Pyrene-Labeled Base-Discriminating Fluorescent DNA Probes for Homogeneous SNP Typing

Article abstract of DOI:10.1021/ja039625y

This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides, ^PyU and ^PyC, which contain a pyrenecar

Full text of DOI:10.1021/ja039625y

Published on Web 03/23/2004
Pyrene-Labeled Base-Discriminating Fluorescent DNA Probes
for Homogeneous SNP Typing
Akimitsu Okamoto, Keiichiro Kanatani, and Isao Saito*
Contribution from the Department of Synthetic Chemistry and Biological Chemistry,
Faculty of Engineering, Kyoto UniVersity and SORST, Japan Science and
Technology Corporation, Kyoto 615-8510, Japan
Received November 16, 2003; E-mail: saito@sbchem.kyoto-u.ac.jp
Abstract: This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases
and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides,
PyU and PyC, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The
fluorescence spectrum of the duplex containing a PyU/A base pair showed a strong emission at 397 nm on
327 nm excitation. In contrast, the fluorescence of duplexes containing PyU/N base pairs (N ) C, G, or T)
was considerably weaker. The proposed structure of the duplex containing a matched PyU/A base pair
suggests that the high polarity near the pyrenecarboxamide group is responsible for the strong A-selective
fluorescence emission. Moreover, the fluorescence of the duplex containing a PyU/A base pair was not
quenched by a flanking C/G base pair. The fluorescence properties are quite different from previous BDF
nucleobases, where fluorescence is quenchable by flanking C/G base pairs. The duplex containing the C
derivative, PyC, selectively emitted fluorescence when the base opposite PyC was G. The drastic change of
fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. PyU-
and PyC-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing
of DNA samples containing c-Ha-ras and BRCA2 SNP sites.
Introduction
Fluorescence-labeled DNA probes have wide applications that
range from genomic sequencing to the rapid detection of
infections and genetic diseases.1 Almost all previous assays
ultimately rely upon sequence recognition events associated with
DNA hybridization. For the detection of mismatched base pairs,
such hybridization assays have inherent limitations in terms of
their sensitivity. For example, the detection of single nucleotide
alterations, such as single nucleotide polymorphisms (SNPs) and
point mutations, in target sequences using a conventional
hybridization assay requires a distinguishable difference in the
base-pairing energies between full-matched versus mismatched
sequences. These differences are usually very small when only
a single mismatched base pair in an extended oligodeoxynucle-
otide (ODN) is involved. Furthermore, the duplex stabilities of
ODNs with a fixed length sometimes vary considerably with
the base content.
Fluorescence-labeled DNA probes have the potential to
simplify DNA probe assays if the fluorescence label exhibits a
drastic change in fluorescence intensity when the labeled probe
hybridizes with a target sequence. Due to this fluorescence
change, the bases on the complementary strands can be
fluorometrically read out without separation and washing. We
have recently demonstrated a new concept for base-discriminat-
ing fluorescent (BDF) nucleobases, which can clearly distinguish
Figure 1. Schematic illustration of a new homogeneous SNP typing method
using a base-discriminating fluorescent (BDF) probe. BDF probes give
strong fluorescence only when the base opposite BDF base is a target base
(e.g., C for MDA,2c T for MDI,2c and A for NPP2d and BPP2a).
the type of base opposite the BDF base by a fluorescence change
(Figure 1).2 Although the ODNs containing BDF bases are
actually used as tools for the detection of single nucleotide
alterations, there are still problems to be solved, such as the
quenching of the BDF fluorescence by the flanking base pairs.
For example, when the flanking base pair of the BDF base,
methoxybenzodeazaadenine (MDA), is a G/C base pair, the
fluorescence of the BDF base is considerably suppressed.2c
A
SNP typing method employing such a BDF base would be
inaccurate for the sequence containing a G/C base pair near the
SNP site. Thus, BDF bases that are more base-selective and
insensitive to flanking base pairs are highly desirable.
