Nile Red nucleoside: Design of a solvatofluorochromic nucleoside as an indicator of micropolarity around DNA

Article abstract of DOI:10.1021/jo060168o

The fluorophore, Nile Red, effectively works as a polarity-sensitive fluorescence probe. We have designed a new nucleoside modified by Nile Red for examining the change in the polarity of the microenvironment surrounding DNA. We synthesized a Nile Red nuc

Full text of DOI:10.1021/jo060168o

Nile Red Nucleoside: Design of a Solvatofluorochromic Nucleoside
as an Indicator of Micropolarity around DNA
Akimitsu Okamoto,* Kazuki Tainaka, and Yoshimasa Fujiwara
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto UniVersity,
Kyoto 615-8510, Japan
okamoto@sbchem.kyoto-u.ac.jp
ReceiVed January 25, 2006
The fluorophore, Nile Red, effectively works as a polarity-sensitive fluorescence probe. We have designed
a new nucleoside modified by Nile Red for examining the change in the polarity of the microenvironment
surrounding DNA. We synthesized a Nile Red nucleoside (1), formed by replacing nucleobases with
Nile Red, through the coupling of a 2-hydroxylated Nile Red derivative and 1,2-dideoxyglycan. This
nucleoside showed a high solvatofluorochromicity. The fluorescence of 1 incorporated into DNA was
greatly shifted to shorter wavelength by the addition of â-cyclodextrin. The photophysical function of
the Nile Red nucleoside will be a good optical indicator for monitoring the change in the micropolarity
properties at a specific site on target sequences with interaction between DNA and DNA-binding molecules.
Introduction
Solvatochromic fluorophores, which show shifts in absorption
and emission bands induced by a change in solvent nature or
composition, are useful for monitoring the structural transforma-
tion and intermolecular interaction of biomolecules with mi-
cropolarity change in the surroundings.5 The well-known
fluorophore, Nile Red, is a hydrophobic, highly fluorescent,
Local variations in the structure and dynamics of biomol-
ecules play important roles in mediating the interaction events
between biomolecules. Monitoring of the microenvironmental
change in DNA and its surroundings is extremely important
because the structure of DNA itself and the formation of DNA-
protein complexes are strongly influenced by their microenvi-
ronmental conditions. For such a purpose, fluorescent probes
that can be site-specifically incorporated into the DNA sequence
of interest and are sensitive to the change in its microenviron-
ment are highly valuable and desirable.1 Nucleosides possessing
various fluorophores have been explored for studying the
structures and dynamics of nucleic acids, including fluorescent
nucleoside analogues, as exemplified by 2-aminopurine,2 ethy-
nyl-extended nucleobase derivatives,3 and nucleoside analogues
replaced by flat aromatic fluorophores.4
(2) (a) Ward, D. C.; Reich, E.; Stryer, L. J. Biol. Chem. 1969, 244, 1228-
1237. (b) Menger, M.; Tuschl, T.; Eckstein, F.; Porschke, D. Biochemistry
1996, 35, 14710-14716. (c) Lacourciere, K. A.; Stivers, J. T.; Marino, J.
P. Biochemistry 2000, 39, 5630-5641.
(3) (a) Seela, F.; Zulauf, M.; Sauer, M.; Deimel, M. HelV. Chim. Acta
2000, 83, 910-927. (b) Hurley, D. J.; Seaman, S. E.; Mazura, J. C.; Tor,
Y. Org. Lett. 2002, 4, 2305-2308. (c) Okamoto, A.; Kanatani, K.; Saito,
I. J. Am. Chem. Soc. 2004, 126, 4820-4827. (d) Okamoto, A.; Tainaka,
K.; Saito, I. Bioconjugate Chem. 2005, 16, 1105-1111. (e) Okamoto, A.;
Tainaka, K.; Nishiza, K.-i.; Saito, I. J. Am. Chem. Soc. 2005, 127, 13128-
13129.
(4) (a) Stra¨ssler, C.; Davis, N. E.; Kool, E. T. HelV. Chim. Acta 1999,
82, 2160-2171. (b) Kool, E. T. Acc. Chem. Res. 2002, 35, 936-943. (c)
Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125, 4972-
4973. (d) Okamoto, A.; Tainaka, K.; Saito, I. Tetrahedron Lett. 2003, 44,
6871-6874.
* To whom correspondence should be addressed. Phone: +81-75-383-2755.
FAX: +81-75-383-2759.
(1) (a) Wojczewski, C.; Stolze, K.; Engels, J. W. Synlett 1999, 1667-
1678. (b) Hawkins, M. E. Cell Biochem. Biophys. 2001, 34, 257-281. (c)
Okamoto, A.; Saito, Y.; Saito, I. J. Photochem. Photobiol. C 2005, 6, 108-
122.
