PRODAN-conjugated DNA: Synthesis and photochemical properties

Article abstract of DOI:10.1021/ja069156a

A solvatochromic fluorophore, PRODAN, has been used as a microenvironment-sensitive reporter. Based on the chemistry of PRODAN, we designed and synthesized four novel fluorescent nucleosides, ^PDNX (X = U, C, A, and G), to which a PRODAN fluorop

Full text of DOI:10.1021/ja069156a

Published on Web 03/23/2007
PRODAN-Conjugated DNA: Synthesis and Photochemical
Properties
Kazuki Tainaka,† Kazuo Tanaka,† Shuji Ikeda,† Ken-ichiro Nishiza,‡ Tomo Unzai,‡
Yoshimasa Fujiwara,‡ Isao Saito,§ and Akimitsu Okamoto*,†,‡
Contribution from the Frontier Research System, RIKEN (The Institute of Physical and Chemical
Research), Wako, Saitama 351-1098, Japan, Department of Synthetic Chemistry and Biological
Chemistry, Faculty of Engineering, Kyoto UniVersity, Kyoto 615-8510, Japan, and NEWCAT
Institute, School of Engineering, Nihon UniVersity, and SORST, Japan Science and Technology
Agency, Tamura, Koriyama 963-8642, Japan
Received December 21, 2006; E-mail: aki-okamoto@riken.jp
Abstract: A solvatochromic fluorophore, PRODAN, has been used as a microenvironment-sensitive reporter.
Based on the chemistry of PRODAN, we designed and synthesized four novel fluorescent nucleosides,
PDNX (X ) U, C, A, and G), to which a PRODAN fluorophore was attached at pyrimidine C5 or purine C8.
The fluorescent nucleosides sensitively varied the Stokes shift values depending on the orientational
polarizability of the solvent. The PDNX incorporated into DNA also changed the Stokes shift values depending
on the DNA structure. In particular, the excitation spectrum of the PDNX-containing duplex shifted to a longer
wavelength and gave a smaller Stokes shift value when the base opposite PDNX could form a Watson-
Crick base pair with PDNX. A lower energy excitation of PDNX-containing DNA resulted in a strong fluorescence
emission selective to the Watson-Crick pairing base. This unique photochemical character was applicable
to the efficient typing of single-nucleotide polymorphisms of genes.
with micropolarity change in the surroundings.5 The most
important fluorescent characteristic of a solvatochromic fluoro-
Introduction
Local variations in the structure and dynamics of biomol-
ecules play important roles in mediating the interactions between
biomolecules. In particular, monitoring the microenvironmental
change in conformation, polarity, viscosity, and charge around
the DNA surface is essential in order to understand the great
variety of biological events involving DNA in cells. For such a
purpose, fluorescent probes that can be site-specifically incor-
porated into the DNA sequence of interest and are sensitive to
the change in its microenvironment are highly valuable and
desirable.1 Nucleosides possessing various fluorophores have
been explored to study the structures and dynamics of nucleic
acids, including fluorescent nucleoside analogues, as exemplified
by 2-aminopurine,2 ethynyl-extended nucleobase derivatives,3
and nucleoside analogues replaced by flat aromatic fluoro-
phores.4
phore is the linear correlation between the Stokes shifts of the
fluorophores and the orientational polarizabilities of the solvents,
based on the dipole interaction theory of Lippert and Mataga.6
The well-known solvatochromic fluorophore 6-propionyl-2-
dimethylaminonaphthalene (PRODAN) and its derivatives are
among the most promising candidates for such a microenvi-
ronment-sensitive fluorescent probe.7 The electronic transitions
(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) Hwang, G. T.; Seo, Y. J.;
Kim, S. J.; Kim, B. H. Tetrahedron Lett. 2004, 45, 3543-3546. (e) Saito,
Y.; Miyauchi, Y.; Okamoto, A.; Saito, I. Chem. Commun. 2004, 1704-
1705, (f) Okamoto, A.; Tainaka, K.; Saito, I. Bioconjugate Chem. 2005,
16, 1105-1111. (g) Wagner, C.; Rist, M.; Mayer-Enthart, E.; Wagenknecht,
H. A. Org. Biomol. Chem. 2005, 3, 2062-2063.
(4) (a) Stra¨ssler, C.; Davis, N. E.; Kool, E. T. HelV. Chim. Acta 1999, 82,
2160-2171. (b) Arzumanov, A.; Godde, F.; Moreau, S.; Toulme´, J.-J.;
Weeds, A.; Gait, M. J. HelV. Chim. Acta 2000, 83, 1424-1436. (c) Kool,
E. T. Acc. Chem. Res. 2002, 35, 936-943. (d) Okamoto, A.; Tainaka, K.;
Saito, I. J. Am. Chem. Soc. 2003, 125, 4972-4973. (e) Okamoto, A.;
Tainaka, K.; Saito, I. Tetrahedron Lett. 2003, 44, 6871-6874. (f) Okamoto,
A.; Tainaka, K.; Saito, I. Chem. Lett. 2003, 32, 684-685. (g) Gaied, N.
B.; Glasser, N.; Ramalanjaona, N.; Beltz, H.; Wolff, P.; Marquet, R.; Burger,
A.; Me´ly, Y. Nucleic Acids Res. 2005, 33, 1031-1039. (h) Okamoto, A.;
Tainaka, K.; Fujiwara, Y. J. Org. Chem. 2006, 71, 3592-3598.
(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.
Solvatochromic fluorophores, which show shifts in absorption
and emission bands induced by a change in solvent nature or
composition, may be applicable to monitoring the structural
transformation and intermolecular interaction of biomolecules
† RIKEN (The Institute of Physical and Chemical Research).
‡ Kyoto University.
§ Nihon University and SORST, Japan Science and Technology Agency.
(1) (a) Wojczewski, C.; Stolze, K.; Engels, J. W. Synlett 1999, 1667-1678.
(b) Hawkins, M. E. Cell Biochem. Biophys. 2001, 34, 257-281.
(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. (d) Kimura, T.; Kawai, K.; Fujitsuka,
M.; Majima, T. Chem. Commun. 2004, 1438-1439. (e) Tashiro, R.;
Sugiyama, H. J. Am. Chem. Soc. 2005, 127, 2094-2097.
(6) (a) Lippert, E. Z. Electrochem. 1957, 61, 962-975. (b) Mataga, N.; Kaifu,
Y.; Koizumi, M. Bull. Chem. Soc. Jpn. 1956, 29, 465-470.
(7) (a) Weber, G.; Farris, F. J. Biochemistry 1979, 18, 3075-3078. (b)
MacGregor, R. B.; Weber, G. Ann. N.Y. Acad. Sci. 1981, 366, 140-154.
(c) Prendergast, F. G.; Meyer, M.; Carlson, G. L.; Iida, S.; Potter, J. D. J.
Biol. Chem. 1983, 258, 7541-7544. (d) MacGregor, R. B.; Weber, G.
Nature 1986, 319, 70-73.
9
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J. AM. CHEM. SOC. 2007, 129, 4776-4784
10.1021/ja069156a CCC: $37.00 © 2007 American Chemical Society

PRODAN-Conjugated DNA
A R T I C L E S
Results and Discussion
Synthesis and Photochemical Properties of PDNU Nucleo-
side. Synthesis of the novel deoxyuridine-based fluorescent
nucleoside is outlined in Scheme 1. 6-Dimethylamino-2-
naphthaldehyde 1 was prepared according to the method
reported in the literature.10 Addition of lithium acetylide to 1
provided the corresponding coupling product, which was
subsequently deprotected with sodium methoxide to give 2. A
3′,5′-protected 5-iodo-2′-deoxyuridine was condensed with 2 to
yield compound 3 via Sonogashira coupling. Hydrogenation of
3 followed by oxidation with tetrapropylammonium perruthenate
produced 4, which was then desilylated to afford the free
nucleoside 5 (PDNU).