(2) (a) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125, 4972-
4973. (b) Okamoto, A.; Tainaka, K.; Saito, I. Chem. Lett. 2003, 32, 684-
685. (c) Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem. Soc.
2003, 125, 9296-9297. (d) Okamoto, A.; Tainaka, K.; Saito, I. Tetrahedron
Lett. 2003, 44, 6871-6874.
(1) (a) Skogerboe, K. J. Anal. Chem. 1995, 67, 449R-454R. (b) Southern, E.
M. Trends Genet. 1996, 12, 110-115. (c) Fodor, S. P. A. Science 1997,
277, 393. (d) Eng, C.; Vijg, J. Nat. Biotechnol. 1997, 15, 422-426.
9
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J. AM. CHEM. SOC. 2004, 126, 4820-4827
10.1021/ja039625y CCC: $27.50 © 2004 American Chemical Society

Base-Discriminating Fluorescent DNA Probes
A R T I C L E S
The fluorescence of pyrene-1-carboxaldehyde shows a strong
dependence on the solvent polarity.3 For example, the fluores-
cence in nonpolar solvents, such as n-hexane, is very weak (ΦF
< 0.001).3a This particular fluorescence arises from the n-π*
transition. However, on increasing the solvent polarity, the
π-π* level, which lies close to the n-π* level, is lowered
below the n-π* level during the lifetime of the excited state
by solvent relaxation. Thus, in polar solvents, the π-π* state
becomes a fluorescence emitting state, and pyrene-1-carboxal-
dehyde shows a strong fluorescence (ΦF in ethanol ) 0.15).3a
Pyrene-1-carboxaldehyde has thus been used for probing polarity
at the micelle-water interface, where it is located.3a Such
fluorescent probes would be valuable for monitoring the
microenvironment of DNA duplexes, for example, for sensing
the difference in polarities between the inside and the outside
of DNA duplexes. By using such fluorescence changes induced
by the polarity change near the fluorophore, we propose it is
feasible to experimentally sense nucleobases opposite the BDF
base in a target strand. We assumed that if the pyrenecarbonyl
fluorophore is attached to uracil at the C-6 position, via a rigid
propargyl linker, the fluorophore would be extruded to the
outside of the groove, a highly polar aqueous phase, due to base
pairing with A (matched). In such a case, the pyrene-labeled
BDF probe should exhibit a strong A-selective fluorescence.
In contrast, when the pyrene fluorophore is intercalated into a
DNA duplex due to the lack of base-pairing (mismatched), the
BDF base would exhibit no emission due to the location of the
fluorophore at a highly hydrophobic site in the groove. On the
basis of this concept, we designed a new type of pyrene-labeled
BDF nucleoside.
Figure 2. Absorption and fluorescence spectra of 2.5 µM PyU monomer
(50 mM sodium phosphate, 0.1 M sodium chloride, pH ) 7.0, 25 °C).
Excitation wavelength was 345 nm. λmax ) 341 nm (ꢀ 20,800). λem ) 397
nm. ΦF ) 0.209.
Scheme 1 a
In this paper, we report on the synthesis and fluorescence
properties of DNA probes containing a new type of pyrene-
labeled BDF nucleoside. These BDF bases, PyU and PyC,
exhibited unique fluorescence properties depending on the nature
of the base on the complementary strand, and distinguished A
and G opposite a BDF base, respectively, by a sharp fluores-
cence change. Unlike previously reported pyrene-labeled ODN
probes,4 PyU and PyC are the first nucleobases whose fluores-
cence intensities vary sharply depending on the nature of the
complementary base. These PyU- and PyC-containing BDF
probes could be effective reporter probes for homogeneous SNP
typing.
a Reagents and conditions: (a) 5a or 5b, Pd(Ph3P)4, CuI, triethylamine,
N,N-dimethylformamide, room temperature, 12 h, 60% for 2a, 60% for 2b;
(b) (iPr2N)2PO(CH2)2CN, 1H-tetrazole, acetonitrile, room temperature, 1
h, quant.; (c) trichloroacetic acid, dichloromethane, room temperature, 5
min, 27% for 4a, 75% for 4b.