(5) (a) Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem. Soc.
2003, 125, 9296-9297. (b) Jadhav, V. R.; Barawkar, D. A.; Ganesh, K. N.
J. Phys. Chem. B 1999, 103, 7383-7385. (c) Kimura, K.; Kawai, K.;
Majima, T. Org. Lett. 2005, 7, 5829-5832.
10.1021/jo060168o CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/04/2006
3592
J. Org. Chem. 2006, 71, 3592-3598

Nile Red Nucleoside
SCHEME 1. Synthesis of Nile Red Nucleoside (1) and 1-Containing ODN
solvatochromic dye that has been extensively applied to examine
the local polarity in zeolites6 and micelles7 and is among the
most promising candidates for a microenvironment-sensitive
fluorescent probe.8 Thus, Nile Red derivatives incorporated into
DNA would also be useful as polarity-sensitive fluorescence
probes for examining the change in polarity of the microenvi-
ronments around DNA. In particular, when Nile Red is fixed
in the place of a nucleobase to oligodeoxynucleotide (ODN)
probes, the change in the micropolarity properties around a
specific site on the target sequences would be easily monitored.
We herein report the sequence-selective fluorescence labeling
of DNA with Nile Red and the solvatochromic function of Nile
Red fixed to DNA as a fluorescent reporter for micropolarity.
We synthesized a Nile Red nucleoside (1), produced by the
replacement of a nucleobase by Nile Red. This nucleoside
showed a high solvatofluorochromicity, just like free Nile Red,
and the fluorescence of 1 incorporated to DNA also greatly
varied with micropolarity to function as an effective micropo-
larity indicator.
2-nitroso-1-phenol according to a protocol reported pre-
viously (56%).9 Compound 2 was converted into a triflate
derivative (75%). Subsequently, we obtained compound 4 â-
selectively (79%) through Heck reaction between 2 and 1,2-
dideoxyglycan 3 given by depyrimidination of thymidine by
means of fragmentation at elevated temperatures in the presence
of hexamethyldisilazane.10 A carbonyl group at 3′-C of 4
was then stereoselectively reduced with tetramethyl-
ammonium triacetoxyborohydride to give Nile Red nucleoside
(1) (97%).
We initially examined the photochemical properties of 1 in
nine different solvents. Both absorption maximum and fluores-
cence emission wavelength shifted to longer wavelength as the
polarity parameter ET of the solvents11 increased (Figure 1 and
Table 1). The absorption maximum greatly changed with solvent
polarity, for example, from 506 nm in diethyl ether to 524 nm
in ethyl acetate, 537 nm in acetonitrile, and 573 nm in
formamide (Figure 1a). The shift of the absorption maximum
to longer wavelength with higher polarity indicates that 1 retains
the solvatochromic property observed for Nile Red. The
fluorescence emission peak was observed at 561 nm in diethyl
ether, 584 nm in ethyl acetate, 606 nm in acetonitrile, and 637
nm in formamide (Figure 1b). The fluorescence emission peaks
were also red-shifted with higher solvent polarity, suggesting
that 1 works as a highly solvatofluorochromic nucleoside. As
Results and Discussion
The outline of the synthesis of a Nile Red nucleoside is shown
in Scheme 1. We prepared a 2-hydroxylated Nile Red deriva-
tive 2 from 1,6-dihydroxynaphthalene and 5-diethylamino-
(6) (a) Uppili, S.; Thomas, K. J.; Crompton, E. M.; Ramamurthy, V.
Langmuir 2000, 16, 265-274. (b) Meinershagen, J. L.; Bein, T. J. Am.
Chem. Soc. 1999, 121, 448-449.
(7) (a) Datta, A.; Mandal, D.; Pal, S. K.; Bhattacharyya, K. J. Phys. Chem.
B 1997, 101, 10221-10225. (b) Maiti, N. C.; Krishna, M. M. G.; Britto, P.
J.; Periasamy, N. J. Phys. Chem. B 1997, 101, 11051-11060.
(8) (a) Goloni, C. M.; Williams, B. W.; Foresman, J. B. J. Fluorescence
1998, 8, 395-404. (b) Deye, J. F.; Berger, T. A.; Anderson, A. G. Anal.
Chem. 1990, 62, 615-622.
(9) (a) Nagy, K.; Go¨ktu¨rk, S.; Biczo´k, L. J. Phys. Chem. A 2003, 107,
8784-8790. (b) Briggs, M. S. J.; Bruce, I.; Miller, J. N.; Moody, C. J.;
Simmonds, A. C.; Swann, E. J. Chem. Soc., Perkin Trans. 1 1997, 1051-
1058.
(10) Coleman. R. S.; Madaras, M. L. J. Org. Chem. 1998, 63, 5700-
5703.
