Molecular design, chemical synthesis, and evaluation of cytosine-carbohydrate hybrids for selective recognition of a single guanine bulged duplex DNA

Article abstract of DOI:10.1016/j.bmcl.2005.06.073

The designed cytosine-carbohydrate hybrid molecule selectively recognized and stabilized the bulged duplex DNA possessing the complementary bulged DNA base, guanine, while the nucleotide base itself did not exhibit any such ability. It was also found that

Full text of DOI:10.1016/j.bmcl.2005.06.073

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4332–4335
Molecular design, chemical synthesis, and evaluation of
cytosine–carbohydrate hybrids for selective recognition of a
single guanine bulged duplex DNA
Yusuke Idutsu, Ayaka Sasaki, Shuichi Matsumura and Kazunobu Toshima*
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi,
Kohoku-ku, Yokohama 223-8522, Japan
Received 17 May 2005; revised 11 June 2005; accepted 14 June 2005
Available online 2 August 2005
Abstract—The designed cytosine–carbohydrate hybrid molecule selectively recognized and stabilized the bulged duplex DNA pos-
sessing the complementary bulged DNA base, guanine, while the nucleotide base itself did not exhibit any such ability. It was also
found that the assistance of the carbohydrate to stabilize the interaction between the nucleotide base and the complementally bulge
DNA base is very helpful for the selective recognition and stabilization of the single-bulged duplex DNA.
Ó 2005 Elsevier Ltd. All rights reserved.
A DNA bulge is the site where one or more extra nucle-
otide bases remain unpaired in the duplex DNA.1 Sin-
gle-base bulges arising from the slippage of DNA can
result in mutation hotspots due to DNA strand mis-
alignment during replication.2 This irregular DNA site
is considered to play an important role in the recogni-
tion by DNA repair proteins,3 and shows a unique reac-
tivity toward drug binding.4 The ligands selectively
binding to the bulged site have been acknowledged as
novel reagents for the chemical-typing of genetic disor-
ders, such as single nucleotide polymorphisms (SNPs).
A successful representative is 2-amino-1,8-naphthyri-
dine reported by Nakatani and Saito.5 However, a
chemical reagent or a device, which can selectively detect
and discriminate four bulged DNA sites (A, C, G, and
T-bulges), has never been reported.
that the deoxyamino sugar exhibited an affinity to the
duplex DNA with no site-specificity (Fig. 1). Based on
these observations, we expected that although a nucleo-
tide base itself could not stabilize a bulged duplex DNA
by the interaction with the complementary bulged DNA
base, a hybrid molecule constructed from a nucleotide
base and the deoxyamino sugar would selectively inter-
act with the complementary bulged DNA base, and sta-
bilize the bulged duplex DNA. In this case, standard
Watson–Crick type hydrogen bondings between the
nucleotide base and the complementary bulged DNA
base would induce the interaction with a base selectivity,
and the deoxyamino sugar would cause a nonspecific
affinity to the duplex DNA and assist in the selective
interaction between the nucleotide base and the comple-
mentary bulged DNA base. These effects would be very
useful for the selective recognition of a bulged duplex
DNA with a specific interaction (Fig. 2). Furthermore,
In our previous studies on artificial DNA interactive
small molecules, we have found that the artificial hybrid
molecules consisting of an intercalator and a deoxy-
amino sugar strongly interacted with a duplex DNA,6
because the deoxyamino sugar functioned as a DNA
groove binder, and significantly enhanced the intercalat-
ing ability of the intercalator. In addition, it was found
R
Strong and nonspecific
Me
Interaction
O
HO
Me2N
O
DNA intercalator
Deoxyamino sugar
intercalator-carbohydrate hybrid
Keywords: DNA; Bulge; Nucleotide base; Carbohydrate; Molecular
recognition.
Duplex DNA
*
Figure 1. Interaction between intercalator–carbohydrate hybrid and
duplex DNA.
Corresponding author. Tel.: +81 45 566 1576; fax: +81 45 566
1576; e-mail: toshima@applc.keio.ac.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.073