Results and Discussion
Synthesis of the novel deoxyuridine-based fluorescent nucleo-
side is outlined in Scheme 1. 5′-protected 5-iodouridine 1 was
prepared according to a method reported in the literature,5 and
then coupled with pyrene-substituted propargylamines, 5a and
5b, to yield compounds 2a and 2b, respectively. Subsequently,
these were converted to phosphoramidites 3a and 3b for ODN
synthesis. The acid hydrolysis of 2a and 2b afforded free
nucleosides 4a (PyU) and 4b (Py(amino)U), respectively. ODNs
containing PyU and Py(amino)U were synthesized using 3a and
3b according to conventional DNA synthesis.
(3) (a) Kalyanasundaram, K.; Thomas, J. K. J. Phys. Chem. 1977, 81, 2176-
2180. (b) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
97, 1515-1566.
First, the absorption and fluorescence spectra of nucleoside
PyU were measured in sodium phosphate buffer (pH ) 7.0)
(Figure 2). The absorption maximum λmax of PyU was observed
at 341 nm, characteristic of the pyrene chromophore. A strong
fluorescence at 397 nm was observed on excitation at 345 nm
(ΦF ) 0.209).
(4) (a) Mann, J. S.; Shibata, Y.; Meehan, T. Bioconjugate Chem. 1992, 3, 554-
558. (b) Korshun, V. A.; Prokhorenko, I. A.; Gontarev, S. V.; Skorobogatyi,
M. V.; Balakin, K. V.; Manasova, E. V.; Malakhov, A. D.; Berlin, Y. A.
Nucleosides & Nucleotides 1997, 16, 1461-1464. (c) Lewis, F. D.; Zhang,
Y.; Letsinger, R. L. J. Am. Chem. Soc. 1997, 119, 5451-5452. (d) Paris,
P. L.; Langenhan, J. M.; Kool, E. T. Nucleic Acids Res. 1998, 26, 3789-
3793. (e) Yamana, K.; Zako, H.; Azuma, K.; Iwase, R.; Nakano, H.;
Murakami, A. Angew. Chem., Int. Ed. 2001, 40, 1104-1106. (f) Amann,
N.; Pandurski, E.; Fiebig, E.; Wagenknecht, H.-A. Chem. Eur. J. 2002, 8,
4877-4883.
Before measuring the absorption and fluorescence of PyU-
containing ODN, we examined the duplex stabilities of PyU-
containing ODN 5′-d(CGCAATPyUTAACGC)-3′ (ODN1-
(PyU)) hybridized with complementary strand 5′-d(GC-
(5) Sheardy, R. D.; Seeman, N. C. J. Org. Chem. 1986, 51, 4301-4303.
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A R T I C L E S
Okamoto et al.
Table 1. Melting Temperatures and Photophysical Properties of Pyrene-Labeled ODNsa
sequencesb
N
Tm/°C
λmax/nm
ꢀmax
ꢀ c
λ
em/nm
ΦF
d
ODN1(PyU)
350
342
350
350
20 400
23 200
18 800
18 000
11 600 (327)
16 400 (327)
10 800 (327)
9 600 (327)
8 800 (327)
25 200 (350)
24 000 (350)
22 000 (350)
24 000 (350)
22 400 (350)
20 400 (344)
24 800 (344)
18 400 (344)
17 600 (344)
17 200 (344)
13 600 (329)
17 200 (329)
11 600 (329)
16 000 (329)
14 000 (329)
402
397
395
399
0.077
0.203
0.136
0.070
0.045
0.0015
0.0010
0.0014
0.0011
0.0004
0.048
0.151
0.023
0.029
0.035
0.068
0.035
0.016
0.147
0.027
ODN1(PyU)/ODN1′(N)
A
C
G
T
49.8
42.5
42.5
40.8
350
13 200
393
ODN1(Py(amino)U)
335, 351
336, 353
335, 352
335, 352
335, 352
350
343
350
350
350
18 800, 26 800
21 200, 26 800
18 000, 23 600
20 400, 25 600
19 200, 24 000
22 800
24 800
21 600
20 400
19 200
380, 397
381, 396
380, 397
381, 397
381, 396
399
395
399
400
396
ODN1(Py(amino)U)/ODN1′(N)
A
C
G
T
58.2
51.2