(11) Mulov, S. L.; Carmichael, I.; Hug, G. L. Handbook of Photochem-
istry; Marcel Dekker: New York, 1993.
J. Org. Chem, Vol. 71, No. 9, 2006 3593

Okamoto et al.
TABLE 1. Absorption and Fluorescence Properties of 1 in
Different Solventsa
λab
(nm)
ꢀmax
(×103)
λem
(nm)
b
solvents
ET
Φ
diethyl ether
dioxane
ethyl acetate
chloroform
N,N-dimethylformamide
dimethyl sulfoxide
acetonitrile
34.6
36.0
38.1
39.1
43.8
45.0
46.0
55.5
56.6
506
522
524
546
545
555
537
557
573
4.12
3.56
3.93
4.47
3.79
3.63
4.16
3.38
3.32
561
572
584
596
613
618
606
632
637
0.35
0.31
0.30
0.22
0.28
0.19
0.20
0.09
0.10
methanol
formamide
a Condition: 10 µM 1 in different solvents at 20 °C. b Reported in ref
10.
TABLE 2. Melting Temperatures (Tm) of Duplexes Containing
Nile Red
Tm
-∆H
-∆S
b
b
b
N
(°C)a
(kcal mol-1
)
(cal mol-1 K-1
)
-∆G298
A
G
C
T
44.8
47.6
50.4
50.8
54.4
45.5
52.3
59.5
75.6
73.9
77.9
55.7
135
156
207
200
211
147
12.1
13.1
13.8
14.8
15.0
11.8
Ap
no base
a 2.5 µM 5′-d(GCGTTA1ATTGCG)-3′/5′-d(CGCAATNTAACGC)-3′ in
50 mM sodium phosphate (pH ) 7.0) and 0.1 M sodium chloride.
b Thermodynamic parameters were calculated from Tm values of 10, 5, 2.5,
and 1.25 µM duplex in 50 mM sodium phosphate (pH ) 7.0) and 0.1 M
sodium chloride.
∆S were -74 to -78 kcal mol-1 and -200 to -211 cal mol-1
K-1, respectively. Group II, which consists of duplexes pos-
sessing A or G as a base opposite 1 and a duplex possessing a
single-base bulge of 1 (“no base” in Table 2), has Tm values
ca. 5 °C lower than that of Group I (45-48 °C). The ∆H and
∆S calculated were -52 to -60 kcal mol-1 and -135 to -156
cal mol-1 K-1, respectively. The values of Tm and ∆H suggest
that the Nile Red fluorophore of Group I would bind to the
duplex tightly and contribute to duplex stabilization, as com-
pared with the fluorophore of Group II.
FIGURE 1. Absorption and fluorescence of 1. Spectra were measured
using 10 µM 1 in different solvents at 20 °C. (a) Absorption spectra of
1 in different solvents. Peak 1, in diethyl ether; peak 2, in ethyl acetate;
peak 3, in acetonitrile; peak 4, in formamide. (b) Fluorescence emission
spectra of 1 in different solvents. Peak 1, in diethyl ether; peak 2, in
ethyl acetate; peak 3, in acetonitrile; peak 4, in formamide. (c)
Fluorescence colors of 1 in different solvents. The sample solutions
were illuminated through a 450 nm long pass filter.
These duplexes showed only a small difference in their
photophysical properties (Figure 2a). The absorption maxima
of single-stranded and double-helical ODNs, except for a bulge
duplex, were observed at 607-609 nm. The absorption maxi-
mum of a bulge duplex was observed at 603 nm, but it is a
very slight shift. The fluorescence emission peak of 1-containing
ODN was also almost constant at ∼650 nm regardless of the
type of hybridized strands (Figure 2b). The very small shift of
absorption maximum and fluorescence emission peak suggests
that the micropolarity around 1 in duplexes is not significantly
changed by the nature of bases opposite 1 in the duplex.
â-Cyclodextrin has been recognized as one of the most
important host materials for organic molecules in aqueous media
and as the donor of a hydrophobic microenvironment.12 This
host molecule is known to form an inclusion complex with Nile
Red.13 In this complex, the electron-donating diethylamino group
of Nile Red is placed in the nanocavity of â-cyclodextrin, and
shown in Figure 1c, the fluorescence color of 1 vividly changed
according to the polarity of solvents.
Nucleoside 1 was incorporated efficiently into an ODN
according to a conventional phosphoramidite method after
protection of 1 (96%) and conversion into the phosphoramidite
form (95%). Using the ODN 5′-d(GCGTTA1ATTGCG)-3′, we
examined the melting temperatures (Tm) and thermodynamic
parameters of six different duplexes with the complement 5′-
d(CGCAATNTAACGC)-3′ (N ) A, G, C, T, and abasic
nucleotide (1′,2′-dideoxyribose, Ap)) or 5′-d(CGCAATTAA-
CGC)-3′ in a buffer solution of 50 mM sodium phosphate (pH
) 7.0) and 100 mM sodium chloride. The results were
unequivocally separable into two types of duplexes (Table 2).