We initially examined the photochemical properties of PDN
U
nucleoside 5 in different solvents. The fluorescence excitation
spectra shifted depending on the nature of the solvent (Figure
2a and Table 1). From molecular orbital calculations, the first
absorption band of PRODAN has been characterized by three
different electronic transitions.11 Excitation bands around 360
and 380 nm of PDNU correspond to the π* f π transition and
the π* f n transition, respectively. The higher energy band
predominates in nonpolar solvents, whereas the lower energy
band contributes progressively more to the excitation spectra
in protic solvents. These spectral changes can be attributed to
specific interactions, such as hydrogen bonds, between PDNU
and solvent molecules. A series of emission spectra showed
solvent-dependent shifts of the fluorescence emission. Thus, the
Stokes shift, the difference in wavenumbers between excitation
maximum and emission maximum, greatly changed with solvent
polarity (Figure 2b and Table 1). The change in the Stokes shift
(∆ν) was roughly proportional to the orientational polarizability
(∆f) of the solvent, obeying the dipole interaction theory of
Lippert and Mataga (Figure 2c).6 PDNU in protic solvents showed
a larger solvatochromic shift, suggesting the existence of specific
solute-solvent interactions, such as hydrogen bonding.12 Such
solvatochromic behavior of PDNU would make possible the
fluorometric detection of the microstructural changes in DNA
through incorporation of PDNU into DNA and subsequent
monitoring of the photochemical behavior.
Figure 1. Structure of PRODAN-labeled nucleosides PDNX.
of PRODAN derivatives are strongly influenced by heteroge-
neous environmental factors in contact with the chromophore
in the ground state.8
The inside of the grooves of the DNA helix is more
hydrophobic than bulk water, and electron-rich heteroatoms of
the natural bases are exposed to the surface of the major groove.9
We presumed that a heterogeneous environmental change at a
specific site of DNA could be read out as fluorescent signals if
the PRODAN is anchored to the surface of the major groove.
Therefore, we have designed novel fluorescent nucleosides
possessing structures such that PRODAN is located at the
surface of the major groove of the duplex.
In this paper, we report the synthesis of a new type of
PRODAN-labeled nucleoside (PDNX, X ) U, C, A, and G) and
the photochemical properties of oligodeoxyribonucleotides
(ODNs) containing them (Figure 1). PDNX incorporated into
DNA exhibited unique photochemical behavior in sensitive
response to the nature of the complementary base of PDNX. The
shift of the excitation spectra of PRODAN as a function of the
base opposite PDNX resulted in the base-selective fluorescence
emission by PRODAN as the result of lower energy excitation.
This unique photochemical character is applicable to the efficient
typing of single-nucleotide polymorphisms in genes.
Scheme 1 a
a
Reagents and conditions: (a) trimethylsilylacetylene, 1.6 M n-BuLi, THF, -78 °C, 30 min, 92%; (b) 0.1 M sodium methoxide, methanol, room
temperature, 1 h, 97%; (c) 5-iodo-3′O,5′O-bis(tert-butyldimethylsilyl)-2′-deoxyuridine, tetrakis(triphenylphosphine)palladium(0), copper(I) iodide, triethylamine,
DMF, room temperature, 5 h, 93%; (d) 10% Pd/C, methanol, H2, room temperature, 9 h, 72%; (e) tetrapropylammonium perruthenate, 4-methylmorpholine
N-oxide, 4 Å MS, dichloromethane, room temperature, 1 h, 85%; (f) 1 M tetrabutylammonium fluoride, room temperature, 12 h, 82%; (g) 4,4′-dimethoxytrityl
chloride, pyridine, room temperature, 4 h, 93%; (h) (iPr2N)2PO(CH2)2CN, 1H-tetrazole, acetonitrile, room temperature, 30 min.
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A R T I C L E S
Tainaka et al.
Table 1. Photochemical Properties of PDNU Nucleoside 5 in
Different Solventsa
1
solvents
λex.max (nm)
λem.max (nm)
ΦF
∆ν (cm-
)
∆
f b
aprotic
449
465
462
472
HMPA
DMSO
DMF
acetonitrile
362
371
363
360
0.553
0.413
0.361
0.417
5373
5487
5918
6609
0.260
0.265
0.275
0.306
protic
481
508
491
524
2-propanol
glycol
ethanol
water
371
382
371
380
0.325
0.168
0.281
0.033
6164
6479
6600
7238
0.275
0.276
0.289
0.320
a Conditions: 2.5 µM PDNU nucleoside 5 in different solvents at 25 °C.
bOrientational polarizabilitiy (∆f) is defined by ∆f ) (ꢀ - 1)/(2ꢀ + 1) -
(n2 - 1)/(2n2 + 1).
Table 2. ODNs Used in This Study
sequences
ODN1(PDNU)
ODN1(PDNC)
ODN1(PDNA)
ODN1(PDNG)
ODN1′(N)
5′-d(CGCAACPDNUCAACGC)-3′
5′-d(CGCAACPDNCCAACGC)-3′
5′-d(CGCAACPDNACAACGC)-3′
5′-d(CGCAACPDNGCAACGC)-3′
5′-d(GCGTTGNGTTGCG)-3′
(N ) A, G, T, or C)
ODN2(PDNU)
ODN2′(N)
5′-d(CGCAATPDNUTAACGC)-3′
5′-d(GCGTTANATTGCG)-3′
(N ) A, G, T, or C)
ODNMTHFR
(
(
PDNU)
PyC)
5′-d(GAGPDNUCGATTTCAT)-3′
5′-d(GAGPyCCGATTTCAT)-3′
ODNMTHFR
between the chromophore and surroundings in the ground state
in addition to interaction with water molecules. As described
above, the higher energy band around 380 nm in the excitation
spectra corresponds to the π* f n transition with charge-transfer
character. Previous reports have suggested that this electronic
transition band might be greatly stabilized by interactions
between the dimethylamino group or carbonyl group of the
PRODAN chromophore and specific functional groups, such
as ester linkage, ether linkage, hydroxy residue, and amide
linkage.8 In the duplex, the PRODAN chromophore that is
attached to the uracil base at the C5 position via a short linker
could be anchored to the vicinity of the surface in the major
groove because of stable base pairing with an adenine base.
The bathochromic shift of the excitation spectra in the fully
matched duplex might originate from the electronic interactions
between the PRODAN chromophore and the heteroatoms
exposed on the surface of the DNA duplex. Although PDNU in
the single strand would also interact with the heteroatoms of
the DNA bases, a smaller bathochromic effect could result
because of the lack of a tight interaction by effective interchain
diffusion in the single-stranded state. The emission maxima of
the free nucleoside, the single-stranded ODN1(PDNU), and the
Figure 2. Photochemical behavior of PDNU nucleoside 5. (a) Normalized
excitation spectra in different solvents. Signal 1, HMPA; signal 2,
acetonitrile; signal 3, ethanol; signal 4, ethylene glycol. (b) Normalized
emission spectra in different solvents. Signal 1, HMPA; signal 2, acetonitrile;
signal 3, ethanol; signal 4, ethylene glycol. (c) Lippert plot of Stokes shift
in wavenumbers (∆ν) against the orientational polarizability (∆f) of aprotic
(b) and protic (2) solvents.