49.1
53.2
ODN2(PyU)
ODN2(PyU)/ODN2′(N)
A
C
G
T
58.9
51.2
50.4
49.9
ODN2(PyC)
350
350
350
343
18 400
21 600
17 200
20 800
400
403
400
393
ODN2(PyC)/ODN2′(N)
A
C
G
T
57.9
50.0
65.1
55.5
350
18 400
401
a The duplexes (2.5 µM) were measured in 50 mM sodium phosphate and 0.1 M sodium chloride (pH ) 7.0) at 25 °C. b ODN1(PyU) )
5′-d(CGCAATPyUTAACGC)-3′, ODN1′(N) ) 5′-d(GCGTTANATTGCG)-3′ (N ) A, C, G or T), ODN1(Py(amino)U) ) 5′-d(CGCAATPy(amino)UTAACGC)-
3′, ODN2(PyU) ) 5′-d(CGCAACPyUCAACGC)-3′, ODN2′(N) ) 5′-d(GCGTTGNGTTGCG)-3′ (N ) A, C, G or T), ODN2(PyC) ) 5′-d(CGCAACPyC-
CAACGC)-3′. c ꢀ is the molar extinction coefficient given at the wavelength shown in parentheses, which are the excitation wavelength in fluorescence
measurements. d The fluorescence quantum yields (ΦF) were calculated according to ref 6.
GTTANATTGCG)-3′ (ODN1′(N), N ) A, C, G, or T) (Table
1). The melting temperature of ODN1(PyU)/ODN1′(A) was 7
to 9 °C higher than that observed for other mismatched duplexes
in sodium phosphate buffer (pH 7.0). The high stability of
duplex ODN1(PyU)/ODN1′(A) suggests that PyU forms a stable
base pair only with A.
on the fluorescence of PyU nucleoside 4a. The quantum yield
of 4a increased with increasing solvent polarity (ΦF ) 0.068
in carbon tetrachloride and 0.211 in methanol). In contrast, the
fluorescence of Py(amino)U nucleoside 4b, which does not possess
an amide carbonyl group, was very weak, and little affected by
the solvent polarity (ΦF ) 0.027 in carbon tetrachloride and
0.057 in methanol). This fluorescence behavior indicates that
the pyrenecarboxamide chromophore of PyU can act as an
antenna that is highly sensitive to the solvent polarity. The
fluorescence change of Py(amino)U-containing duplexes ODN1-
Measurements of the absorption and fluorescence spectra of
2.5 µM of ODN1(PyU)/ODN1′(N) duplex in sodium phosphate
buffer (pH ) 7.0) were performed. The results are summarized
in Figure 3 and Table 1. The absorption maximum λmax of the
matched duplex ODN1(PyU)/ODN1′(A) was 342 nm, which was
close to the λmax observed for PyU nucleoside (Figure 3a). In
contrast, when the complementary base of PyU was a mis-
matched base (i.e., N ) C, G, and T), the absorption maximum
was at 350 nm, which was 9 nm higher than that of the PyU
nucleoside. The absorption data suggests that the pyrenecar-
boxamide chromophore of ODN1(PyU)/ODN1′(N) duplexes,
except for that of ODN1(PyU)/ODN1′(A), strongly interacts with
the duplex strand. The fluorescence spectrum of ODN1(PyU)/
ODN1′(A) showed a strong fluorescence at 397 nm at 327 nm
excitation (ΦF ) 0.203). In contrast to the high fluorescence
intensity for full-matched duplex, the fluorescence of single-
stranded ODN1(PyU) and mismatched ODN1(PyU)/ODN1′(N)
duplexes was much weaker. The fluorescence excitation spectra
exhibited strong peaks arising from PyU in the duplexes (Figure
3b). This suggests that the change in microenvironment near
the pyrenecarboxamide chromophore of PyU in each duplex
would lead to these remarkable differences in fluorescence
intensities.