Group I consists of duplexes possessing C, T, or Ap as a base
opposite 1. Tm was relatively high (50-54 °C), and ∆H and
(12) (a) Breslow, R. Acc. Chem. Res. 1995, 28, 146-153. (b) Szejtli, J.
Chem. ReV. 1998, 98, 1743-1753. (c) Connors, K. A. Chem. ReV. 1997,
97, 1325-1357.
(13) (a) Hazra, P.; Chakrabarty, D.; Chakraborty, A.; Sarkar, N. Chem.
Phys. Lett. 2004, 388, 150-157. (b) Srivatsavoy, V. J. P. J. Lumin. 1999,
82, 17-23.
3594 J. Org. Chem., Vol. 71, No. 9, 2006

Nile Red Nucleoside
FIGURE 2. Absorption and fluorescence spectra for different structural
contexts of 1-containing ODN. Spectra were measured using a 2.5 µM
sample in 50 mM sodium phosphate (pH ) 7.0) and 100 mM sodium
chloride at 20 °C. A, G, C, and T, 5′-d(GCGTTA1ATTGCG)-3′/5′-
d(CGCAATNTAACGC)-3′ (N ) A, G, C, and T, respectively); ss,
5′-d(GCGTTA1ATTGCG)-3′; Ap, 5′-d(GCGTTA1ATTGCG)-3′/5′-
d(CGCAATApTAACGC)-3′ (Ap ) abasic site); No, 5′-d(GC-
GTTA1ATTGCG)-3′/5′-d(CGCAATTAACGC)-3′. (a) Absorption spec-
tra. (b) Fluorescence emission spectra. Excitation wavelength was 600
nm.
FIGURE 3. Changes in absorption and fluorescence spectra of 1
dependent on â-cyclodextrin concentration. Spectra of 10 µM 1 in water
were measured when â-cyclodextrin concentration was 0, 0.125, 0.25,
0.5, 1, 2, 5, 10, 15, and 20 mM. (a) Absorption spectra. (b) Fluorescence
emission spectra. Excitation wavelength was 560 nm.
slope and intercept of a straight line obtained from the plot of
1/(I - I0) versus the reciprocal of the â-cyclodextrin concentra-
tion.14 Additionally, on addition of â-cyclodextrin the fluores-
cence intensity increased to 6.2 times the original intensity. This
is probably due to retardation of the nonradiative rate in the
â-cyclodextrin cavity because of the prevention of the rotational
freedom of the diethylamino group and the decrease in the
polarity inside the cavity.15
Having established the blue shift of the fluorescence spectrum
of 1 by the formation of a 1-â-cyclodextrin inclusion complex,
we measured the fluorescence emission spectra of the duplexes
of 1-containing ODN, 5′-d(GCGTTA1ATTGCG)-3′, and ODNs
containing different bases to form a pair with 1, 5′-d(CGCAAT-
NTAACGC)-3′, in the presence of 10 mM â-cyclodextrin. When
N was C, T, or Ap, the fluorescence emission peak was observed
at approximately 650 nm, which was only slightly shifted from
the fluorescence spectra of duplex samples without â-cyclo-
dextrin shown in Figure 2b (Figure 4a). This result suggests
that â-cyclodextrin added to sample solutions does not interact
with 1 in these duplexes. On the other hand, when N was A, G,
or missing, a shoulder at the shorter wavelength side of the
spectra appeared (Figure 4b). Particularly, for a bulge duplex,
the fluorescence emission maximum shifted to 620 nm. The
peak shift to a shorter wavelength suggests that a complex of
the 1-containing ODN and â-cyclodextrin was formed. The
thus â-cyclodextrin would work as an additive for effectively
controlling the micropolarity around nucleoside 1. We measured
the photophysical properties of 1 (10 µM) in water with
gradually changing concentration of â-cyclodextrin (0 to 20
mM) (Figure 3a). The absorption spectrum was broad and the
maximum was observed at 580 nm before the addition of
â-cyclodextrin. The absorption spectra became sharper as
â-cyclodextrin was added, and the maximum was slightly blue
shifted to 578 nm in the presence of 20 mM â-cyclodextrin.
