Specific induced circular dichroism and enhanced B to Z transitions of duplexes stabilized by chromophore-linked alkynylnucleoside residues

Article abstract of DOI:10.1002/chem.200902882

We have developed an induced circular dichroism (ICD) probe with a chromophore-linked alkynyldeoxyribose skeleton for analyzing higher-order structures of DNA duplexes in the visible-light region. When CG-repeated oligonucleotides (ODNs) with the probe at their 5' ends adopted Z-form duplexes at a high NaCl concentration, strong ICD signals were observed at the absorptive region of the chromophore. On the other hand, their B-form duplexes, formed at a low NaCl concentration, produced a faint ICD signal. The specific ICD for the Z-form duplexes was found to appear only when the chromophores were attached at the 5' ends of each of the ODNs. Furthermore, the chromophoric alkynylnucleoside residues effectively promoted the B to Z transition of the ODN.

Full text of DOI:10.1002/chem.200902882

FULL PAPER
DOI: 10.1002/chem.200902882
Specific Induced Circular Dichroism and Enhanced B to Z Transitions of
Duplexes Stabilized by Chromophore-Linked Alkynylnucleoside Residues
Kazuhisa Fujimoto,* Sayaka Aizawa, Ikko Oota, Junya Chiba, and Masahiko Inouye*[a]
Abstract: We have developed an in-
duced circular dichroism (ICD) probe
centration, strong ICD signals were ob-
served at the absorptive region of the
chromophore. On the other hand, their
B-form duplexes, formed at a low NaCl
concentration, produced a faint ICD
signal. The specific ICD for the Z-form
duplexes was found to appear only
when the chromophores were attached
at the 5’ ends of each of the ODNs.
Furthermore, the chromophoric alky-
nylnucleoside residues effectively pro-
moted the B to Z transition of the
ODN.
with
a chromophore-linked alkynyl-
deoxyribose skeleton for analyzing
higher-order structures of DNA du-
plexes in the visible-light region. When
CG-repeated oligonucleotides (ODNs)
with the probe at their 5’ ends adopted
Z-form duplexes at a high NaCl con-
Keywords: chromophore-labeled
DNA · circular dichroism · duplex
transitions · nucleosides
Introduction
between B- and Z-DNA duplexes at the visible-light
region.[4] For instance, Purrello et al. have recently reported
that anionic nickel(II) porphyrins selectively bind to Z-form
duplexes of poly-CG sequences in the presence of spermine
such that the B- and Z-form duplexes can be discriminated
by CD spectroscopy.[4a] Although this type of sensor mole-
cules based on intermolecular interactions are favorable for
detecting Z-form duplexes as they are, the sensors hardly
avoid the inherent problem that their detection abilities are
seriously dependent on their binding constants with DNA.
This situation may disturb the quantitative assessment for
the content of Z-form duplexes against B-form ones on the
basis of the CD signals. In addition, these exogenous sensor
molecules are likely to interfere with the interaction of Z-
DNAs with proteins.
Covalent labeling of ODNs with a chromophore at their
5’ and/or 3’ ends will surmount the problems mentioned
above and have the potential to assess the interaction of Z-
DNAs with proteins quantitatively.[5,6] Berova et al. have
used a tetraarylporphyrin derivative as an ODN-labeling
chromopore and have investigated the B to Z transition of
CG-repeated 8-mer ODN labeled with the chromophore by
CD spectroscopy.[5] Upon self-hybridization of the labeled
ODN, strong induced CD (ICD) signals appeared from the
porphyrin at a high NaCl concentration condition that pro-
motes Z-duplex formation, whereas moderate ICD signals
were observed at a low NaCl concentration. The two por-
phyrins attached at the 5’ ends of each of ODNs might com-
municate with each other through the p stacking, resulting
in an exciton-type ICD. They claimed that the labeled ODN
In nucleic acid chemistry, left-handed Z-DNAs are one of
the conundrums that confront chemists and biologists be-
cause their functions are still obscured in vivo.[1] Prior to the
exploration of their latent biological functions, their physio-
logical characteristics such as intermolecular interactions
with Z-DNA-binding proteins[2] should be comprehensively
studied at the oligonucleotide (ODN) level in vitro. Circular
dichroism (CD) spectroscopy works well for detecting Z-
DNA duplexes at their absorptive region, but this strategy is
inappropriate to analyze interactions of DNAs with proteins
owing to the overlap between DNA and protein absorptive
regions.[3] Therefore, discrimination between B- and Z-DNA
duplexes should be carried out at a visible-light region
where biological molecules scarcely absorb. This methodolo-
gy may allow the intermolecular interactions of Z-DNAs
with proteins to be easily detected.