(
Py(amino)U)/ODN1′(N) was also examined (Figure 4). The
fluorescence quantum yields were very low (ΦF < 0.002), and
the selectivity of ODN1(Py(amino)U)/ODN1′(N) against the four
bases (N ) A, C, G and T) was also very low. The results
obtained from comparative experiments using PyU and Py(amino)
U
clearly indicate that the base-selective fluorescence emission
by ODN1(PyU) strongly correlates with the polarity sensitivity
of the PyU fluorophore.
To confirm the proposed principle of A-selective fluorescence
emission of PyU, we performed a molecular modeling study of
the PyU-containing duplexes (Figure 5). The structure of the
PyU-containing duplexes was obtained from optimization of 5′-
d(AAACPyUCAAA)-3′/5′-d(TTTGNGTTT)-3′ (N ) A or G)
using the AMBER* force field in water employing MacroModel
(ver. 6.0). In the energy-minimized structures for the duplex
containing a PyU/A base pair, the pyrenecarboxamide chro-
mophore of PyU was extruded to the outside of the duplex, and
exposed to a highly polar aqueous phase, as shown in Figure
5a. In contrast, the duplex containing a PyU/G mismatched base
pair showed a structure in which the glycosyl bond of uridine
was rotated to the syn conformation, as shown in Figure 5b.
The propargyl linker of PyU was stacked into the minor groove
The fluorescence of pyrene-1-carboxaldehyde is known to
be highly dependent on the solvent polarity.3,7 The fluorescence
intensity of pyrene-1-carboxaldehyde is very strong in polar
solvents due to the preferred π-π* transition. In nonpolar
solvents, a weak fluorescence, attributed to a nonemissive n-π*
transition, is observed.7b,8 We examined the effect of solvent
(7) (a) Lianos, P.; Cremel, G. Photochem. Photobiol. 1980, 31, 429-434. (b)
Kumar, C. V.; Chattopadhyay, S. K.; Das, P. K. Photochem. Photobiol.
1983, 38, 141-152. (c) Cundall, R. B. Photochemistry 1992, 23, 3-8. (d)
Ahuja, R. C.; Moebius, D. Langmuir 1992, 8, 1136-1144.
(8) Murov. S. L.; Carmichael, I.; Hug, G. L. Handbook of Photochemistry,
2nd ed.; Marcel Dekker: New York, 1993.
(6) Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976, 80,
969-974.
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4822 J. AM. CHEM. SOC. VOL. 126, NO. 15, 2004

Base-Discriminating Fluorescent DNA Probes
A R T I C L E S
Figure 4. Fluorescence spectra of 2.5 µM ODN1(Py(amino)U) hybridized
with 2.5 µM ODN1′(N) (N ) A, C, G, or T) (50 mM sodium phosphate,
0.1 M sodium chloride, pH ) 7.0, 25 °C). Excitation wavelength was 350
nm.
Figure 3. Spectral change of 2.5 µM ODN1(PyU) hybridized with 2.5 µM
ODN1′(N) (N ) A, C, G, or T) (50 mM sodium phosphate, 0.1 M sodium
chloride, pH ) 7.0, 25 °C). “ss” denotes single-stranded ODN1(PyU). (a)
Absorption and fluorescence spectra. Excitation wavelength was 327 nm.
(b) Fluorescence excitation spectra for 347 nm emission.
and the pyrenecarboxamide unit was bound to the duplex along
the minor groove. In this conformation, the pyrenecarboxamide
group was located at a hydrophobic site of the duplex. The
polarity of the microenvironments near the PyU pyrenecarboxa-
mide group in both cases was quite different. Thus, our modeling
study suggests that the polarity difference around the pyrene-
carboxamide group of PyU directly contributed to the A-selective
fluorescence emission of ODN1(PyU)/ODN1′(N) duplexes.
