in the presence of appreciated cations, forming a duplex through
stacking of bases as well as hydrogen bonding. Duplexes with a
dangling end are widely used to estimate the energy of the stacking
interaction,1,2 because it is believed that the energy contribution
from the dangling end results from the stacking interaction between
the dangling end residue and the bases of a neighboring base pair.
For DNA, stabilization of the core duplex by a single dangling
end with free energy contributions from 0.48 to -0.96 kcal mol-1
depending on the base type, neighboring base pair, and position of
the dangle (5′ or 3′ end) has been reported, and adenine dangling
ends are more or equally as stabilizing as other dangling ends.2 To
understand the factors contributing to stacking and to develop
nucleotides with enhanced properties, nonnatural residues with an
aromatic hydrocarbon group as a single dangling end have been
examined.3 Nevertheless, it is difficult to develop nonnatural
aromatic residues that can adopt a conformation suitable for stacking
with neighboring bases, and increasing the size of the aromatic
hydrocarbon eliminates solubility in water. We report here the
synthesis and thermodynamic properties of deoxyadenosine deriva-
tives tethering a phenyl or naphthyl group by means of an amido
linker (Scheme 1). The presence of a linker that covalently connects
deoxyadenosine with an aromatic hydrocarbon group allows adop-
tion of a conformation suitable for stacking on the end of a DNA
duplex, providing free energy contributions equal to or greater than
the Watson-Crick A/T base pair (Scheme 2).
Synthesis of the phenylurea and naphthylurea derivatives of
deoxyadenosine was started with 2′-deoxyadenosine (see Supporting
Information).4 Preparation of the oligonucleotides was carried out
as was previously reported,5 and all oligonucleotides were confirmed
by MALDI-TOF MS (PE Biosystems Voyager). To evaluate the
ability of the deoxyadenosine derivatives to stack on the end of a
DNA duplex, we measured the thermodynamic parameters for the
formation of DNA duplexes with 5′ or 3′ dangling ends by a self-
complementary oligonucleotide (Table 1). All of the duplexes in
Table 1 except (AATGCGCATT)2 showed a two-state transition
in thermal melting (see Supporting Information).6 As is shown in
Table 1, the single phenylurea or naphthylurea derivative of
deoxyadenosine at 5′ ends next to a 5′A/3′T base pair enhanced
the core duplex stability by 1.8 and 2.2 kcal mol-1 in ∆G°37,
respectively, greater than an adenine dangling end (0.9 kcal mol-1).
These stabilization energies were comparable to the free energy
for the terminal A/T base pair formations of (AATGCGCATT)2
Scheme 2
(-∆∆G°37 ) 1.9 kcal mol-1). Intriguingly, the stabilization by the
aromatic hydrocarbon groups originates from the entropy term,
implying preorganization of the DNA strand in the single-stranded
state7 or contributions from desolvation. The deoxyadenosine
derivatives at 5′ ends of (TGCGCA)2 also increased the duplex
stability by 2.9 and 3.3 kcal mol-1, respectively, which were greater
than these derivatives next to 5′A/3′T. It is noteworthy that these
free energy contributions were much larger than the terminal A/T
base pair formations of (ATGCGCAT)2 (-∆∆G°37 ) 1.7 kcal
mol-1). Because the prediction parameters for an adenine dangling
end on 5′A/3′T and 5′T/3′A base pairs are the same (-0.51 and
-0.50 kcal mol-1 in ∆G°37, respectively),2 the phenyl and naphthyl
groups next to 5′T/3′A interact more efficiently than the same
groups next to 5′A/3′T. Addition of a second dangling residue to
the 5′ dangling nucleotide increased the duplex stability, while such
stabilization was not observed for an adenine dangling end.8 The
duplex with the highest stability in Table 1 is (AZATGCGCAT)2,
which provides a ∆G°37 that is 4.3 kcal mol-1 greater than that of
(ATGCGCAT)2. In contrast, a single phenylurea derivative of
deoxyadenosine at 3′ ends enhanced the duplex stability to the same
degree as adenine (0.7-0.8 kcal mol-1), and the second dangling
residue did not increase stability. This observation demonstrates
the lesser ability of the phenyl group to stack on the 3′ end of the
duplex. On the other hand, stabilization by a single naphthylurea
derivative of deoxyadenosine at 3′ ends was slightly greater (-1.0
kcal mol-1), and the second dangling residue increased the duplex
stability. As compared to the XX or ZZ dangling end, a smaller
stabilization energy was provided by the addition of an adenine to
the 3′ X or Z dangling end, respectively, demonstrating the stacking
interaction between the hydrocarbon groups in the XX and ZZ
dangling end. This observation is different from that at the 5′ end,
suggesting the position-dependent manner. Table 1 also shows that
the stabilities of (XTGCGCAT)2 and (ZTGCGCAT)2 are 0.5-0.6
kcal mol-1 greater than that of (ATGCGCAT)2. The results imply
† High Technology Research Center, Konan University.