Photochemistry of PDNU-Containing DNA. PDNU nucleoside
5 was incorporated efficiently into an ODN according to a
conventional phosphoramidite method after protection of 5 and
conversion into the phosphoramidite form 6 (Scheme 1). We
examined the photochemical properties of the single-stranded
ODN1(PDNU), 5′-d(CGCAACPDNUCAACGC)-3′, and the du-
plex formed by hybridization with ODN1′(A), 5′-d(GCGT-
TGAGTTGCG)-3′ (Table 2). Excitation maxima of PDNU were
observed at 380 nm in the free nucleoside, at 389 nm in single-
stranded ODN, and at 404 nm in the duplex form. The excitation
spectra of PDNU-containing ODNs compared with the free
nucleoside PDNU in aqueous solution showed larger batho-
chromic shifts (Figure 3a and Table 3). The origin of the shift
in the excitation spectra was ascribed to additional interactions
(8) (a) Viard, M.; Gallay, J.; Vincent, M.; Meyer, O.; Robert, B.; Paternostre,
M. Biophys. J. 1997, 73, 2221-2234. (b) Parasassi, T.; Krasnowska, E.
K.; Bagatolli, L.; Gratton, E. J. Fluorescence 1998, 8, 365-373. (c)
Bagatolli, L. A.; Parasassi, T.; Fidelio, G. D.; Gratton, E. Photochem.
Photobiol. 1999, 70, 557-564. (d) Sire, O.; Alpert, B.; Royer, C. A.
Biophys. J. 1996, 70, 2903-2914.
(9) (a) Lamm, G.; Pack, G. R. J. Phys. Chem. 1997, 101, 959-965. (b) Young,
M. A.; Jayaram, B.; Beveridge, D. L. J. Phys. Chem. B 1998, 102, 7666-
7669.
(10) List, B.; Barbas, C. F., III; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 15351-15355.
(11) Ilich, P.; Prendergast, F. G. J. Phys. Chem. 1989, 93, 4441-4447.
(12) (a) Jozefowicz, M.; Kozyra, K. A.; Heldt, J. R.; Heldt, J. Chem. Phys.
2005, 320, 45-53. (b) Zurawsky, W. P.; Scarlata, S. F. J. Phys. Chem.
1992, 96, 6012-6016.
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PRODAN-Conjugated DNA
A R T I C L E S
spectra on emission wavelength suggests that the photochemical
behavior of the DNA-tethered chromophore would be affected
by heterogeneous interactions not only with bulk water mol-
ecules but also with other factors, such as the identity of the
heteroatoms of the DNA bases.
The effect on the Stokes shift of the heterogeneity in the
microenvironment around PDNU may be increased by a large
restriction in mobility due to stable base pair formation of PDN
U
incorporated into a fully matched duplex. Therefore, we assumed
that when the complementary base of PDNU is a mismatched
base (i.e., N ) C, G, and T), an increase in the flexibility of
PDNU due to the failure of base pairing would result in a decrease
in heterogeneous interactions. Before measuring the excitation
and emission spectra, we examined the duplex stabilities of
ODN1(PDNU) hybridized with the complementary strand
ODN1′(N) (N ) A, C, G, or T) (Table 3). The melting
temperature (Tm) of ODN1(PDNU)/ODN1′(A) (60 °C) was close
to the Tm of the “native” T/A duplex (61 °C), showing that the
PRODAN-labeled DNA formed a thermally stable duplex. On
the other hand, the Tm values observed for mismatched duplexes
were 4 to 9 °C lower than that of ODN1(PDNU)/ODN1′(A), and
the stability of mismatched duplexes decreased in the order N
) G, T, and C of ODN1′(N).
The destabilization of duplexes due to the failure of base
pairing influenced the photochemical behavior of ODN1(PDNU).
Measurements of the excitation spectra of 2.5 µM of the
ODN1(PDNU)/ODN1′(N) duplex in sodium phosphate buffer
(pH ) 7.0) were performed at the emission wavelength of 520
nm (Figure 4). The excitation spectra of the mismatched duplex
were changed significantly by the nature of the complementary
base. When the base opposite PDNU is C, G, and T, excitation
maxima were observed at 390, 400, and 398 nm, respectively.
These wavelengths were shorter than the excitation maximum
of the matched duplex ODN1(PDNU)/ODN1′ (A). The unique
spectral shifts in the excitation spectra are valuable for
discrimination of the nature of the base located at the opposite
site of PDNU by use of red-edge excitation. The absorbance of
ODN1(PDNU)/ODN1′ (A) at 450 nm was considerably larger
than those of other duplexes. The fluorescence spectrum of
ODN1(PDNU)/ODN1′(A) exhibited a strong fluorescence at 522
nm on excitation at 450 nm (ΦF ) 0.201), whereas the
fluorescence of single-stranded ODN1(PDNU) and mismatched
ODN1(PDNU)/ODN1′(N) (N ) C, G, or T) duplexes were much
weaker (Figure 4). The emission maximum in mismatched
duplexes was slightly affected by alteration of the complemen-
tary base of PDNU (λem ) 518-521 nm), and based on these
data, the Stokes shifts of mismatched duplexes were calculated
as 6373, 5806, and 5858 cm-1 for N ) C, G, and T, respectively.
The Stokes shift increased with an increase in the thermal
stability of the duplexes. This relationship indicates that the
formation of a stable base pair may be correlated with the
generation of interactions between the PRODAN of PDNU and
heteroatoms of the groove surface affecting the decrease in
Stokes shift.
Figure 3. Comparison of the fluorescence excitation and emission spectra
of PDNU nucleoside, PDNU in a single-stranded DNA, and PDNU in a duplex
DNA. (a) Fluorescence excitation (left) and emission (right) spectra of
nucleoside 5 (1, blue), single-stranded (2, black), and duplex (3, red).
Excitation spectra were measured for emission at a wavelength of 520 nm,
and emission spectra were excited at 380, 389, and 404 nm for nucleoside,
single-stranded, and duplex, respectively. (b) Blue edge effect. Fluorescence
excitation maxima of nucleoside 5 (1, blue), single-stranded (2, black), and
duplex (3, red) are plotted against the emission wavelength at the blue edge
of the fluorescence spectra.
duplex ODN1(PDNU)/ODN1′(A) were observed at 524, 520, and
522 nm, respectively, and thus their Stokes shift values showed
great variation, corresponding to 7238, 6476, and 5559 cm-1
,
respectively. Smaller Stokes shifts might indicate that the
position of the PRODAN chromophore would be restricted to
the vicinity of the hydrophobic surface in the major groove.
The restriction in mobility of the fluorophore upon binding to
DNA gives rise to the viscosity effect in the photochemical
properties.13 In general, when the fluorophore positioned in rigid
and viscous media can be stabilized by heterogeneous interac-
tions with the surroundings in the ground state, the excitation
spectra measured at the blue edge of the fluorescence signal is
shifted to shorter wavelengths as a function of emission
wavelength.14 Figure 3b shows a plot of excitation maximum
against each wavelength at the blue edge region of the
fluorescence spectra. Excitation spectra of the PDNU nucleoside
exhibited little change, whereas large bathochromic shifts of
the excitation maximum were observed in the case of PDNU
incorporated into DNA (∆λex ) 5 nm in the single-stranded
state, ∆λex ) 11 nm in the duplex). The dependence of excitation
The Stokes shift of PDNU varied greatly depending on the
nature of the base opposite PDNU. On the other hand, the effect
of the flanking base pair of PDNU on the Stokes shift was small.
ODNs containing the -TPDNUT- sequence, ODN2(PDNU), were
prepared, and the fluorescence of duplexes hybridized with the
complementary strand ODN2′(N) was measured. The photo-
(13) (a) Okamoto, A.; Tainaka, K.; Nishiza, K.-i.; Saito, I. J. Am. Chem. Soc.
2005, 127, 13128-13129. (b) Balter, A. J. Fluorescence 1997, 7, S99-
105. (c) Bajorek, A.; Pa¸czkowski, J. Macromolecules 1998, 31, 86-
95.
(14) (a) Demchenko, A. P. Luminescence 2002, 17, 19-42. (b) Pavlovich, V.
S. Zh. Prikl. Spektr. 1976, 25, 480-487.
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A R T I C L E S
Tainaka et al.