Figure 5. Molecular modeling of the simulated conformations of duplexes
5′-d(AAACPyUCAAA)-3′/5′-d(TTTGNGTTT)-3′ (N ) A or G). The models
were optimized by AMBER* force field in water using MacroModel version
5.0. Top views (top) and side views from the major groove (bottom) are
shown. PyU and complementary N base are represented by blue and red,
respectively. Pyrene group of PyU is shown in green. (a) N ) A. (b) N )
G.
the flanking base pair on the fluorescence emission, we prepared
a BDF probe containing the -CPyUC- sequence, 5′-d(CG-
CAACPyUCAACGC)-3′ (ODN2(PyU)), and measured the fluo-
rescence of duplexes hybridized with the complementary strand
5′-d(GCGTTGNGTTGCG)-3′ (ODN2′(N), N ) A, C, G, or T).
The fluorescence quantum yield of ODN2(PyU)/ODN2′(A) was
relatively high (ΦF ) 0.151), whereas the fluorescence emis-
sions of the single-stranded ODN2(PyU) and duplex ODN2-
(PyU)/ODN2′(N) were strongly suppressed (Figure 6). The
fluorescence of ODN2(PyU)/ODN2′(N) was more A-selective
Next, we examined the effect of the flanking base pair on
the fluorescence intensity. The C/G base pair is well-known to
stabilize intercalated structures.9 The stacking of the flanking
C/G base pair to PyU, and particularly to the PyU side chain-
intercalating structure, may be the reason for the duplex
stabilization (Figure 4b). Therefore, to investigate the effect of
(9) (a) Nakatani, K.; Okamoto, A.; Matsuno, T.; Saito, I. J. Am. Chem. Soc.
1998, 120, 11 219-11 225. (b) Nakatani, K.; Sando, S.; Saito, I. Nat.
Biotechnol. 2001, 19, 51-55.
than ODN1(PyU)/ODN1′(N). These results suggest that the Py
U
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J. AM. CHEM. SOC. VOL. 126, NO. 15, 2004 4823

A R T I C L E S
Okamoto et al.
Figure 6. Fluorescence spectra of 2.5 µM ODN2(PyU) hybridized with
2.5 µM ODN2′(N) (N ) A, C, G, or T) (50 mM sodium phosphate, 0.1 M
sodium chloride, pH ) 7.0, 25 °C). Excitation wavelength was 344 nm
Figure 7. Fluorescence spectra of 2.5 µM ODN2(PyC) hybridized with
2.5 µM ODN2′(N) (N ) A, C, G, or T) (50 mM sodium phosphate, 0.1 M
sodium chloride, pH ) 7.0, 25 °C). Excitation wavelength was 329 nm.
Scheme 2 a
results are similar to the absorption behavior of PyU-containing
ODN duplexes. The fluorescence emission from ODN2(PyC)/
ODN2′(N) was highly G-selective, as shown in Figure 6. BDF
nucleoside that emits fluorescence selectively for G is unusual,
and PyC is the first BDF nucleoside that can detect G base on
the complementary strand.
The fluorescence change induced by the nature of a comple-
mentary base would be very useful for SNP typing. Therefore,
we examined the discrimination of SNPs in human SNP
sequences using PyU- and PyC-containing BDF probes. 15-mer
DNA strands containing c-Ha-ras10 and BRCA2 SNP sites11
were used as target sequences (Figure 8). For the ras gene c-Ha-
ras sequence, which possesses a C/A SNP site, we designed a
PyU-containing BDF probe, 5′-d(GGCGCCGPyUCGGTGTG)-
3′ (ODNras(PyU)). On hybridization with the target sequence,
ODNras(PyU) showed an A-allele-specific fluorescence at 398
nm using an excitation wavelength of 344 nm, as shown in
Figure 8a and Table 2. The fluorescence of a nonhybridized
ODN or an ODN probe hybridized with a C-allele sequence
was negligible. Similarly, for a BRCA2 sequence possessing a
T/G SNP site, the PyC-containing BDF probe, 5′-d(GCAGC-
a Reagents and conditions: (a) N,N-dimethylformamide dimethylacetal,
N,N-dimethylformamide, 55 °C, 2 h; (b) 4,4′-dimethoxytrityl chloride,
pyridine, room temperature, 5 h, 50% (two steps); (c) 5a, Pd(Ph3P)4, CuI,
triethylamine, N,N-dimethylformamide, room temperature, 12 h, 47%; (d)
(iPr2N)2PO(CH2)2CN, 1H-tetrazole, acetonitrile, room temperature, 1 h,
quant.