With this in mind, several research groups have developed
chromophore-based sensor molecules that can discriminate
[a] Dr. K. Fujimoto, S. Aizawa, I. Oota, Dr. J. Chiba,
Prof. Dr. M. Inouye
Graduate School of Pharmaceutical Sciences
University of Toyama, Sugitani 2630
Toyama 930-0194 (Japan)
Fax : (+81)76-434-5049
E-mail: fujimoto@pha.u-toyama.ac.jp
inouye@pha.u-toyama.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902882.
Chem. Eur. J. 2010, 16, 2401 – 2406
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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predominantly adopts the Z-form duplex at a saturated
NaCl concentration on the basis of the CD spectral change.
However, in this case the content of the Z-form duplex
seems to be partial even at the saturated condition.[7] We
thought that the tetraarylporphyrin moieties on the duplex
structure may hinder the B to Z transition owing to their
large p planes. Taking these results into account, we de-
signed a chromophoric ICD probe 1 with an alkynyldeoxyri-
bose skeleton that has previously reported by us for analyz-
ing the B to Z transition of DNAs in the visible-light region.
Here we report strong ICDs that are specific for Z-form du-
plexes of the chromophore-labeled ODNs and promotion of
their B to Z transitions by the attached chromophores.
molar extinction coefficient (e) of the chromophore is high
enough to give an intense ICD signal at its absorptive
region. Indeed, e of the deprotected 3 is approximately
80000 molꢀ1 Lcmꢀ1 in a Tris-HCl buffer (100 mm, pH 7.0),
which is comparable to that of fluorescein. Second, the p-
plane size of its xanthene moiety is smaller than that of por-
phyrin, which leads to the expectation of a smooth B to Z
transition in the labeled ODNs.
Scheme 1 shows the synthesis of the probe 1 and the
ODN sequences used in this study. A xanthone derivative
4[9] was allowed to react with the mono-lithiated product of
2,5-dibromo-p-xylene to yield 5 as a basic chromophore
skeleton.[10] The cross-coupling reaction of 5 with the pub-
lished alkynyldeoxyribose 6[8] gave 7, which was protected
on the xanthene moiety by pivaloylation to provide the
probe 1. CG-repeated ODNs were modified with the phos-
phoramidite 2 at their ends with a DNA synthesizer. Five la-
beled ODNs, fa, fa’, fb, fc, and fd, were used in this study in
which the chromophore containing the ethynyl group is ab-
breviated as F.
Results and Discussion
The alkynyldeoxyribose-based ICD probe 1 (Piv=pivaloyl;
DMTr=4,4’-dimethoxytrityl) with a fluorescein-like chro-
mophore was converted into the phosphoramidite 2 and
then introduced into ODNs by solid-phase DNA synthesis.