Absorbance in 20 mM â-cyclodextrin increased to 2.2 times
its original value. An isosbestic point appeared at 638 nm on
addition of â-cyclodextrin, suggesting that â-cyclodextrin binds
to 1 in a single binding mode. Changes in the fluorescence
emission spectra were larger than in the absorption spectra. The
fluorescence emission spectra of 1 in water with gradually
changing concentration of â-cyclodextrin on excitation at 560
nm are shown in Figure 3b. A fluorescence peak was observed
at 651 nm before the addition of â-cyclodextrin. On successive
addition of â-cyclodextrin, the spectrum gradually exhibited a
hypsochromic shift, and finally at 20 mM â-cyclodextrin the
peak maximum was at ∼637 nm. The 14-nm blue shift in
emission spectra and the increase in fluorescent intensity indicate
the formation of an inclusion complex between 1 and â-cyclo-
dextrin. The binding of â-cyclodextrin to 1 results in the
exclusion of solvent molecules from the surrounding of Nile
Red and a decrease of the micropolarity around 1. The binding
constant Kb was determined as 1.26 M-1 at 298 K, from the
(14) de la Pena, A. M.; Ndou, T.; Zung, J. B.; Warner, I. M. J. Phys.
Chem. 1991, 95, 3330-3334.
(15) (a) Krishna, M. M. G. J. Phys. Chem. A 1999, 103, 3589-3595.
(b) Sarkar, N.; Das, K.; Nath, D. N.; Bhattacharyya, K. Langmuir 1994,
10, 326-329.
J. Org. Chem, Vol. 71, No. 9, 2006 3595

Okamoto et al.
FIGURE 4. Changes in fluorescence spectra of 1-containing ODN
dependent on the nature of the complementary base in the presence of
â-cyclodextrin. Spectra were measured using a 2.5 µM sample and 10
mM â-cyclodextrin in 50 mM sodium phosphate (pH ) 7.0) and 100
mM sodium chloride at 20 °C. Excitation wavelength was 560 nm. A,
G, C, and T, 5′-d(GCGTTA1ATTGCG)-3′/5′-d(CGCAATNTAACGC)-
3′ (N ) A, G, C, and T, respectively); ss, 5′-d(GCGTTA1ATTGCG)-
3′; Ap, 5′-d(GCGTTA1ATTGCG)-3′/5′-d(CGCAATApTAACGC)-3′
(Ap ) abasic site); No, 5′-d(GCGTTA1ATTGCG)-3′/5′-d(CGCAAT-
TAACGC)-3′. (a) Small peak shift observed for a duplex where
the base opposite 1 was C, T, or abasic nucleotide. (b) Large peak
shift observed for a duplex where the base opposite 1 was A, G, or
missing.
classification of duplexes based on the changes in fluorescence
spectra by the addition of â-cyclodextrin was in good agreement
with the two groups classified based on thermodynamic stabili-
ties shown in Table 2.
FIGURE 5. Fluorescence spectra deconvoluted into two peaks.
Fluorescence spectra of 1-containing ODN in the presence of â-cy-
clodextrin in Figure 4 were analyzed. (a) 5′-d(GCGTTA1ATTGCG)-
3′/5′-d(CGCAATATAACGC)-3′. (b) 5′-d(GCGTTA1ATTGCG)-3′/5′-
d(CGCAATGTAACGC)-3′. (c) 5′-d(GCGTTA1ATTGCG)-3′/5′-d(CGC-
AATTAACGC)-3′.
The fluorescence spectra of Group II, shown in Figure 4b,
were deconvoluted into two overlapping signals. When a 650-
nm-centered fluorescence peak was subtracted from the original
fluorescence spectrum, a peak whose maximum wavelength was
615 nm appeared (Figure 5). The peak at 615 nm is a new signal
that appears as a result of â-cyclodextrin addition to a sample
solution. The appearance of a new peak with a shorter
wavelength indicates the formation of an inclusion complex
between 1 in the duplex and â-cyclodextrin and a significant
decrease of the micropolarity around 1 in the duplex. The
fluorescence at 615 nm roughly corresponds to the polarity
of DMF as shown in Table 1. The 1 of Group II would slide
out of a π-stacking array in a double-helical structure because
of the existence of the purine base opposite 1 or the forma-
tion of a bulge structure. Actually, the Tm of Group II was lower
than that for Group I as described above, suggesting that any
intercalation of 1 to a double-helical structure was lost or
very slight. Therefore, 1 in these structures could easily be
included by â-cyclodextrin possessing an interior space of low
polarity, resulting in a fluorescence spectrum with a shorter
wavelength.