CTPyCAGGCAGC)-3′ (ODNBRCA2(PyC)), was prepared. The
side chain-intercalating conformation in the mismatched du-
plexes was stabilized by the flanking C/G base pair, resulting
in a suppression of their fluorescence intensity. In addition, it
should be noted that the fluorescence of ODN2(PyU)/ODN2′-
(A) was not quenched by the flanking C/G base pair. These
fluorescence properties are quite different from those observed
in previous BDF nucleobases,2 where the fluorescence is
efficiently quenched by flanking C/G base pairs.
Since PyU shows a highly A-selective fluorescence emission,
we expected that the analogous C derivative (PyC) would exhibit
a G-selective fluorescence emission. Thus, we prepared PyC
according to Scheme 2, and measured the absorption and
fluorescence spectra of the PyC-containing ODN, 5′-d(CG-
CAACPyCCAACGC)-3′ (ODN2(PyC)), hybridized with the
complementary strand ODN2′(N) (Figure 7). The absorption
maxima λmax of ODN2(PyC)/ODN2′(N) were 343 nm for
matched (N ) G) and 350 nm for mismatched bases. These
Py
fluorescence of ODN
( C) hybridized with the target
BRCA2
sequence was highly G-allele-selective (Figure 8b and Table
3). Thus, these BDF probes act as effective fluorescence probes
for the detection of single nucleotide alterations. The difference
in fluorescence intensities was more conveniently and precisely
detectable using a fluorescence imager, as shown in Figure 8.
The use of PyU- and PyC-containing BDF probes made it
possible to judge the type of base located at a specific site on
the target DNA by simple mixing.
(10) Reddy, E. P.; Reynold, R. K.; Santo, E.; Barbacid, M. Nature 1982, 300,
149-152.
(11) Ford, D.; Easton, D. F.; Stratton, M.; Narod, S.; Goldgar, D.; Devilee, P.;
Bishop, D. T.; Weber, B.; Lenoir, G.; Chang-Claude, J.; Sobol, H.; Teare,
M. D.; Struewing, J.; Arason, A.; Scherneck, S.; Peto, J.; Rebbeck, T. R.;
Tonin, P.; Neuhausen, S.; Barkardottir, R.; Eyfjord, J.; Lynch, H.; Ponder,
B. A. J.; Gayther, S. A.; Birch, J. M.; Lindblom, A.; Stoppa-Lyonnet, D.;
Bignon, Y.; Borg, A.; Hamann, U.; Haites, N.; Scott, R. J.; Maugard, C.
M.; Vasen, H.; Seitz, S.; Cannon-Albright, L. A.; Schofield, A.; Zelada-
Hedman, M. Am. J. Hum. Genet. 1998, 62, 676-689.
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A R T I C L E S
Figure 8. Fluorescence spectra (top) and images (bottom) of 15-mer SNP sequences of human gene hybridized with PyU- or PyC-containing BDF probes.
(a) c-Ha-ras C/A SNP sequence (2.5 µM) was hybridized with 2.5 µM ODNras
PyU) (50 mM sodium phosphate, 0.1 M sodium chloride, pH ) 7.0, 25 °C).
Excitation wavelength was 344 nm. “ss” denotes single-stranded ODNras
PyU). For fluorescence imaging, the sample solutions were illuminated with a UV
transilluminator (290-365 nm). The image was taken through a 380-nm long pass emission filter by means of CCD camera. (b) BRCA2 T/G SNP sequence
(2.5 µM) was hybridized with 2.5 µM ODNBRCA2
PyC) (50 mM sodium phosphate, 0.1 M sodium chloride, pH ) 7.0, 25 °C). Excitation wavelength was
345 nm. “ss” denotes single-stranded ODNBRCA2
PyC). For fluorescence imaging, the sample solutions were illuminated under a UV transilluminator (290-
365 nm). The image was taken as described above.