The alkynyldeoxyribose skeleton has been previously devel-
oped by us and can be linked to various aromatic groups by
Sonogashira coupling reactions.[8] We expected that the
chromophore of 1 would be a superior reporter for duplex
analysis by CD spectroscopy for several reasons. First, the
Rich et al. have reported that
a
hairpin ODN,
d(CG)3T4(CG)3, can bind to a set of the two Za domains of
the human RNA editing enzyme, double-stranded RNA de-
aminase I (ADAR1).[2d] This research reveals the possibility
that left-handed short duplexes may play a role as the bind-
ing site to ADAR1 in a gene. Therefore, we measured the
CD spectra for the palindromic ODNn and ODNfa of 6-
mer to assess their B to Z transitions in the presence of
NaCl. The nonlabeled ODNn was found to be on its way to
the B to Z transition at 258C in the presence of NaCl (5.0m)
by comparing it with the CD spectra between 258C and 58C
(Figure S1 in the Supporting Information). For ODNfa in
the presence of NaCl (0.1m), CD bands of typical double-
stranded B-form duplexes were observed at the absorptive
region of nucleobases (Figure 1, left), whereas no meaning-
ful signals existed at ꢁ330 nm (Figure 1, right). In the pres-
ence of 5.0m NaCl, the characteristic positive and negative
CD signals for Z-form duplexes clearly appeared at 269 and
293 nm at 258C, respectively, accompanied with the disap-
pearance of the negative CD signal around 250 nm. This
Scheme 1. a) A synthetic scheme for the alkynyldeoxyribose-based ICD probe. b) The ODN sequences used in this study (F and M represent the residue
of 3 and 5-methylcytosine, respectively).
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Chem. Eur. J. 2010, 16, 2401 – 2406

Enhanced B to Z Transitions of Duplexes
FULL PAPER
Figure 1. CD spectra of ODNfa (20 mm) in a Tris-HCl buffer (100 mm,
pH 7.0) in the presence of NaCl (0.1–5.0m) at 258C (c=5.0, g=2.0,
and a=0.1m NaCl).
Figure 3. UV/Vis spectra of ODNfa (20 mm) in
(100 mm, pH 7.0) in the presence of NaCl (0.1–5.0m) at 258C
(c=5.0m, g=2.0m, and a=0.1m NaCl).
a Tris-HCl buffer
spectral change at the absorptive region of the nucleobases
means that the Z-form duplex of ODNfa exclusively exists
in the presence of 5.0m NaCl. Note that under these condi-
tions two new intense bands appeared at long wavelengths
with a plus-minus pattern near 530 and 480 nm (Figure 1,
right). The emerging bands are assigned to exciton-type
ICD between the two chromophoric residues of 1.[5,11] The
meaningless ICD signal in the B-form duplex was switched
to a strong one in the Z-form duplex. To correlate the inten-
sity of the ICD with the content of the Z-form duplex, the
spectral changes of the CD intensities at 529 nm and 293 nm
were both plotted as a function of NaCl concentration
(Figure 2). The two concentration-dependent transition
curves coincide well with each other. Therefore, the concen-
tration dependence of the ICD intensity was found to direct-
ly reflect the content of the Z-form duplex.
only two chromophoric species, probably the B- and Z-form
duplexes. The hypochromicity observed during the transition
is attributed to the enforced p stacking between the chro-
mophore and the nucleobase pairs in the Z-form duplex
compared with the B-form duplex. A conformational search
by using computational chemistry was performed for the B-
and Z-duplexes of ODNfa for reinforcing the contribution
of the p stacking to the hypochromicity. Figure 4 displays
Figure 4. Plausible structures for the Z- and B-duplexes of ODNfa. The
CPK models in these structures represent the p planes of the xanthene
moieties and the guanine bases at the 5’ and 3’ ends of ODNfa, respec-
tively.
plausible structures for the Z and B forms, which were opti-
mized in water without NaCl by MacroModel 9.5
(Mestro 9.0 interface) by using an OPLS 2005 force field.
In the optimized structure for the Z form, the p plane of the
xanthene moiety is stacked with that of the guanine base at
the 3’ end of the complementary strand. On the other hand,
in the case of the B form, the xanthene p plane adopts an
almost perpendicular conformation against that of the
Figure 2. Normalized intensities derived from the CD spectra of ODNfa
at 293 nm (a) and 529 nm (c) plotted against NaCl concentration.