‡ Department of Chemistry, Faculty of Science and Engineering, Konan
University.
§ Kinki University.
| Present address: National Institute of Radiological Sciences, 4-9-1, Anagawa,
Inage-ku, Chiba, 263-8555, Japan.
9
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J. AM. CHEM. SOC. 2003, 125, 8086-8087
10.1021/ja034465b CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
end residue increased the DNA duplex stability up to 2.2 kcal mol-1
per modification, which is greater than an increase in stability
reported for pyrene as a DNA base of the 5′ dangling end (1.7
kcal mol-1 per modification).3a The deoxyadenosine derivatives at
helical termini providing large stabilization energy may be useful
for stabilizing a hybridization with designed DNA. Also, because
the configuration of the amido linker determines the position of
the aromatic hydrocarbon group mediated by noncovalent interac-
tions, the derivatives might also be applied as an environmental
response material.
Table 1. Thermodynamic Parameters for the Self-Complementary
Duplexesa
b
c
∆H°
∆S°
∆G°37
∆∆G°37
(kcal mol-1
Tm
sequence
(kcal mol-1
)
(kcal mol-1
)
(kcal mol-1
)
)
(°C)
ATGCGCATd
AATGCGCATTe
ATGCGCAA
-62.0
-75.8
-49.1
-64.6
-65.0
-53.9
-63.5
-71.1
-56.8
-64.9
-70.4
-63.1
-64.0
-56.2
-60.5
-66.2
-53.1
-64.3
-59.7
-47.7
-55.7
-47.6
-55.7
-44.4
-171
-208
-131
-175
-176
-138
-165
-187
-146
-168
-183
-171
-173
-148
-161
-181
-138
-166
-156
-129
-145
-122
-144
-111
-9.3
-11.2
-8.5
0
54.3
61.2
56.3
60.9
61.3
72.5
72.8
72.5
72.7
76.2
76.9
60.1
61.3
64.1
64.8
59.5
66.8
74.8
70.1
50.3
67.0
67.0
69.7
70.0
-1.9
0.8
AATGCGCATd
AAATGCGCATd
XATGCGCAT
XXATGCGCAT
AXATGCGCAT
ZATGCGCAT
ZZATGCGCAT
AZATGCGCAT
ATGCGCATAd
ATGCGCATAAd
ATGCGCATX
ATGCGCATXX
ATGCGCATXA
ATGCGCATZ
ATGCGCATZZ
ATGCGCATZA
TGCGCA
-10.2
-10.3
-11.1
-12.2
-12.9
-11.5
-12.9
-13.6
-10.0
-10.3
-10.1
-10.6
-10.1
-10.3
-12.6
-11.4
-7.6
-0.9
-1.0
-1.8
-2.9
-3.6
-2.2
-3.6
-4.3
-0.7
-1.0
-0.8
-1.3
-0.8
-1.0
-3.3
-2.1
1.7
Acknowledgment. This work was supported in part by Grants-
in-Aid for Scientific Research from the Ministry of Education,
Science, Sports and Culture, Japan.
Supporting Information Available: Synthetic procedures of the
phenylurea and naphthylurea derivatives of deoxyadenosine, their
calculated and measured mass data, and melting curves for the duplexes
with the 5′ single dangling end next to the 5′A/3′T base pair (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
XTGCGCA
XTGCGCAT
ZTGCGCA
ZTGCGCAT
-10.5
-9.8
-10.9
-9.9
-1.2
-0.5
-1.6
-0.6
References
(1) Sugimoto, N.; Kierzek, R.; Turner, D. H. Biochemistry 1987, 26, 6, 4554-
a The parameters were determined by Tm-1 versus log(Ct) plot and curve
fittings.8 The average errors in ∆H°, ∆S°, and ∆G°37 by the two methods
are (4.7%, (4.7%, and (3.4%, respectively. All experiments were done
in a buffer containing 1 M NaCl, 10 mM Na2HPO4, 1 mM EDTA at pH
7.0. Underline indicates the nucleotides not forming a canonical Watson-
Crick base pair. b ∆∆G°37 ) ∆G°37 - ∆G°37(ATGCGCAT). c Tm is
calculated at 100 µM. d Data are from ref 8. e Because accurate parameters
could not be obtained due to a non-two-state transition, the values indicated
are the predicted ones according to the nearest-neighbor parameters.5
4558.