Table 3. Photochemical Propertiesa and Melting Temperatures (Tm)b of PDNU-Containing ODNs
c
1
entry
ODN
λ
ex. max (nm)
λ
em. max (nm)
Irel
ΦF
∆ν (cm-
)
Tm (°C)
1
2
3
4
5
ODN1(PDNU)
389
404
390
400
398
520
522
518
521
519
0.273
1
0.289
0.392
0.290
0.179
0.201
0.115
0.068
0.073
6476
5559
6373
5806
5858
ODN1(PDNU)/ODN1′(A)
ODN1(PDNU)/ODN1′(C)
ODN1(PDNU)/ODN1′(G)
ODN1(PDNU)/ODN1′(T)
60.0
50.7
56.3
53.5
6
7
8
9
ODN2(PDNU)
389
406
395
401
399
517
518
518
518
516
0.243
1
0.346
0.344
0.406
0.077
0.212
0.143
0.073
0.091
6365
5326
6011
5634
5689
ODN2(PDNU)/ODN2′(A)
ODN2(PDNU)/ODN2′(C)
ODN2(PDNU)/ODN2′(G)
ODN2(PDNU)/ODN2′(T)
50.6
42.7
46.1
45.9
10
b
a Conditions: The duplexes (2.5 µM) were measured in 50 mM sodium phosphate (pH ) 7.0) and 0.1 M sodium chloride at 25 °C. The absorbance of
c
the samples was monitored at 260 nm from 10 to 90 °C. Relative fluorescence intensities were monitored at 520 nm after excitation at 450 nm.
Figure 5. Fluorescence excitation and emission spectra of ODN1(PDNC)/
Figure 4. Fluorescence excitation and emission spectra of ODN1(PDNU)/
ODN1′(N). Normalized excitation (left) and relative emission (right) spectra
of 2.5 µM ODN1(PDNC) hybridized with 2.5 µM ODN1′(N) were measured
ODN1′(N). Normalized excitation (left) and relative emission (right) spectra
of 2.5 µM ODN1(PDNU) hybridized with 2.5 µM ODN1′(N) were measured
in 50 mM sodium phosphate (pH ) 7.0) and 0.1 M sodium chloride at 25
°C. “s.s.” denotes a single-stranded ODN1(PDNC). Excitation spectra were
measured for the 450 nm emission wavelength, and excitation spectra were
measured for the 520 nm emission wavelength.
in 50 mM sodium phosphate (pH ) 7.0) and 0.1 M sodium chloride at 25
°C. “s.s.” denotes a single-stranded ODN1(PDNU). Excitation spectra were
measured for the 450 nm emission wavelength, and excitation spectra were
measured for the 520 nm emission wavelength.
Oxidation of 8 with tetrapropylammonium perruthenate pro-
duced PRODAN-labeled C derivative 9. The protective group
of 9 was converted from a labile formamidine group15 to an
acetyl group, which was subsequently desilylated to give 10.
The deoxyribose 5′-hydroxy group of 10 was protected with
the 4,4′-dimethoxytrityl group, and then the resulting nucleoside
was converted into the phosphoramidite 11. The PDNC-containing
ODN, ODN1(PDNC), was synthesized using 11, and then the
excitation and emission spectra of ODN1(PDNC) hybridized with
ODN1′(N) were measured (Figure 5). The excitation maxima
of single-stranded ODN1(PDNC) and mismatched duplexes were
observed at 388-391 nm. On the other hand, the excitation
maximum of ODN1(PDNC)/ODN1′(G) was observed at 410 nm,
a shift to a significantly longer wavelength of approximately
20 nm. In addition, the bathochromic effect on the excitation
spectrum in a fully matched duplex resulted in the lowest value
of Stokes shift (∆ν ) 5522 cm-1), while the Stokes shifts of
other duplexes were calculated to be within the range 6491 to
6762 cm-1. The melting temperature of ODN1(PDNC)/ODN1′(G)
was 9 to 11 °C higher than that of mismatched duplexes (Table
4), suggesting that the base pair stability affects the variation
of Stokes shift through heterogeneous microenvironmental
factors in contact with the PRODAN chromophore. The use of
chemical properties of the duplexes are summarized in Table
3. The melting temperature of ODN2(PDNU)/ODN2′(A) was 4
to 8 °C higher than those of mismatched duplexes. The higher
thermal stability of the duplex was followed by the bathochromic
shift of the excitation spectra and the decrease in the value of
the Stokes shift in a similar fashion to the photochemical
behavior observed for ODN1(PDNU)/ODN1′(A). The fluores-
cence intensity of the fully matched ODN2(PDNU)/ODN2′(A)
duplex was relatively high (ΦF ) 0.212) on excitation at 450
nm, whereas the fluorescence emissions of the single-stranded
ODN2(PDNU) and mismatched ODN2(PDNU)/ODN2′(N) (N )
C, G, or T) duplexes were suppressed. The fluorescence behavior
of ODN2(PDNU) suggests that the effect of the nature of the
neighboring base pair on the photochemical behavior of PDNU
is not significant.
Photochemistry of PDNC-Containing DNA. Because the
novel fluorescent deoxyuridine PDNU displayed a sharp change
in the Stokes shift in response to the nature of the complemen-
tary base, we assumed that the analogous C derivative (PDNC)
would also exhibit a complementary base-dependent Stokes shift
change in a similar fashion as PDNU. The synthetic route for
PDNC is outlined in Scheme S1 (Supporting Information). The
fluorophore precursor 2 was coupled with 3′,5′-bissilyl-5-iodo-
2′-deoxycytidine under Sonogashira coupling conditions to
produce 7. After hydrogenation of 7, the C4 amino group of
cytosine was protected by a formamidine group to yield 8.
(15) (a) Zemlicka, J.; Chla´dek, S.; Holy, A.; Smrt, J. Collect. Czech. Chem.
Commun. 1966, 31, 3198-3211. (b) Vincent, S.; Mioskowski, C.; Lebeau,
L. J. Org. Chem. 1999, 64, 991-997.
9
4780 J. AM. CHEM. SOC. VOL. 129, NO. 15, 2007

PRODAN-Conjugated DNA
A R T I C L E S
Table 4. Photochemical Propertiesa and Melting Temperatures (Tm)b of PDNX-Containing ODNs
c
1
entry
ODN
λex.max (nm)
λem.max (nm)
Irel
ΦF
∆ν (cm-
)
Tm(°C)
1
2
3
4
5
ODN1(PDNC)
390
391
388
410
391
525
525
526
530
524
0.410
0.328
0.365
1
0.070
0.053
0.055
0.077
0.065
6593
6528
6762
5522
6491
ODN1(PDNC)/ODN1′(A)
ODN1(PDNC)/ODN1′(C)
ODN1(PDNC)/ODN1′(G)
ODN1(PDNC)/ODN1′(T)
55.6
54.5
65.3
56.2
0.471
6
7
8
9
ODN1(PDNA)
391
382
394
397
405
522
526
526
522
517
0.260
0.179
0.322
0.664
1
0.096
0.080
0.069
0.167
0.202
6418
7167
6369
6032
5349
ODN1(PDNA)/ODN1′(A)
ODN1(PDNA)/ODN1′(C)
ODN1(PDNA)/ODN1′(G)
ODN1(PDNA)/ODN1′(T)
57.6
55.8
56.7
60.3
10
11
12
13
14
15
ODN1(PDNG)
391
373
406
381
398
524
515
519
521
524
0.290
0.167
1
0.191
0.750
0.121
0.072
0.128
0.097
0.113
6491
7392
5363
7053
6042
ODN1(PDNG)/ODN1′(A)
ODN1(PDNG)/ODN1′(C)
ODN1(PDNG)/ODN1′(G)
ODN1(PDNG)/ODN1′(T)
57.5
64.4
60.3
57.9
b
a Conditions: The duplexes (2.5 µM) were measured in 50 mM sodium phosphate (pH ) 7.0) and 0.1 M sodium chloride at 25 °C. The absorbance of
c
the samples was monitored at 260 nm from 10 to 90 °C. Relative fluorescence intensities were monitored at 520 nm after excitation at 450 nm.
excitation at a longer wavelength (λex ) 450 nm) gave a highly
G-selective fluorescence emission from ODN1(PDNC)/ODN1′(N)
(Figure 5).