Y. Idutsu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4332–4335
4333
H
NH2
NHTMS
For example
Me
a
N
H
H
O
N
HN
N
N
N
N
N
N G
C
N
O
deoxyribose
HO
O
OTMS
N
Me2N
N
H
O
H
3
6
NH2
Me
Me
RO
Me2N
O
O
BzO
Specific interaction
N
N
N
N
N
N
Me2N
OAc
b
Bulged DNA base
Me
5
O
O
HO
7: R=Bz
1: R=H
c
Me2N
N
N
N
N
DNA base
Deoxyamino sugar
Scheme 1. Synthesis of 1. Regents and conditions: (a) HMDS, TMSCl,
reflux, 6 h, 93%; (b) TMSOTf, ClCH2CH2Cl, reflux, 2 h, 74%; (c)
NaOMe, MeOH, 60 °C, 1.5 h, 87%.
Nucleotide base-carbohydrate hybrid
Bulged duplex DNA
Figure 2. Interaction between nucleotide base–carbohydrate hybrid
and bulged duplex DNA.
smoothly coupled to the trimethylsilylated cytosine 6,8
prepared from cytosine (3), hexamethyldisilazane
(HMDS), and trimethylsilyl chloride (TMSCl), in the
presence of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) in ClCH2CH2Cl under reflux for 2 h to
give the cytosine glycoside 7 in 74% yield with a very
high b-stereoselectivity. Deprotection of the benzoyl
group in 7 using NaOMe in MeOH at 60 °C for 1.5 h
furnished the desired hybrid 1 in 87% yield. Another
hybrid 2 was also obtained by a similar procedure involv-
ing a glycosidation reaction using ethyleneglycol.9,10
if this new concept that a hybrid molecule consisting of a
nucleotide base and a nonspecific binder, such as a
deoxyamino sugar, could selectively interact with the
only complementary bulged DNA base and then stabi-
lize the bulged duplex DNA, all four kinds of single
bulged duplex DNAs would be selectively detected by
the expanded methods using four nucleotide bases based
on this concept. In this communication, we report that
the designed cytosine–carbohydrate hybrid selectively
recognized the guanine bulged DNA base and stabilized
the guanine bulged duplex DNA.
With the designed cytosine–carbohydrate hybrids 1 and
2 in hand, base selective stabilization of the bulged
duplex DNA was examined by measuring the melting
temperature (Tm) of Nakatani and SaitoÕs four duplex
To confirm our hypothesis, we newly synthesized two
cytosine–carbohydrate hybrids 1 and 2, and chose cyto-
sine (3) and cytidine (4) as the reference materials in this
preliminary study (Fig. 3). The designed hybrid 1 pos-
sesses a hybrid structure in which cytosine and the
deoxyamino sugar are directly connected to each other,
while the artificial 2 has another hybrid structure in
which cytosine and the deoxyamino sugar are linked
through a C2 linker, –OCH2CH2–. These hybrids are dif-
ferent in terms of the distance between cytosine and the
deoxyamino sugar. Needless to say, cytosine (3) is a
nucleotide base itself without possessing a carbohydrate,
and cytidine (4) possesses another carbohydrate, ribose,
instead of the deoxyamino sugar found in 1 and 2.
DNAs
(4.77 lM),
d(TCCAGGCAAC)/d(GTTGC
XCTGGA) involving a single nucleotide bulge (X = A,
C, G, or T), in the presence of each molecule
(5.00 lM) in sodium cacodylate buffer (10 mM,
pH 7.0) containing NaCl (100 mM).5a,b The measure-
ments were carried out at least three times and these re-
sults are summarized in Table 1. In the case of 1, the Tm
was significantly increased by 2.6 °C for the G bulged
duplex DNA, d(TCCAGGCAAC)/d(GTTGCGCTGGA).
However, in drastic contrast, no increase in the Tm was
observed for the A, C, and T bulged duplex DNAs as
well as for the fully complementary duplex DNA,
d(TCCAGGCAAC)/d(GTTGCCTGGA). These results
clearly indicated that the hybrid 1 selectively stabilized
only the G bulged duplex DNA. Furthermore, the 1:1
ratio of hybrid 1 and the G bulged duplex DNA was suf-
ficient to observe the detectable DTm. To obtain more
information on the structure and activity relationship
for stabilizing the G bulged duplex DNA, more Tm mea-
surements were conducted in the presence of the other
compounds 2–4. The DTm values in the presence of these
compounds were 0.9, 0.5, and 0.3 °C for 2, 3, and 4,
respectively, in sharp contrast to the DTm of 2.6 °C in
the presence of hybrid 1. These results showed that the
hybrid structure consisting of the cytosine and the
deoxyamino sugar is indispensable for stabilizing the
G bulged DNA, and the deoxyamino sugar could not
be replaced by another sugar, ribose, for this purpose.
Both the suitably hydrophobic nature11 of the deoxy-
amino sugar and the N,N-dimethyl group positively
charged under neutral conditions must be very helpful
The chemical synthesis of the hybrid 1 is outlined in
Scheme 1. Thus, the known glycosyl acetate 57 was
NH2
Me
NH2
Me
O
O
HO
O
O
HO
N
N
Me2N
N
N
Me2N
1
O
N
2
O
NH2
N
NH2
HO
N
O
HN
O
HO OH
Figure 3. Nucleotide base–carbohydrate hybrids 1 and 2, cytosine (3),
and cytidine (4).