(2) Bommarito, S.; Peyret, N.; SantaLucia, J., Jr. Nucleic Acids Res. 2000,
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(4) Protection of hydroxyl groups of 2′-deoxyadenosine was performed by
reaction with tert-butyldimethylsilyl chloride (TBDMS-Cl) to give the
bis-TBDMS derivative in 73% yield. The compound was reacted with
phenyl isocyanate at room temperature, and the phenylurea derivative was
obtained in 77% yield. Removal of the TBMDS groups on 3′ and 5′-
hydroxyl groups by treatment with tetrabutylammonium fluoride gave N6-
(N′-phenylcarbamoyl)-2′-deoxyadenosine (X) in 93% yield. Each com-
pound was purified by column chromatography on silica gel (CH2Cl2/
MeOH). Protection of the 5′-hydroxyl group of nucleoside X with a 4,4′-
dimethoxytrityl (DMT) group (89%) and phosphitilation of the 3′-hydroxyl
group with cyanoethyl-N,N,N′,N′-tetraisopropyl phosphoramidite (82%)
yielded the phosphoramidite derivative which can be readily used for
automated DNA synthesis.5,8,10 Synthesis of N6-(N′-naphthylcarbamoyl)-
2′-deoxyadenosine (Z) was achieved using the same process. Synthesis
of the phenyl and naphthyl derivatives was confirmed by identification
with 1H NMR (Varian INOVA 400 NMR) and ESI MS (Finnigan Mat
LCQ) (see Supporting Information).
Figure 1. Illustrations for the phenylurea derivative of deoxyadenosine
(black) stacking on the neighboring 5′A/3′T base pair (gray) at the 5′ end
(A) and 3′ end (B) of a B-DNA duplex.
that the X and Z pair with thymine in the duplexes. However, their
enthalpy terms (-47.6 and -44.4 kcal mol-1) are comparable to
those of (TGCGCA)2 and (ATGCGCAA)2. Obviously, more data
are required to confirm the base pair formation.
It is reported that the amido linker portion of the self-assembled
dendrimers by ureido-s-triazine derivatives forms a hydrogen bond
with an intramolecular ring nitrogen atom in the triazine group,
forming a planar configuration in water.9 In the same fashion, the
amido linker of the deoxyadenosine derivatives may interact with
N1 of adenine, and the aromatic hydrocarbon group stacks on the
interstrand base of a neighboring base pair. Assuming the config-
uration above, the phenylurea derivative may adopt a base pair-
mimic geometry as illustrated in Figure 1, that is consistent with
our thermodynamic observations. The phenyl group at the 5′ end
of B-DNA overlaps with the interstrand base of a neighboring base
pair, but there is less overlap at the 3′ end. With the naphthylurea
derivative of deoxyadenosine, stacking with the neighboring base
pair may be possible at the 3′ end as well as at the 5′ end. From
the configuration shown in Figure 1, we may also expect that the
stacking increases when the neighboring base pair is 5′T/3′A.
The base-pair mimic nucleotides developed here stabilized the
DNA duplex equally or more than Watson-Crick A/T base pairs.
Incorporation of the single deoxyadenosine derivative as a dangling
(5) Sugimoto, N.; Nakano, S.; Yoneyama, M.; Honda, K. Nucleic Acids Res.
1996, 24, 4501-4505.
(6) Melting experiments were conducted on Shimadzu 1650 and 1700
spectrophotometers, and the heating and cooling rates were 0.5 and 2 °C/
min, respectively. At ∼7 µM, the melting temperatures (Tm’s) of
(XATGCGCAT)2 and (ZATGCGCAT)2 monitored at 260 nm were 58.0
and 60.0 °C, respectively, both higher than that of the octanucleotide
duplex of (ATGCGCAT)2 (45.3 °C), and much higher than that of adenine
dangling ends of (AATGCGCAT)2 (48.3 °C). The melting curve for
(ZATGCGCAT)2 monitored at 325 nm revealed a decrease in absorption
when the duplex was dissociated, and its Tm was identical to that at 260
nm. These observations imply that the aromatic hydrocarbon group stacks
efficiently on the 5′ end of the duplex and the dangling residues are not
frayed before the cooperative transition. The non-two-state transition for
(AATGCGCATT)2 might have originated from a hairpin-loop formation.
(7) Kool, E. T. Chem. ReV. 1997, 97, 1473-1487.
(8) Ohmichi, T.; Nakano, S.; Miyoshi, D.; Sugimoto, N. J. Am. Chem. Soc.
2002, 124, 10367-10372.
(9) Brunsveld, L.; Vekemans, J. A.; Hirschberg, J. H.; Sijbesma, R. P.; Meijer,
E. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4977-4982.
(10) Sugimoto, N.; Nakano, M.; Nakano, S. Biochemistry 2000, 39,
11270-11281.
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