Photochemistry of PDNA- and PDNG-Containing DNA.
PRODAN tethered to pyrimidine C6 resided in a duplex major
groove and resulted in a base-selective decrease in the Stokes
shift. Therefore, because substituents attached at the purine C8
position are placed at the side of the major groove, the purine
derivatives, PDNA and PDNG, in which PRODAN was attached
to the purine C8 position, should exhibit photochemical character
in a similar fashion to those observed for PDNU and PDNC. These
fluorescent purines were synthesized according to Scheme S2
(Supporting Information), and the fluorescence and thermal
denaturation experiments were conducted under the conditions
described above. The Stokes shifts of PDNA and PDNG were also
significantly changed depending on the nature of the opposite
base (Table 4 and Figure 6). The bathochromic effect on the
excitation spectra in fully matched duplexes resulted in the
lowest value of Stokes shift (∆ν ) 5349 and 5363 cm-1 for
PDNA and PDNG, respectively). On the other hand, the maxima
of the excitation spectra and the Stokes shift values of
mismatched duplexes varied widely. The ODN1(PDNA)/
ODN1′(A) and ODN1(PDNG)/ODN1′(A) duplexes exhibited
very large Stokes shifts, whereas the relatively small Stokes
shifts of ODN1(PDNA)/ODN1′(G) and ODN1(PDNG)/ODN1′(T)
showed significantly high fluorescence intensities on excitation
at 450 nm. Substitution at purine C8 may induce the formation
of wobble Watson-Crick base pairs or Hoogsteen base pairs
by a conformational transition,16 which modulates the interaction
between the chromophore and the microenvironment factors.
Figure 6. Fluorescence excitation and emission spectra of ODN1(PDNX)/
Application to Multicolored Genotyping. These marked
ODN1′(N) (X ) (a) A and (b) G). Normalized excitation (left) and relative
changes in the photochemical behavior of PDNX-containing
DNAs that are dependent on the nature of the complementary
base may play a key role in a facile genotyping assay. We have
previously developed base-discriminating fluorescent nucleo-
sides (BDF nucleosides) that change their fluorescence intensity
depending on the nature of the nucleoside pairing with the BDF
nucleoside.3c,17 By hybridization of a probe containing a BDF
nucleoside with a target DNA, the type of single-nucleotide
polymorphism (SNP) base in the target DNA was fluorometri-
emission (right) spectra of 2.5 µM ODN1(PDNX) hybridized with 2.5 µM
ODN1′(N) were measured in 50 mM sodium phosphate (pH ) 7.0) and
0.1 M sodium chloride at 25 °C. “s.s.” denotes a single-stranded ODN1(PDNX).
Excitation spectra were measured for the 450 nm emission wavelength,
and excitation spectra were measured for the 520 nm emission wavelength.
cally identified with high accuracy. A DNA probe containing a
typical BDF nucleoside PyC exhibits a strong fluorescence when
a
PyC/G base pair is formed by hybridization with a comple-
(17) (a) Okamoto, A.; Saito, Y.; Saito, I. J. Photochem. Photobiol. C 2005, 6,
108-122. (b) Okamoto, A. Bull. Chem. Soc. Jpn. 2005, 78, 2083-2097.
(c) Okamoto, A.; Tainaka, K.; Ochi, Y.; Kanatani, K.; Saito, I. Mol. BioSyst.
2006, 2, 122-127.
(16) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I., Jr. Nucleic Acids: Structures,
Properties, and Functions; University Science Books: Sausalito, CA, 2000.
9
J. AM. CHEM. SOC. VOL. 129, NO. 15, 2007 4781

A R T I C L E S
Tainaka et al.
(Figure 7). The SNP typing results obtained using the BDF
method were in good agreement with those obtained by direct
sequencing.
In conclusion, we have synthesized four PRODAN-labeled
nucleosides PDNX and examined their photochemical properties.
Their Stokes shift values significantly changed depending on
the solvent orientational polarizability and DNA structure. PDN
X
fixed on a fully matched duplex exhibited an excitation spectrum
at a longer wavelength and a smaller Stokes shift value,
compared with those of PDNX in a single-stranded state and
mismatched duplexes. The bathochromic shift of the excitation
spectrum of PDNX and lower energy excitation resulted in
fluorescence emissions selective to the Watson-Crick pairing
base. The base-selective emission of PDNU facilitated multi-
colored SNP typing through the combination with BDF probes
with a shorter emission wavelength. The photochemical property
exhibited by PRODAN fixed on DNA is not only very unique
but also applicable to the design of new fluorescent DNA probes
for DNA sequencing, genotyping, monitoring of DNA structural
transition, and observation of microenvironmental change at-
tended by biomolecular interaction.
Figure 7. Structure of PyC and cluster diagram showing the genotype
assignment for an hMTHFR C677T SNP in 25 samples using PDNU- and
PyC-probes. The samples amplified by asymmetric PCR were added to a
solution of 2.5 mM probes in a 50 mM sodium phosphate (pH ) 7.0) and
100 mM sodium chloride on a 1536-well microtiter plate. Equipment
used: a 440 ( 10 nm excitation band-pass filter and a 510 ( 10 nm
Experimental Section
General. NMR spectra were measured with Varian Mercury 400
and JEOL JNM-GX 400 spectrometers. Coupling constants (J value)
are reported in hertz. The chemical shifts are shown in ppm downfield
from tetramethylsilane, using residual chloroform (δ ) 7.26 in 1H NMR,
emission band-pass filter for ODNMTHFR(
PDNU); a 340 ( 10 nm excitation
δ ) 77.0 in 13C NMR) and dimethylsulfoxide (δ ) 2.48 in H NMR,
1
band-pass filter and a 410 ( 5 nm emission band-pass filter for
δ ) 39.5 in 13C NMR) as an internal standard. FAB mass spectra were
recorded on a JEOL JMS HX-110A spectrometer. Masses of ODNs
were determined with a MALDI-TOF MS (Perseptive Voyager Elite,
acceleration voltage 21 kV, negative mode) with 2′,3′,4′-trihydroxy-
acetophenone as a matrix, using T8 ([M - H]- 2370.61) and T17 ([M
- H]- 5108.37) as an internal standard for ODNs. Calf intestine alkaline
phosphatase (Promega), Crotalus adamanteus venom phosphodiesterase
I (USB), and Penicillium citrinum nuclease P1 (Roche) were used for
the enzymatic digestion of ODNs.
ODNMTHFR
0.1 s.
(
PyC); lamp energy ) 7000 (× 75 × 2-16 W); count time )
mentary strand (λex ) 329 nm, λem ) 393 nm), whereas the
fluorescence is very weak when PyC forms a base pair with A,
C, or T.3c A mixture of probes that have different wavelengths
of excitation and emission would enable the use of multicolored
sensing of gene SNP sites. By addition of the mixture of PDNU-
and PyC-labeled probes to a single well containing a gene
sample, the type of base at a specific SNP site will be fluoro-
metrically determinable in a single cuvette. Using PDNU- and
PyC-containing BDF probes, we tested a BDF genotyping assay
for a human methylenetetrahydrofolate reductase (hMTHFR)
gene containing a C677T mutation, 5 ′-d(...ATGAAATCG-
PCTC...)-3′ (P ) G/A SNP site), which is associated with cor-
onary artery disease.18 We prepared BDF probes ODNMTHFR(B)
5′-d(GAGBCGATTTCAT)-3′ (B ) PDNU or PyC) for typing of
an hMTHFR C677T SNP site. An SNP site containing a 109-
nt fragment of the antisense strand of hMTHFR was amplified
by asymmetric PCR and was added to a mixture containing both
probes in 50 mM sodium phosphate (pH ) 7.0) and 100 mM
6-Dimethylamino-2-(1-hydroxy-2-propyn-1-yl)naphthalene (2).