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Y. Idutsu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4332–4335
Table 1. Melting temperature (Tm) of bulged-containing duplex DNAs in the presence and absence of compounda
Duplex DNA
Tm(ꢀ)
32.0
Compound
Tm(+)
2Tm
2.6
50-TCCAG_GCAAC-30
30-AGGTCGCGTTG-50
1
34.6
2
3
4
1
32.9
32.5
32.3
31.9
0.9
0.5
0.3
ꢀ0.3
50-TCCAG_GCAAC-30
30-AGGTCACGTTG-50
50-TCCAG_GCAAC-30
30-AGGTCCCGTTG-50
50-TCCAG_GCAAC-30
30-AGGTCTCGTTG-50
50-TCCAGGCAAC-30
30-AGGTCCGTTG-50
32.2
33.6
31.1
45.3
1
1
1
33.5
30.8
44.9
ꢀ0.1
ꢀ0.3
ꢀ0.4
a Measurement of the melting temperature was conducted using bulged duplex DNA (4.77 lM, strand concentration) and compound (5.00 lM). The
absorbance at 260 nm was measured in sodium cacodylate buffer (10 mM, 7.0) containing NaCl (100 mM). Temperature was increased at a rate of
1 °C/min from 15 to 60 °C. Measurements were carried out at least three times.
3. (a) Wang, Y.-H.; Bortner, C. D.; Griffith, J. J. Biol. Chem.
1993, 268, 17571; (b) Malkov, V. A.; Biswas, I.; Camerini-
Otero, R. D.; Hsieh, P. J. Biol. Chem. 1997, 272, 23811.
4. (a) Nelson, J. W.; Tinoco, I., Jr. Biochemistry 1985, 24,
6416; (b) White, S. A.; Draper, D. E. Nucleic Acids Res.
1987, 15, 4049; (c) Woodson, S. A.; Crothers, D. M.
Biochemistry 1988, 27, 8904; (d) Williams, L. D.; Gold-
berg, I. H. Biochemistry 1988, 27, 3004; (e) Cortes, C. J.;
Wang, H.-J. Biochemistry 1996, 35, 616; (f) Nakatani, K.;
Okamoto, A.; Saito, I. Angew. Chem., Int. Ed. 1999, 38,
3378; (g) Xi, Z.; Hwang, G.-S.; Goldberg, I. H.; Harris, J.
L.; Pennington, W. T.; Fouad, F. S.; Qabaja, G.; Wright,
J. M.; Jones, G. B. Chem. Biol. 2002, 9, 925; (h) Colgrave,
M. L.; Williams, H. E. L.; Searle, M. S. Angew. Chem., Int.
Ed. 2002, 41, 4754.
5. (a) Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. Soc.
2000, 122, 2172; (b) Nakatani, K.; Horie, S.; Murase, T.;
Hagihara, S.; Saito, I. Bioorg. Med. Chem. 2003, 11, 2347;
(c) Nakatani, K.; Horie, S.; Saito, I. J. Am. Chem. Soc.
2003, 125, 8972.
6. (a) Toshima, K.; Ouchi, H.; Okazaki, Y.; Kano, T.;
Moriguchi, M.; Asai, A.; Matusumura, S. Angew. Chem.,
Int. Ed. 1997, 36, 2748; (b) Toshima, K.; Takano, R.;
Maeda, Y.; Suzuki, M.; Asai, A.; Matsumura, S. Angew.
Chem., Int. Ed. 1999, 38, 3733; (c) Toshima, K.; Maeda,
Y.; Ouchi, H.; Asai, A.; Matsumura, S. Bioorg. Med.
Chem. Lett. 2000, 10, 2163; (d) Toshima, K.; Okuno, Y.;
Nakajima, Y.; Matsumura, S. Bioorg. Med. Chem. Lett.
2002, 12, 671; (e) Toshima, K.; Takano, R.; Ozawa, T.;
Matsumura, S. Chem. Commun. 2002, 212.
when interacting with the duplex DNA and assist in the
selective interaction between the cytosine and the com-
plementary bulged DNA base, guanine.12 In addition,
the length between the cytosine and the deoxyamino su-
gar was also found to be very important for inducing
such an effect. The longer linker leading to the more flex-
ible structure of 2 probably lost the suitable fitting form