Each CD intensity was normalized on the basis of the intensities at the
NaCl concentration of 5.0m.
To elucidate the origin of the specific ICD, we measured
the UV/Vis spectra of ODNfa. As the concentration of
NaCl was increased, large hypochromicity arose both in the
nucleobase and in the chromophore absorption bands ac-
companied with isosbestic points at 283, 414, and 513 nm
(Figure 3). These isosbestic points indicate the presence of
nucleoACHTNUTRGNEbUNG ase. These findings support the fact that the two
chromophores may communicate with each other through
the base pairs, resulting in the observed exciton-type ICD in
the Z-form duplex.
Chem. Eur. J. 2010, 16, 2401 – 2406
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K. Fujimoto, M. Inouye et al.
However, a large amount of NaCl could somewhat affect
the UV/Vis spectral changes at the absorptive region of the
chromophore in ODNfa. Therefore, we performed the fol-
lowing control experiments to rule out this ambiguity. In the
UV/Vis spectra of the deprotected 3, the influence of NaCl
was negligible (Figure S2 in the Supporting Information).
Subsequently, ODNfb, which consists of 5-methylcytosine
instead of cytosine, was subjected to the same measurements
because this substitution is well known to promote the for-
mation of Z-form duplexes at a relatively low NaCl concen-
tration (Figure S3 in the Supporting Information).[12] Indeed,
the NaCl concentration of 2.0m is enough to complete the
Z-duplex formation in ODNfb to afford a strong ICD signal
at the same level of that for ODNfa in the presence of 5.0m
NaCl. These experiments support the fact that the large hy-
pochromicity mentioned above is only attributed to the tight
p stacking arising between the chromophore and the nucleo-
base pairs and not to the presence of a large amount of
NaCl.[13]
from the cooling process). Similar hysteresis phenomena are
known to be observed in DNA triplex structures.[14] In the
triplexes, the slow chemical equilibrium between the triplex
and duplex structures causes a hysteresis curve in the pro-
cesses. In our system, the stabilization of the Z-form duplex
by the two chromophores might slow down the renaturation
to afford the observed hysteresis phenomenon.
The longer CG-repeated palindromic ODNfc that consists
of 12 bases showed a similar spectral tendency to ODNfa
(Figure S4 in the Supporting Information). The ICD intensi-
ty of the Z-form duplex in ODNfc was approximately
1.6 times as large as that in ODNfa under the same condi-
tion despite the longer distance between the two chromo-
phores. Lewis et al. have reported that the ICD intensity of
labeled ODNs with two stilbenes placed on the edges of
their duplex structures is sensitive not only to the distance
between the two stilbenes but also to the orientation of the
dipole moments in them.[15] Because one turn in Z-DNA du-
plexes involves 12 base pairs, ODNfc is just able to form
one turn duplex, whereas ODNfa corresponds to half of this
size.[16] The orientation of the dipole moments in the two
chromophores on ODNfc is considered to be more favor-
For exploration of the thermal influence of the chromo-
phore upon Z-form duplex formation, we measured the CD
spectra of ODNfa in the presence of NaCl (5.0m) at various
temperatures. Figure 5 shows the CD spectral change for the
AHCTUNGTREGaNNUN ble for yielding the ICD than that in ODNfa. Thus, the
well-fitted orientation of the two chromophores in ODNfc
not only compensated for the distance disadvantage but it
also enhanced the intensity of the ICD signal.
Several CG-sequenced ODNs with the chromophores
were analyzed by CD spectroscopy to examine the influence
of the number and the position of the chromophore upon
the specific ICD and the structural stability in the Z-form
duplexes of the labeled ODNs. Figure S5 in the Supporting
Information exhibits the CD spectra of the combination of
ODNn’/ODNfa’ and palindromic ODNfd. The duplex
structure of the former has only a single chromophore at the
5’ end, and the latter bears the chromophore at the 3’ end.