To a solution of trimethylsilylacetylene (453 µL, 3.3 mmol) in THF
(10 mL) was added n-butyllithium (1.6 M solution in hexane, 2.0 mL,
3.3 mmol) at -78 °C, and the reaction mixture was stirred at room
temperature for 30 min. After addition of 6-dimethylamino-2-formyl-
naphthalene (500 mg, 2.5 mmol) in THF (20 mL) at -78 °C, the
reaction mixture was stirred at 0 °C for 30 min. The resulting mixture
was diluted with sat. aq. NH4Cl at 0 °C, extracted with ethyl acetate.
The organic phase was washed with brine, dried over anhydrous
MgSO4, filtered, and concentrated in Vacuo. The crude product was
purified by silica gel column chromatography (hexane-ethyl acetate
) 5:1) to yield the adduct (683 mg, 2.3 mmol, 92%) as a yellow solid:
1H NMR (CDCl3, 400 MHz) δ 7.80 (s, 1H), 7.69-7.64 (m, 2H), 7.52
(dd, 1H, J ) 1.7, 8.5 Hz), 7.15 (dd, 1H, J ) 2.5, 9.1 Hz), 6.91 (s, 1H),
3.03 (s, 6H), 2.33 (brs, 1H), 0.21 (s, 9H); 13C NMR (CDCl3, 100 MHz)
δ 148.9, 134.8, 133.9, 129.0, 126.8, 126.3, 125.4, 125.1, 116.6, 106.4,
105.3, 91.4, 65.3, 40.9, -0.1; FABMS (NBA/CHCl3), m/z 297 (M+),
HRMS calcd for C18H23ONSi (M+) 297.1549, found 297.1550.
A mixture of the adduct (683 mg, 2.3 mmol) and sodium methoxide
(100 mM in methanol, 20 mL) was stirred at room temperature for 1
h. The reaction mixture was neutralized with 1 M HCl and extracted
with ethyl acetate. The organic phase was washed with brine, dried
over anhydrous MgSO4, filtered, and concentrated in Vacuo. The crude
product was purified by silica gel column chromatography (hexane-
ethyl acetate ) 3:1) to yield 2 (500 mg, 2.2 mmol, 97%) as a yellow
syrup: 1H NMR (CDCl3, 400 MHz) δ 7.85 (s, 1H), 7.72 (d, 1H, J )
9.0 Hz), 7.68 (d, 1H, J ) 8.6 Hz), 7.54 (dd, 1H, J ) 1.6, 8.4 Hz), 7.18
sodium chloride. The fluorescence from ODNMTHFR(
PDNU)
was detected through a 440 ( 10 nm excitation filter and a
510 ( 10 nm emission filter, and the fluorescence from
Py
ODN
( C) was detected through a 340 ( 10 nm excita-
MTHFR
tion filter and a 410 ( 5 nm emission filter. When the
fluorescence intensity of each probe was plotted on an x-y plot,
all the samples clearly separated into three clusters, which were
identified as G-homozygotes, A-homozygotes, and heterozygotes
(18) (a) Girelli, D.; Friso, S.; Trabetti, E.; Olivieri, O.; Russo, C.; Pessotto, R.;
Faccini, G.; Pignatti, P. F.; Mazzucco, A.; Corrocher, R. Blood 1998, 91,
4158-4163. (b) Brody, L. C.; Conley, M.; Cox, C.; Kirke, P. N.; McKeever,
M. P.; Mills, J. L.; Molloy, A. M.; O’Leary, V. B.; Parle-McDermott, A.;
Scott, J. M.; Swanson, D. A. Am. J. Hum. Genet. 2002, 71, 1207-1215.
9
4782 J. AM. CHEM. SOC. VOL. 129, NO. 15, 2007

PRODAN-Conjugated DNA
A R T I C L E S
(dd, 1H, J ) 2.2, 9.0 Hz), 6.92 (s, 1H), 5.57 (s, 1H), 3.06 (s, 6H), 2.71
(d, 1H, J ) 2.2 Hz), 2.25 (brs, 1H); 13C NMR (CDCl3, 100 MHz) δ
149.0, 134.9, 133.5, 129.0, 126.9, 126.3, 125.3, 124.9, 116.7, 106.3,
83.9, 74.6, 64.7, 40.8; FABMS (NBA/CHCl3) m/z 225 (M+), HRMS
calcd for C15H15ON (M+) 225.1154, found 225.1150.
1H NMR (CDCl3, 400 MHz) δ 8.33 (s, 1H), 8.16 (brs, 1H), 7.90 (dd,
1H, J ) 1.7, 8.7 Hz), 7.78 (d, 1H, J ) 9.2 Hz), 7.63-7.59 (m, 2H),
7.16 (dd, 1H, J ) 2.5, 9.1 Hz), 6.86 (d, 1H, J ) 2.2 Hz), 6.31 (dd, 1H,
J ) 5.9, 7.7 Hz), 4.42 (dt, 1H, J ) 2.7, 5.7 Hz), 3.94 (q, 1H, J ) 3.0
Hz), 3.84 (dd, 1H, J ) 3.6, 11.3 Hz), 3.78 (dd, 1H, J ) 3.3, 11.2 Hz),
3.41-3.24 (m, 2H), 3.10 (s, 6H), 2.79 (t, 2H, J ) 7.3 Hz), 2.23 (ddd,
1H, J ) 2.7, 5.8, 13.1 Hz), 2.02 (ddd, 1H, J ) 5.9, 7.7, 13.4 Hz),
0.92-0.90 (m, 18H), 0.11-0.07 (m, 12H); 13C NMR (CDCl3, 100
MHz) δ 198.3, 163.2, 150.2, 150.0, 137.6, 137.2, 130.7, 130.3, 130.0,
126.1, 125.1, 124.4, 116.3, 113.8, 105.3, 87.8, 84.9, 72.3, 63.1, 40.9,
40.4, 36.9, 25.9, 25.7, 22.7, 18.4, 18.0, -4.7, -4.8, -5.3, -5.4;
FABMS (NBA/CHCl3) m/z 682 ([M + H]+), HRMS calcd for
C36H56O6N3Si2 ([M + H]+) 682.3708, found 682.3704.
5-PRODAN-2′-deoxyuridine (5). To a solution of 4 (343 mg, 0.50
mmol) in THF (5 mL) was added tetrabutylammonium fluoride (1 M
solution in THF, 1.1 mL, 1.1 mmol). The mixture was stirred at room
temperature for 12 h. The resulting mixture was evaporated and purified
by silica gel column chromatography (chloroform-methanol ) 20:1)
to yield 5 (186 mg, 0.41 mmol, 82%) as a lemon yellow solid: 1H
NMR (DMSO-d6, 400 MHz) δ 11.4 (brs, 1H), 8.46 (s, 1H), 7.89 (d,
1H, J ) 9.2 Hz), 7.82 (dd, 1H, J ) 1.7, 8.7 Hz), 7.79 (s, 1H), 7.67 (d,
1H, J ) 8.8 Hz), 7.27 (dd, 1H, J ) 2.6, 9.2 Hz), 6.94 (d, 1H, J ) 2.4
Hz), 6.17 (t, 1H, J ) 6.9 Hz), 5.27 (brs, 1H), 5.09 (brs, 1H), 4.24 (m,
1H), 3.76 (q, 1H, J ) 3.5 Hz), 3.61-3.53 (m, 2H), 3.27-3.22 (m,
2H), 3.05 (s, 6H), 2.57 (t, 2H, J ) 7.6 Hz), 2.14-2.03 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 198.2, 163.4, 150.3, 150.1, 137.2, 136.7,
130.6, 129.9, 129.6, 125.9, 124.5, 123.8, 116.4, 112.7, 104.7, 87.3,
83.9, 70.3, 61.2, 39.9, 39.4, 36.6, 21.8; FABMS (NBA/DMSO) m/z
454 ([M + H]+), HRMS calcd for C24H28O6N3 ([M + H]+) 454.1978,
found 454.1978.