Conclusion
In conclusion, we have synthesized a polarity-sensitive Nile
Red nucleoside and a nucleic acid containing it. These sensi-
tively changed fluorescence wavelength according to the change
of micropolarity. The fluorescence of the Nile Red fixed in a
DNA duplex was greatly shifted to shorter wavelength by the
addition of â-cyclodextrin. The photophysical function of the
Nile Red nucleoside will be a good optical indicator for
monitoring the change in the micropolarity properties at a
3596 J. Org. Chem., Vol. 71, No. 9, 2006

Nile Red Nucleoside
was stirred for 2 h at ambient temperature. The reaction mixture
was concentrated and purified by column chromatography on silica
gel, eluting with chloroform-methanol (30:1) to give the 5′-
protected nucleoside (492 mg, 96%) as a purple solid: 1H NMR
(400 MHz, CDCl3) δ ) 8.69 (d, 1H, J ) 0.9 Hz), 8.27 (d, 1H, J
) 8.1 Hz), 7.68 (dd, 1H, J ) 8.2, 1.3 Hz), 7.50 (dd, 2H, J ) 8.1,
1.3 Hz), 7.39 (dd, 4H, J ) 9.0, 1.6 Hz), 7.34 (d, 1H, J ) 9.2 Hz),
7.27 (t, 2H, J ) 7.5 Hz), 7.18 (t, 1H, J ) 7.7 Hz), 6.81 (d, 4H, J
) 9.0 Hz), 6.58 (dd, 1H, J ) 9.2, 2.7 Hz), 6.45 (d, 1H, J ) 2.7
Hz), 6.36 (s, 1H), 5.39 (dd, 1H, J ) 10.1, 5.7 Hz), 4.48-4.46 (m,
1H), 4.14 (dd, 1H, J ) 7.3, 4.6 Hz), 3.75 (s, 6H), 3.45 (q, 4H, J )
7.1 Hz), 3.43-3.36 (m, 2H), 2.39 (ddd, 1H, J ) 13.2, 5.7, 1.8
Hz), 2.15 (ddd, 1H, J ) 13.0, 10.1, 6.0 Hz), 2.00 (br. s, 1H), 1.25
(t, 6H, J ) 7.0 Hz); 13C NMR (100 MHz, CDCl3) δ ) 183.5, 158.5,
152.2, 150.7, 146.7, 145.7, 144.8, 136.1, 132.2, 131.2, 131.1, 130.1,
128.3, 127.8, 127.5, 126.8, 126.0, 124.9, 121.1, 113.2, 109.6, 105.7,
96.2, 86.6, 86.3, 79.8, 74.6, 64.4, 55.2, 45.0, 44.1, 12.6; FABMS,
m/z 736 [M+]; HRMS calcd for C46H44N2O7 [M+] 736.3149, found
736.3147.
specific site on target sequences with interaction between DNA
and other DNA-binding molecules.
Experimental Section
1′-C-(2-Nile Red)-2′-deoxy-3′-ketoriboside (4). To a solution
of 2 (4.01 g, 12 mmol), which was synthesized according to ref 9
(56% yield), in dichloromethane (300 mL) and triethylamine (50
mL) was added N-phenyl bis(trifluoromethanesulfonimide) (4.29
g, 12 mmol) at 0 °C, and the mixture was stirred for 3 h at ambient
temperature. After concentration, the crude product was purified
by column chromatography on silica gel, eluting with hexanes-
ethyl acetate (3:1) to give a triflate (4.2 g, 75%) as a dark purple
solid: 1H NMR (400 MHz, CDCl3) δ ) 8.54 (d, 1H, J ) 2.6),
8.41 (d, 1H, J ) 8.6), 7.66 (d, 1H, J ) 9.0), 7.50 (dd, 1H, J ) 8.6,
2.6), 6.72 (dd, 1H, J ) 9.2, 2.7), 6.50 (d, 1H, J ) 2.6), 6.41 (s,
1H), 3.50 (quartet, 4H, J ) 7.1 Hz), 1.29 (t, 6H, J ) 7.1 Hz); 13
C
NMR (100 MHz, CDCl3) δ ) 182.0, 152.7, 151.6, 151.4, 147.1,
134.3, 131.7, 129.5, 128.6, 126.9, 125.3, 123.2, 122.1, 116.3, 105.3,
96.2, 45.3, 12.6; FABMS, m/z 466 [M+]; HRMS calcd for
C21H17N2O5F3S [M+] 466.0810, found 466.0806.