The chromophores, independently of the NaCl concentra-
tions, provided no ICDs in both of these cases.[17] Next, the
CD signals at the absorptive region of nucleobases were
compared between the four duplexes, ODNfa, ODNn,
ODNn’/ODNfa’, and ODNfd, to evaluate the contents of
their Z-form duplexes. In the positive CD signals around
270 nm, the lmax in ODNfa was 268 nm, whereas those in
the others were ꢁ273 nm. Furthermore, ODNfa showed a
much higher absolute molar ellipticity, j[q]j, of 10400 in the
negative CD signal around 290 nm than others (2300–2700).
In the complexes of CG-sequenced ODNs with the Z-DNA-
binding proteins, their absolute molar ellipticities were re-
ported to be beyond 10000.[2] Therefore, our data imply that
a set of two chromophores attached at the 5’ ends effectively
stabilized the Z-form duplexes and produced the specific
ICD.
Figure 5. CD spectra of ODNfa (10 mm) in a Tris-HCl buffer (100 mm,
pH 7.0) in the presence of NaCl (5.0m) at temperatures ranging from
58C to 608C.
Z-duplex at temperatures ranging from 58C to 608C. As the
temperature increased, both of the CD intensities from the
nucleobases and from the chromophore gradually decreased.
Surprisingly, the Z-form duplex partially survived at 608C
on the basis of the negative CD band around 290 nm,
whereas the CD spectra of ODNn lacking the chromophore
showed the complete melting of its Z-form duplex at the
same temperature (Figure S1 in the Supporting Informa-
tion). This finding indicates that the attached chromophore
provides the Z-form duplexes of the labeled ODNs with an
extra thermal stability by strong p stacking. To ensure the
thermal effect of the chromophore, melting points (Tm) for
the duplexes of ODNfa were measured by using UV/Vis
spectroscopy at 260 nm. In the heating and cooling processes
between 208C and 808C, a hysteresis curve was observed
only for the Z-form duplex of ODNfa. The Tms for the B
and Z forms were estimated to be 45 (45) and 62 (46)8C, re-
spectively (the temperatures in parentheses were obtained
Conclusion
We have developed an ICD probe for DNA duplexes by
using an alkynyldeoxyribose skeleton. The CG-repeated
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Enhanced B to Z Transitions of Duplexes
FULL PAPER
J=9.2, 3.2 Hz, 5H), 7.31–7.25 (m, 2H), 7.21–7.17 (m, 1H), 7.07–6.92 (m,
4H), 6.82 (dd, J=9.0, 2.4 Hz, 5H), 6.60 (dd, J=9.6, 2.0 Hz, 1H), 6.45 (d,
J=1.6 Hz, 1H), 5.14 (t, J=7.8 Hz, 1H), 4.52–4.50 (m, 1H), 4.06–4.00 (m,
1H), 3.77 (s, 6H), 3.30 (d, J=4.8 Hz, 2H), 2.48–2.32 (m, 5H), 1.97 (s,
3H), 1.39 ppm (s, 9H); 13C NMR (CDCl3): d=186.0, 176.3, 158.6, 158.5,
158.4, 155.0, 153.1, 144.9, 138.2, 136.0, 135.9, 134.0, 133.4, 132.1, 130.9,
130.5, 130.1, 129.9, 129.1, 128.9, 128.2, 127.79, 127.77, 127.73, 126.69,
120.1, 118.5, 118.0, 113.11, 113.08, 110.2, 106.2, 93.7, 86.2, 86.0, 83.2, 74.4,
68.2, 64.5, 55.2, 55.1, 42.5, 39.3, 27.0, 20.1, 18.9 ppm; IR (KBr): n˜ =3420,
2930, 1759, 1640, 1601 cmꢀ1; HRMS (ESI): m/z: calcd for C54H51O9:
843.3533; found: 843.3488 [M+H]+.