5-[3-(6-Dimethylaminonaphthalen-2-yl)-3-hydroxy-1-propyn-1-
yl]-3′O,5′O-bis(tert-butyldimethylsilyl)-2′-deoxyuridine (3). To a
solution of 5-iodo-3′ O,5′O-bis(tert-butyldimethylsilyl)-2′-deoxyuridine
(2.9 g, 5.0 mmol), 2 (915 mg, 4.1 mmol), and triethylamine (1.7 mL,
12 mmol) in DMF (20 mL) were added tetrakis(triphenylphosphine)-
palladium(0) (950 mg, 0.82 mmol) and copper(I) iodide (310 mg, 1.6
mmol) under nitrogen. The mixture was stirred at room temperature
for 5 h. The resulting mixture was concentrated in Vacuo and diluted
with ethyl acetate. This solution was washed with 5% (w/v) EDTA
solution and 5% (w/v) sodium bisulfite solution, dried over Na2SO4,
filtered, and evaporated. The crude product was purified by silica gel
column chromatography (toluene-ethyl acetate ) 4:1) to yield 3 (3.2
g, 4.65 mmol, 93%) as a yellow solid: 1H NMR (CDCl3, 400 MHz) δ
8.59 (brs, 1H), 7.98 (d, 1H, J ) 3.3 Hz), 7.88 (s, 1H), 7.71 (d, 1H, J
) 9.0 Hz), 7.64 (d, 1H, J ) 9.6 Hz), 7.56 (dd, 1H, J ) 1.6, 8.5 Hz),
7.15 (dd, 1H, J ) 2.5, 9.1 Hz), 6.90 (d, 1H, J ) 2.0 Hz), 6.27 (dd, 1H,
J ) 5.8, 7.6 Hz), 5.73 (s, 1H), 4.39-4.37 (m, 1H), 3.97-3.96 (m,
1H), 3.85 (ddd, 1H, J ) 2.6, 5.4, 11.4 Hz), 3.74-3.71 (m, 1H), 3.04
(s, 6H), 2.69 (brs, 1H), 2.30 (ddd, 1H, J ) 2.5, 5.8, 13.1 Hz), 2.04-
1.96 (m, 1H), 0.89-0.83 (m, 18H), 0.08-0.02 (m, 12H); 13C NMR
(CDCl3, 100 MHz) δ 161.2, 149.0, 142.5, 134.9, 133.7, 129.1, 126.8,
126.43, 126.42, 125.52, 125.46, 125.12, 125.07, 116.6, 106.3, 99.6,
94.2, 88.4, 85.91, 85.88, 77.6, 77.5, 72.4, 65.3, 63.0, 42.0, 40.8, 25.9,
25.7, 18.3, 18.0, -4.8, -4.7, -5.4, -5.6; FABMS (NBA/CHCl3) m/z
679 (M+), HRMS calcd for C36H53O6N3Si2 (M+) 679.3473, found
679.3475.
5-PRODAN-5′O-(4,4′-dimethoxytrityl)-2′-deoxyuridine 3′O-(2-
cyanoethyl)-N,N-diisopropylphosphoramidite (6). To a solution of
5 (166 mg, 0.40 mmol) in pyridine (5 mL) was added 4,4′-dimethoxy-
trityl chloride (174 mg, 0.51 mmol). The mixture was stirred at room
temperature for 4 h. The resulting mixture was evaporated and purified
by silica gel column chromatography (ethyl acetate) to yield the
tritylated product (278 mg, 0.37 mmol, 93%) as a lemon yellow solid:
1H NMR (DMSO-d6, 400 MHz) δ 11.39 (brs, 1H), 8.31 (s, 1H), 7.83
(d, 1H, J ) 9.2 Hz), 7.71 (dd, 1H, J ) 1.6, 8.6 Hz), 7.63 (d, 1H, J )
8.8 Hz), 7.53 (s, 1H), 7.36 (d, 2H, J ) 7.5 Hz), 7.27-7.21 (m, 7H),
7.12 (t, 1H, J ) 7.3 Hz), 6.93 (d, 1H, J ) 2.4 Hz), 6.82 (d, 4H, J )
8.6 Hz), 6.22 (t, 1H, J ) 6.8 Hz), 5.30 (d, 1H, J ) 4.2 Hz), 4.32-4.27
(m, 1H), 3.88 (q, 1H, J ) 3.8 Hz), 3.64 (s, 6H), 3.20 (d, 2H, J ) 4.0
Hz), 3.05 (s, 6H), 3.03 (t, 2H, J ) 7.7 Hz), 2.34-2.16 (m, 4H); 13C
NMR (DMSO-d6, 100 MHz) δ 197.7, 170.2, 163.2, 158.1, 158.0, 150.2,
150.1, 144.6, 137.1, 136.4, 135.4, 135.3, 130.5, 129.7, 129.5, 127.8,
127.6, 126.6, 125.8, 124.5, 123.7, 116.3, 113.1, 113.0, 104.7, 85.7,
85.5, 83.9, 70.5, 63.8, 54.9, 39.9, 39.3, 36.8, 21.6; FABMS (NBA/
DMSO) m/z 755 ([M + H]+), HRMS calcd for C45H45O8N3 ([M +
H]+) 755.3207, found 755.3212.
5-PRODAN-3′O,5′O-bis(tert-butyldimethylsilyl)-2′-deoxyuri-
dine (4). A mixture of 3 (673 mg, 0.99 mmol) and 10% Pd/C (180
mg) in methanol (10 mL) was stirred under hydrogen atmosphere at
room temperature for 9 h. The mixture was filtered through Celite,
washed with methanol, and evaporated under reduced pressure. The
crude product was purified by silica gel chromatography (hexane-
ethyl acetate ) 3:1) to yield the corresponding reduction product
(diastereomeric mixture, 488 mg, 0.71 mmol, 72%) as a yellow solid:
1H NMR (CDCl3, 400 MHz) δ 8.41 and 8.40 (s × 2, total 1H), 7.67
(d, 1H, J ) 9.0 Hz), 7.63 (s, 1H), 7.62 (d, 1H, J ) 8.6 Hz), 7.431 and
7.428 (s × 2, total 1H), 7.37 and 7.34 (t × 2, total 1H, J ) 1.8 Hz),
7.16 and 7.14 (d × 2, total 1H, J ) 2.4 Hz), 6.904 and 6.898 (s × 2,
total 1H), 6.30 (dd, 1H, J ) 5.7, 8.1 Hz), 4.78-4.72 (m, 1H), 4.37 (dt,
1H, J ) 2.6, 8.2 Hz), 3.92 (q, 1H, J ) 2.5 Hz), 3.79 (ddd, 1H, J )
2.9, 4.4, 11.4 Hz), 3.72 (dt, 1H, J ) 3.0, 11.4 Hz), 3.03 (s, 6H), 2.84
and 2.75 (brs × 2, total 1H), 2.56-2.41 (m, 2H), 2.222 and 2.216
(quintet × 2, total 1H, J ) 2.6 Hz), 2.09-1.90 (m, 3H), 0.89 (s, 9H),
0.86 (d, 9H, J ) 4.8 Hz), 0.08-0.02 (m, 12H); 13C NMR (CDCl3, 100
MHz) δ 163.6, 163.5, 149.93, 149.89, 148.7, 137.84, 137.75, 136.21,
136.16, 134.5, 128.7, 126.6, 124.4, 124.3, 124.2, 116.7, 114.6, 106.5,
87.9, 84.99, 84.96, 73.4, 73.3, 72.41, 72.36, 63.1, 63.0, 41.2, 40.9, 38.39,
38.36, 25.90, 25.88, 25.7, 23.9, 23.8, 18.4, 18.0, -4.5, -4.8, -5.40,
-5.43, -5.5; FABMS (NBA/CHCl3) m/z 681 (M+), HRMS calcd for
C36H55O6N3Si2 (M+) 681.3629, found 681.3632.