To a solution of the 5′-protected nucleoside (74 mg, 0.10 mmol)
and tetrazole (7.0 mg, 0.10 mmol) in 1:1 acetonitrile-dichlo-
romethane (1.0 mL) was added 2-cyanoethyl tetraisopropylphos-
phordiamidite (63.4 µL, 0.20 mmol) under nitrogen. The mixture
was stirred at ambient temperature for 1 h. The resulting mixture
was diluted with ethyl acetate, washed with saturated aqueous
NaHCO3 and brine, and dried over MgSO4. After removal of
solvent, the residue was purified by column chromatography on
silica gel, eluting with hexanes-ethyl acetate (1:1) to give a
diastereomeric mixture of 5 (89 mg, 95%) as a dark purple oil: 1H
NMR (400 MHz, CDCl3) δ ) 8.75 (d, 1H, J ) 1.3 Hz), 8.73 (d,
1H, J ) 1.1 Hz), 8.29 (d, 1H, J ) 8.2 Hz), 8.28 (d, 1H, J ) 8.2
Hz), 7.73 (dd × 2, 1H × 2, J ) 1.8, 8.2 Hz), 7.54-7.51 (2H × 2),
7.42-7.38 (4H × 2), 7.33-7.24 (4H × 2), 7.15-7.20 (1H × 2),
6.82-6.79 (4H × 2), 6.59-6.55 (1H × 2), 6.46 (s, 1H), 6.45 (s,
1H), 6.38 (s × 2, 1H × 2), 5.40-5.35 (1H × 2), 4.57-4.53 (1H
× 2), 4.33-4.28 (1H × 2), 3.91-3.79 (1H × 2), 3.74 (s × 2, 1H
× 2), 3.70-3.57 (2H × 2), 3.45 (quartet, 1H × 2, J ) 7.1 Hz),
3.42-3.38 (1H × 2), 3.32-3.27 (1H × 2), 2.64 (t, 2H, J ) 6.6
Hz), 2.58-2.44 (1H × 2), 2.46 (t, 2H, J ) 6.6 Hz), 2.19-2.10
To a solution of a triflate (141 mg, 0.3 mmol) in N,N-
dimethylformamide (3 mL) were added NaHCO3 (76 mg, 0.9
mmol), n-tetrabutylammonium chloride (84 mg, 0.3 mmol), 4 Å
molecular sieves (141 mg), and palladium(II) acetate (17 mg, 0.075
mmol). A solution of glycal 3 (207 mg, 0.9 mmol), which was
prepared according to a protocol previously reported,9 in N,N-
dimethylformamide (1 mL) was added, and the mixture was stirred
for 3 h at 60 °C. The reaction mixture was filtered through Celite
(chloroform wash), and then the filtrate was extracted with ethyl
acetate and saturated aqueous NaHCO3. After concentration, the
crude product was purified by column chromatography on silica
gel, eluting with hexanes-ethyl acetate (1:2) to give 4 (103 mg,
79%) as a dark purple solid: 1H NMR (400 MHz, CDCl3) δ )
8.63 (d, 1H, J ) 1.3), 8.29 (d, 1H, J ) 8.1), 7.70 (dd, 1H, J ) 8.1,
1.7), 7.59 (d, 1H, J ) 9.2), 6.66 (dd, 1H, J ) 9.2, 2.7), 6.46 (d,
1H, J ) 2.6), 6.36 (s, 1H), 5.41 (dd, 1H, J ) 11.1, 5.8), 4.13 (t,
1H, J ) 3.4), 4.06 (dd, 1H, J ) 12.2, 3.3), 4.02 (dd, 1H, J ) 12.2,
3.6), 3.47 (quartet, 4H, J ) 7.1), 3.00 (dd, 1H, J ) 17.9, 5.9), 2.65
(dd, 1H, J ) 17.9, 11.0), 1.70 (br, 1H), 1.27 (t, 6H, J ) 7.1); 13C
NMR (100 MHz, CDCl3) δ ) 213.2, 183.2, 152.3, 151.0, 146.9,
143.1, 139.4, 132.3, 131.7, 131.2, 127.4, 126.5, 124.9, 121.3, 109.8,
105.8, 96.3, 82.6, 77.5, 61.6, 45.4, 45.1, 12.6; FABMS, m/z 433
[M + H+]; HRMS calcd for C25H25N2O5 [M + H+] 433.1763, found
433.1763.
(1H × 2), 1.25 (t, 6H × 2, J ) 7.1 Hz), 1.27-1.09 (12H × 2); 13
C
NMR (100 MHz, CDCl3) δ ) 183.5, 158.4, 152.1, 150.6, 146.7,
145.5 (d), 144.8 (d), 139.8 (d), 136.1 (m), 132.2 (d), 131.2, 131.1,
130.1 (d), 128.3 (d), 127.7, 127.5, 126.6 (d), 125.9, 124.8, 121.2,
117.4 (d), 113.1, 109.5, 105.6, 96.2, 86.3 (d), 86.2, 86.0 (d), 80.1
(d), 75.9 (m), 64.1 (d), 58.3 (m), 55.1, 45.0, 43.4 (d), 43.2 (m),
29.6, 24.5 (m), 20.3 (d), 20.1 (d), 12.5; 31P NMR (161.7 MHz,
CDCl3, external H3PO4 standard) δ ) 144.2, 143.9; FABMS, m/z
937 [(M + H)+]; HRMS calcd for C55H62N4O8P [(M + H)+]
937.4305, found 937.4307.