ODNs labeled with the chromophores at their 5’ ends self-
hybridized to form stable Z-form duplexes at a high NaCl
concentration. A strong ICD appeared from the absorptive
region of the chromophore only in the case of the Z-form
duplexes, and the attached chromophore was found to pro-
mote the B to Z transition effectively. A good linear rela-
tionship was observed between the contents of Z-form du-
plexes and the intensities of the ICD signals. Therefore, our
probe is expected to be applied in accurate CD analysis for
the interaction of Z-DNA with Z-DNA-binding proteins in
vitro.
Phosphoramidite (2): NEt
ACHTUNGERTN(NUNG iPr)2 (108 mL, 0.62 mmol) and NC-
A
ACHTUNGTRENNUNG
(130 mg, 0.15 mmol) in CH2Cl2 (3 mL) at room temperature. The reaction
mixture was stirred for 30 min at that temperature. After removal of the
solvent by a rotary evaporator, the residue was chromatographed (silica
gel pretreated with CH2Cl2 including 1% (v/v) Et3N; eluent: CH2Cl2) to
give 2 as a yellow foam (yield 97%, 156 mg). M.p.=70–748C; 1H NMR
(CDCl3); d=7.52–7.49 (m, 2H), 7.42–7.36 (m, 6H), 7.29–7.19 (m, 2H),
7.04 (dd, J=7.0, 2.8 Hz, 1H), 6.99–6.90 (m, 3H), 6.82–6.78 (m, 5H), 6.58
(dd, J=9.6, 1.6 Hz, 1H), 6.43 (d, J=2.0 Hz, 1H), 5.13–5.09 (m, 1H),
4.31–4.22 (m, 1H), 3.79 (s, 6H), 3.77–3.68 (m, 2H), 3.62–3.56 (m, 1H),
3.40–3.28 (m, 2H), 3.21–3.15 (m, 1H), 2.67–2.57 (m, 2H), 2.54–2.38 (m,
5H), 1.96 (s, 3H), 1.38 (s, 9H), 1.24–1.09 ppm (m, 12H); 13C NMR
(CD3OD): d=186.0, 176.3, 158.5, 158.4, 154.9, 153.1, 147.5, 144.7, 138.3,
138.2, 135.9, 134.1, 133.5, 131.1, 130.5, 130.2, 130.0, 128.9, 128.3, 128.3,
127.8, 127.8, 126.7, 120.2, 118.4, 118.0, 113.14, 113.08, 110.3, 106.3, 86.4,
86.4, 86.2, 68.6, 68.5, 55.6, 55.20, 55.18, 43.3, 39.3, 30.9, 27.0, 24.7, 24.6,
24.5, 21.0, 20.2, 18.9 ppm; IR (KBr): n˜ =3421, 2927, 1750, 1639,
1602 cmꢀ1; HRMS (ESI): m/z: calcd for C63H67N2NaO10P: 1065.4431;
found: 1065.4480 [M+Na]+.
Experimental Section
General methods and materials: 1H and 13C NMR spectra were recorded
at 400 and 100 MHz, respectively. MALDI-TOF mass spectra were re-
corded with 3-hydroxypicolinic acid as a matrix. High-resolution mass
spectra were obtained by using an ESI-TOF method. Melting points are
uncorrected. Reagents were purchased from commercial sources and
used without further purification. The starting materials, 3,6-bis-O-(tert-
butyldimethylsilyl)xanthone 4,[9] 5-(4,4’-dimethoxytrityl)-1-b-ethynyl-2-
deoxy-d-ribofuranoside 6[8] have been previously reported and were syn-
thesized according to the published procedures.