between 2 and the bulged DNA.
In summary, we demonstrated here, for the first time
that the cytosine–carbohydrate hybrid 1 produced a sta-
ble complex with a G bulged duplex DNA. Although the
DTm value of the G bulged DNA duplex in the presence
of hybrid 1 was not very high, the stabilization phenom-
enon was clearly detectable. The deoxyamino sugar was
essential and the distance from the cytosine was impor-
tant for the selective construction of such a thermody-
namically stable complex. This new strategy using a
hybrid molecule constructed from a nucleotide base
and a nonspecific binder, such as a carbohydrate, will
find wide application for the selective recognition of
other single nucleotide bulged duplex DNAs. To further
improve the ability of hybrid 1, the replacement of the
amino sugar in hybrid 1 with other types of amino sug-
ars and the attachment of oligosaccharides to cytosine
are now under investigation in our laboratories.
7. Toshima, K.; Matsuo, G.; Tatsuta, K. Tetrahedron Lett.
1992, 33, 2175.
Acknowledgments
8. Vorbruggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber.
1981, 114, 1256.
This research was partially supported by a Grant-in-Aid
for the 21st Century COE Program ÔKEIO Life Conju-
gate ChemistryÕ and a Grant-in-Aid for Scientific
Research (C) from the Ministry of Education, Culture,
Sports, Science, and Technology, Japan.
9. The synthesis of 2 will be reported in detail elsewhere.
10. 1H NMR (300 MHz, CD3OD): (d, SiMe4; J Hz) data for 1
and 2. Compound 1: d 1.33 (3H, d, J = 6.0), 1.62 (1H, ddd,
J = 12.0, 12.0, and 10.0), 2.08 (1H, ddd, J = 12.0, 4.0, and
2.0), 2.37 (6H, s), 2.78 (1H, ddd, J = 12.0, 9.0, and 4.0),
3.21 (1H, dd, J = 9.8 and 9.8), 3.47 (1H, dq, J = 9.8 and
6.0), 5.72 (1H, dd, J = 10.0 and 2.0), 5.91 (1H, d, J = 8.0),
7.70 (1H, d, J = 8.0). Compound 2: d 1.29 (3H, d, J = 6.0),
1.66 (1H, ddd, J = 12.0, 12.0, and 9.0), 2.23 (1H, ddd,
J = 12.0, 4.0, and 2.0), 2.75–2.90 (1H, m), 2.79 (3H, s),
2.86 (3H, s), 3.20–3.40 (3H, m), 3.67 (1H, dq, J = 9.8 and
6.0), 3.82–3.95 (1H, m), 4.0–4.10 (1H, m), 4.65 (1H, dd,
J = 9.0 and 2.0), 6.01 (1H, d, J = 8.0), 7.85 (1H, d,
J = 8.0).
References and notes
1. Turner, D. Curr. Opin. Struct. Biol. 1992, 2, 334.
2. (a) Streisinger, G.; Owen, J. Genetics 1985, 109, 633; (b)
Woodson, S. A.; Crothers, D. M. Biochemistry 1987, 26,
904; (c) Woodson, S. A.; Crothers, D. M. Biochemistry
1988, 27, 436.

Y. Idutsu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4332–4335
4335
11. (a) Walker, S.; Valentine, K. G.; Kahne, D. J. Am. Chem.
Soc. 1990, 112, 6428; (b) Ding, W.; Ellestad, G. A. J. Am.
Chem. Soc. 1991, 113, 6617; (c) Uesugi, M.; Sugiura, Y.
Biochemistry 1993, 32, 4622.
12. Nakatani and SaitoÕs group also reported a cytosine deriv-
ative5a possessing an amino group, –CH2CH2CH2NH2. The
DTm value (0.7°) was very similar to those of 2 and 3 and
lower than that of 1 under similar conditions.