1′-C-(2-Nile Red)-2′-deoxyriboside (1). A solution of 4 (48 mg,
0.11 mmol), tetramethylammonium triacetoxyborohydride (162 mg,
0.77 mmol), and acetic acid (20 µL) in 2 mL of tetrahydrofuran
was stirred for 3 h at ambient temperature. The resulting mixture
was concentrated in vacuo and diluted with ethyl acetate. This
solution was washed with saturated aqueous NH4Cl and brine, dried
over MgSO4, filtered, and evaporated. The residue was purified by
column chromatography on silica gel, eluting with chloroform-
methanol (20:1) to give 1 (47 mg, 97%) as a dark purple solid: 1H
NMR (400 MHz, DMSO-d6) δ ) 8.61 (s, 1H), 8.29 (d, 1H, J )
8.2), 7.65-7.62 (m, 2H), 6.69 (d, 1H, J ) 9.2), 6.49 (s, 1H), 6.39
(s, 1H), 5.39 (dd, 1H, J ) 10.3, 5.9), 4.53-4.52 (m, 1H), 4.13-
4.10 (m, 1H), 3.93 (dd, 1H, J ) 11.7, 3.9), 3.84 (dd, 1H, J ) 11.7,
4.9), 3.48 (quartet, 4H, J ) 7.1), 2.41 (ddd, 1H, J ) 13.2, 5.7,
Oligodeoxynucleotide Synthesis and Characterization. Oli-
godeoxynucleosides (ODNs) were synthesized by a conventional
phosphoramidite method using an Applied Biosystems 392 DNA/
RNA synthesizer. ODNs were purified by reversed phase HPLC
on a 5-ODS-H column (10 mm × 150 mm), eluted with a solvent
mixture of 0.1 M triethylammonium acetate (TEAA), pH 7.0, linear
gradient over 30 min from 5% to 25% acetonitrile at a flow rate of
3.0 mL/min. The purity and concentration of the synthesized ODNs
containing modified nucleotides were determined by complete
digestion with calf intestine alkaline phosphatase (50 U/mL), snake
venom phosphodiesterase (0.15 U/mL), and P1 nuclease (50 U/mL)
to 2′-deoxymononucleosides at 37 °C for 3 h.
1.8), 2.14 (ddd, 1H, J ) 13.3, 10.1, 6.3), 1.27 (t, 6H, J ) 7.1); 13
C
NMR (100 MHz, DMSO-d6) δ ) 181.9, 151.9, 150.9, 146.6, 146.4,
138.4, 131.6, 131.0, 130.3, 127.5, 125.3, 124.3, 120.5, 110.3, 104.6,
96.1, 88.2, 79.1, 72.5, 62.5, 44.6, 43.7, 12.6; FABMS, m/z 434
[M+]; HRMS calcd for C25H26N2O5 [M+] 434.1842, found 434.1847.
1′-C-(2-Nile Red)-3′-O-(2-cyanoethyl-N,N′-diisopropylphos-
phoramidite)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyriboside (5). To
a solution of 1 (300 mg, 0.69 mmol) in pyridine (5 mL) was added
4,4′-dimethoxytrityl chloride (155 mg, 0.83 mmol), and the mixture
Melting Temperature (Tm) Measurement. Melting temperature
of the duplex (2.5 µM duplex concentration) was measured with a
spectrophotometer equipped with a Peltier temperature controller
using a 1-cm path length cell in a buffer containing 50 mM sodium
phosphate (pH 7.0) and 100 mM sodium chloride.
Thermodynamics of the Helix-to-coil Transition. The relation-
ship between melting temperatures of heteroduplexes and thermo-
dynamic parameters for the helix-to-coil transition is represented
J. Org. Chem, Vol. 71, No. 9, 2006 3597

Okamoto et al.
by the following equation:
1/Tm ) ∆S/∆H + R‚ln(Ct/4)/∆H
The value of the binding constant Kb for the inclusion complex
was calculated using the following equation:
(1)
1/(I - I0) ) 1/(Kb(I1 - I0)[CD] + 1/(I1 - I0′)
(2)
where [CD] represents the analytical concentration of â-cyclodex-
trin, I0 represents the fluorescence intensity of free Nile Red, I1 is
the fluorescence intensity of the inclusion complex, and I is the
observed fluorescence intensity at its maximum.
where Ct is the total strand concentration. In accordance with
eq 1, ∆H and ∆S were determined by plotting (Tm)-1 versus
ln(Ct/4). We measured the melting temperature of the sample
with varying total ODN strand concentrations; 10, 5, 2.5, 1.25
µM.
Fluorescence Measurement. All fluorescence spectra of ODN
duplexes (2.5 µM, duplex concentration) were taken in a buffer
containing 50 mM sodium phosphate (pH ) 7.0) and 100 mM
sodium chloride.
Supporting Information Available: 1H and 13C NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO060168O
3598 J. Org. Chem., Vol. 71, No. 9, 2006