A basic chromophore skeleton (5): A solution of nBuLi (1.57m) in n-
hexane (6.37 mL, 10 mmol) was added to a solution of 2,5-dibromo-p-
xylene (2.64 g, 10 mmol) in THF (26 mL) at ꢀ788C. The reaction mixture
was stirred for 30 min at that temperature, and a solution of 4[9] (4.57 g,
10 mmol) in THF (45 mL) was added to the mixture. The mixed solution
was heated to room temperature and was quenched by the addition of
HCl (1n, 20 mL). The resulting yellow precipitate was collected by filtra-
tion, washed with a small quantity of n-hexane, and dried in vacuo to
give 5 as a brown solid (yield 84%, 3.3 g). M.p.=284–2878C; 1H NMR
(CD3OD:[D6]DMSO=1:2): d=7.71 (s, 1H), 7.24 (s, 1H), 6.98 (d, J=
9.2 Hz, 2H), 6.66 (d, J=8.8 Hz, 2H), 6.65 (s, 2H), 2.41 (s, 3H), 2.00 ppm
(s, 3H); 13C NMR ([D6]DMSO) d=156.8, 149.1, 135.9, 135.6, 134.0,
132.2, 131.5, 130.5, 125.5, 114.9, 103.8, 38.9, 22.0, 18.4 ppm; IR (KBr): n˜ =
3419, 1566 cmꢀ1; HRMS (ESI): m/z: calcd for C21H16O3Br: 395.0283;
found: 395.0315 [M+H]+.
F nucleoside (3): CCl3CO2H (29 mg, 0.17 mmol) was added to a solution
of 7 (26 mg, 0.034 mmol) in CH2Cl2 (2 mL) at room temperature. The re-
action mixture was stirred for 30 min at that temperature and was
quenched by the addition of NEt3 (1 mL). After removal of the solvent
by using a rotary evaporator, the residue was chromatographed (silica
gel; eluent: CH2Cl2:MeOH=10:1) to give 3 as a red solid (yield 99%,
15 mg). M.p.=192–1948C; 1H NMR (CD3OD): d=7.48 (s, 1H), 7.13 (s,
1H), 7.05 (d, J=8.0 Hz, 2H), 6.72–6.70 (m, 4H), 5.03 (t, J=7.6 Hz, 1H),
4.34 (dd, J=7.1, 4.4 Hz, 1H), 3.89–3.85 (m, 1H), 2.45 (s, 3H), 2.28–2.25
(m, 2H), 1.99 ppm (s, 3H); 13C NMR (CD3OD): d=200.6, 154.9, 139.4,
134.9, 134.8, 134.0, 132.1, 131.1, 125.3, 115.9, 104.5, 94.6, 89.0, 84.2, 73.8,
69.2, 63.9, 43.6, 20.2, 18.9 ppm; IR (KBr): n˜ =1592 cmꢀ1; Ff (Tris-HCl
buffer, 100 mm, pH 7.0) 0.83; HRMS (ESI): m/z: calcd for C28H25O6:
457.1651; found: 457.1654 [M+H]+.
A nucleoside derivative (7): A solution of 6[8] (222 mg, 0.5 mmol) in
DMF (5 mL) was added to a solution of 5 (198 mg, 0.5 mmol), NH
ACHTUNGERTN(NUNG iPr)2
(3 mL), Pd(PPh3)4 (87 mg, 0.075 mmol), and CuI (4.76 mg, 0.025 mmol)
ACHTUNGTRENNUNG
F-modified ODNs: F-modified ODNs were synthesized by using a con-
ventional phosphoramidite method with a DNA synthesizer. The synthe-
sized ODNs were purified by reverse-phase HPLC on a CHEMCO-
BOND 5-ODS-H column (10ꢁ150 mm; Chemco SCIENTIFIC; eluent:
5 mm ammonium formate with the respective CH3CN percentage of
linear gradient over 60 min at a flow rate of 2.0 mLminꢀ1 (5–30% for
ODNfa, ODNfa’, and ODNfd, 0–50% for ODNfb, and 5–50% for
ODNfc)).