To a solution of the tritylated compound (30.2 mg, 40.0 µmol) and
tetrazole (3.08 mg, 43.7 µmol) in anhydrous acetonitrile (400 µL) was
added 2-cyanoethyl tetraisopropylphosphorodiamidite (13.1 µL, 40.0
µmol) under nitrogen. The mixture was stirred at room temperature
for 30 min. The mixture was filtered and used with no further
purification.
To a solution of the previous reaction product (104 mg, 0.15 mmol)
and molecular sieves (4 Å, 100 mg) in dichloromethane (4 mL) were
added 4-methylmorpholine N-oxide (26.4 mg, 0.23 mmol) and tetra-
propylammonium perruthenate (11 mg, 0.03 mmol) at 0 °C, and the
mixture was stirred at room temperature for 1 h. After dilution with
diethyl ether (10 mL), Florisil (60-100 mesh, 100 mg) was added to
the solution and the resulting mixture was stirred at room temperature
for 15 min. The mixture was filtered through Celite, washed with diethyl
ether, and evaporated under reduced pressure. The crude product was
purified by silica gel column chromatography (hexane-ethyl acetate
) 4:1) to yield 4 (88 mg, 0.13 mmol, 85%) as a lemon yellow solid:
ODN Synthesis and Characterization. ODNs were synthesized by
a conventional phosphoramidite method by using an Applied Bio-
systems 392 DNA/RNA synthesizer. Commercially available phos-
phoramidites were used for dA, dG, dC, and dT. The crude mixture
after phosphoramidite synthesis was used for PDNX. Synthesized ODNs
were purified by reversed phase HPLC on a 5-ODS-H column (10 mm
× 150 mm, elution with a solvent mixture of 0.1 M triethylamine acetate
(TEAA), pH 7.0, linear gradient over 30 min from 5% to 30%
acetonitrile at a flow rate of 3.0 mL/min). ODNs containing modified
nucleotides were fully digested with calf intestine alkaline phosphatase
9
J. AM. CHEM. SOC. VOL. 129, NO. 15, 2007 4783

A R T I C L E S
Tainaka et al.
(50 U/mL), snake venom phosphodiesterase (0.15 U/mL), and P1
nuclease (50 U/mL) at 37 °C for 3 h. Digested solutions were analyzed
by HPLC on a CHEMCOBOND 5-ODS-H column (4.6 mm × 150
mm), elution with a solvent mixture of 0.1 M TEAA, pH 7.0, flow
rate of 1.0 mL/min. The concentration of each ODN was determined
by comparing peak areas with a standard solution containing dA, dC,
Multicolored SNP Typing. The amplification reaction of an
MTHFR target DNA sample was performed using a BioRad MyCycler
thermal cycler 96-well sample-loading tray. A 109-mer MTHFR gene
antisense fragment containing a C677T site 5′-d(GCCTTCACAAAG-
CGGAAGAATGTGTCAGCCTCAAAGAAAAGCTGCGTGATGA-
TGAAATCG(A/G)CTCCCGCAGACACCTTCTCCTTCAAGTGCT-
TCAGGTCAGCCTCAAAGC)-3′ (10 ng), Primer 1 5′-d(GCCTTCA-
CAAAGCGGAA)-3′ (400 nM), and Primer 2 5′-d(GCTTTGAGGCT-
GACCTG)-3′ (400 nM) were added to 50 µL of the reaction mixture
containing 7 U HotStarTaq DNA polymerase and HotStarTaq master
mix (Qiagen). The thermal cycling program consisted of an initial
incubation at 95 °C for 15 min, followed by 40 cycles of 30 s at 95
°C, 30 s at 55 °C, and 60 s at 72 °C. After thermal cycling and
subsequent cooling, 15 µL aliquots of the amplified DNA mixtures
were used to carry out a second asymmetric PCR. Primer 1 (400 nM)
and Primer 2 (80 nM) were added to 50 µL of a reaction mixture
containing 7 U HotStarTaq DNA polymerase and HotStarTaq master
mix (Qiagen). The thermal cycling program consisted of an initial
incubation at 95 °C for 15 min, followed by 40 cycles of 30 s at 95
°C, 30 s at 55 °C, and 60 s at 72 °C. After thermal cycling and
subsequent cooling, the amplified DNA was purified using Qiagen Min
Elute. The DNA was mixed with 2.5 µM fluorescent probes 5′-
d(GAGBCGATTTCAT)-3′ (B ) PDNU or PyC) in a buffer solution (pH
) 7.0) consisting of 25 mM sodium phosphate and 50 mM sodium
chloride at room temperature on a 1536-well microtiter plate, and the
fluorescence was measured with a fluorescence reader (Berthold Mithras
LB940). The fluorescence was measured through a 440 ( 10 nm
excitation filter and a 510 ( 10 nm emission filter for the fluorescence
from a PDNU probe and through a 340 ( 10 nm excitation filter and a
410 ( 5 nm emission filter for the fluorescence from a PyC probe. The
count time was 0.1 s, and the lamp energy was 7000 (× 75 × 2-16 W).
dG, and dT at
a concentration of 0.1 mM. MALDI-TOF
ODN1(PDNU): ([M
ODN2(PDNU): ([M
ODN1(PDNC): ([M
ODN1(PDNA): ([M
-
H]-) calcd 4098.81, found 4098.23.
H]-) calcd 4128.83, found 4129.04.
H]-) calcd 4097.83, found 4097.97.
H]-) calcd 4121.85, found 4121.57.
-
-
-
ODN1(PDNG): ([M - H]-) calcd 4137.85, found 4137.95.
Fluorescence Spectra. The fluorescence spectra were obtained using
a Shimadzu RF-5300PC spectrofluorophotometer at 25 °C using a cell
with a 1 cm path length. The excitation and emission bandwidths were
1.5 nm.
The fluorescence quantum yield (ΦF) was determined using 9,10-
diphenylanthracene as a reference, with a known ΦF ) 0.95 in ethanol.
The area of the emission spectrum was integrated using the instru-
mentation software, and the quantum yield was calculated according
to the following equation:
2
Φ
F(S)/ΦF(R) ) [A(S)/A(R)] × [(Abs)(R)/(Abs)(S)] × [n(S)2/n(R)
]
(1)
Here, ΦF(S) and ΦF(R) are the fluorescence quantum yields of the sample
and the reference, respectively. The terms A(S) and A(R) are the areas
under the fluorescence spectra of the sample and the reference,
respectively; (Abs)(S) and (Abs)(R) are the optical densities of the sample
and the reference solution at the wavelength of excitation, respectively;
and n(S) and n(R) are the values of the refractive index for the solvents
used for the sample (n(S) ) 1.333) and the reference (n(R) ) 1.383),
respectively.
Acknowledgment. This research was supported by the
Industrial Technology Research Grant Program in 2006 from
the New Energy and Industrial Technology Development
Organization (NEDO) of Japan.
Melting Temperature (Tm) Measurements. All Tm’s of the ODNs
(2.5 µM, final duplex concentration) were measured in 50 mM sodium
phosphate buffers (pH 7.0) containing 100 mM sodium chloride.
Absorbance vs temperature profiles were measured at 260 nm using a
Shimadzu UV-2550 spectrophotometer equipped with a Peltier tem-
perature controller using a 1 cm path length cell. The absorbance of
the samples was monitored at 260 nm from 5 °C to 90 °C with a heating
rate of 1 °C/min. From these profiles first derivatives were calculated
to determine the Tm value.
Supporting Information Available: Detailed experimental
data on the synthesis of PDNC, PDNA, and PDNG (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA069156A
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4784 J. AM. CHEM. SOC. VOL. 129, NO. 15, 2007