in DMF (10 mL) at 608C under an Ar atmosphere. The reaction mixture
was stirred overnight at that temperature and filtered through a pad of
florisil. The filtrate was evaporated and chromatographed (silica gel;
eluent: from CH2Cl2:MeOH=100:1 to 10:1) to give 7 as a red foam
(yield 76%, 289 mg). M.p.=158–1628C; 1H NMR (CDCl3): d=7.47 (d,
J=8.0 Hz, 2H), 7.37–7.34 (m, 5H), 7.26–7.21 (m, 2H), 7.16–7.13 (m,
1H), 7.04 (d, J=8.4 Hz, 2H), 6.96 (s, 1H), 6.87 (s, 2H), 6.83 (d, J=
9.2 Hz, 2H), 6.77 (dd, J=8.8, 2.0 Hz, 4H), 5.98 (brs, 1H), 5.12 (t, J=
6.8 Hz, 1H), 4.49 (m, 1H), 4.03 (m, 1H), 3.72 (s, 6H), 3.27 (d, J=3.6 Hz,
2H), 2.45–2.36 (m, 5H), 1.89 ppm (s, 3H); 13C NMR (CDCl3): d=175.8,
158.4, 158.0, 154.8, 144.9, 138.0, 136.1, 135.9, 133.2, 132.4, 131.0, 130.1,
128.2, 128.0, 126.7, 124.01, 122.3, 114.7, 113.1, 113.0, 103.8, 93.7, 86.3,
86.1, 83.3, 74.3, 68.2, 64.5, 55.2, 42.5, 20.2, 18.9 ppm; IR (KBr): n˜ =
1594 cmꢀ1; HRMS (ESI): m/z: calcd for C49H43O8: 759.2958; found:
759.2982 [M+H]+.
MALDI-TOF mass data for F-modified ODNs: ODNfa: MS (ESI): m/z:
calcd for C85H97N24O42P6: 2311.46; found: 2311.53 [M+H]+; ODNfa’:
MS (ESI): m/z: calcd for C94H108N27O48P7: 2600.51; found: 2600.17
AHCTUNGTRENNUNG
[M+H]+; ODNfb: MS (ESI): m/z: calcd for C88H103N24O42P6: 2353.51;
found: 2354.00 [M+H]+; ODNfc: MS (ESI): m/z: calcd for
C142H169N48O78P12: 4165.76; found: 4166.05 [M+H]+; ODNfd: MS (ESI):
m/z: calcd for C85H96N24O42P6: 2311.46; found: 2311.17 [M+H]+.
Determination of Tm: Melting curves were measured at 260 nm at tem-
peratures ranging from 208C to 808C by using UV/Vis spectroscopy.
ODNfa (2.0 mm) was dissolved in a solution of Tris-HCl (100 mm, pH 7.0)
including NaCl (0.1m or 5.0m).
An ICD probe (1): A solution of pivaloyl chloride (40 mL, 0.33 mmol) in
CH2Cl2 (1 mL) was added to a solution of 7 (228 mg, 0.3 mmol) and NEt3
(84 mL, 0.6 mmol) in CH2Cl2 (4 mL) at 08C. The reaction mixture was
stirred for 1 h at room temperature, quenched by the addition of a satu-
rated NaHCO3 aqueous solution (2 mL), and extracted with CH2Cl2. The
CH2Cl2 extract was evaporated and chromatographed (silica gel; eluent:
CH2Cl2:MeOH=50:1) to give 1 as a yellow foam (yield 82%, 208 mg).
M.p.=91–948C; 1H NMR (CDCl3): d=7.50 (d, J=7.6 Hz, 2H), 7.39 (dd,
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complete Z-form duplexes of CG-repeated ODNs, the lmax of which
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chromophore hardly changed at various NaCl concentrations (Fig-
ACHTUNGTRENNuUNG re S5c in the Supporting Information).
Received: October 19, 2009
Published online: February 1, 2010
2406
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