(
(
(
(
Table 2. Photophysical Properties of BDF Probe ODNras(
PyU)
quite different from the previously observed behavior of BDF
nucleosides.2 The homogeneous SNP typing method using PyU-
and PyC-containing BDF probes would be a powerful alternative
to conventional SNP typing methods.
Hybridized to c-Ha-ras SNP Sequencea,b
em (nm)c
ΦF
c
allele
λmax (nm)
ꢀλmax
ꢀ344
λ
probe only
C
A
350
350
344
17 200
17 200
21 200
14 800
14 000
21 200
392
390
398
0.004
0.006
0.101
Experimental Section
General. 1H NMR spectra were measured with Varian Mercury 400
(400 MHz) and JEOL JMN R-500 (500 MHz) spectrometers. 13C NMR
spectra were measured with JEOL JMN R-500 (125 MHz) spectrometer.
Coupling constant (J value) are reported in hertz. The chemical shifts
are shown in ppm downfield from tetramethylsilane, using residual
a ODNras PyU) ) 5′-d(GGCGCCGPyUCGGTGTG)-3′, c-Ha-ras SNP
(
sequence ) 5′-d(CACACCGNCGGCGCC)-3′ (N ) C or A). b 2.5 µM
duplex in 50 mM sodium phosphate, 0.1 M sodium chloride (pH ) 7.0) at
c
25 °C. λex ) 344 nm. The fluorescence quantum yields (ΦF) were
calculated according to ref 6.
1
chloroform (δ ) 7.24 in H NMR, δ ) 77.0 in 13C NMR), dimethyl
Table 3. Photophysical Properties of BDF Probe ODNBRCA2
(
PyC)
sulfoxide (δ ) 2.48 in 1H NMR, δ ) 39.5 in 13C NMR), and methanol
(δ ) 3.30 in 1H NMR, δ ) 49.0 in 13C NMR), as an internal standard.
FAB masses were recorded on a JEOL JMS HX-110A spectrometer.
ESI masses were recorded on a Perseptive Mariner ESI-TOF mass
spectrometer.
Hybridized to BRCA2 SNP Sequencea,b
c
allele
λmax (nm)
ꢀλmax
ꢀ345
λ
em (nm)c
ΦF
probe only
T
G
351
351
346
17 200
21 200
16 800
16 400
20 000
16 800
387
390
390
0.035
0.028
0.107
The reagents for DNA synthesis were purchased from Glen Research.
Masses of oligodeoxynucleotides were determined with a MALDI-TOF
MS (Perseptive Voyager Elite, acceleration voltage 21 kV, negative
mode) with 2′,3′,4′-trihydroxyacetophenone as a matrix, using T8 ([M-
H]- 2370.61) and T17 ([M-H]- 5108.37) as an internal standard for
oligodeoxynucleotides. Calf intestinal alkaline phosphatase (Promega),
Crotalus adamanteus venom phosphodiesterase I (USB), and Penicillium
citrinum nuclease P1 (Roche) were used for the enzymatic digestion
of ODNs. All aqueous solutions utilized purified water (Millipore,
Milli-Q sp UF). Reversed-phase HPLC was performed on CHEMCO-
BOND 5-ODS-H columns (10 × 150 mm, 4.6 × 150 mm) with a
Gilson Chromatograph, Model 305, using a UV detector, Model 118,
at 254 or 360 nm.
a
ODNBRCA2(
PyC) ) 5′-d(GCAGCCTPyCAGGCAGC)-3′, BRCA2 SNP
sequence ) 5′-d(GCTGCCTNAGGCTGC)-3′ (N ) T or G). b 2.5 µM
duplex in 50 mM sodium phosphate, 0.1 M sodium chloride (pH ) 7.0) at
c
25 °C. λex ) 345 nm. The fluorescemce quantum yields (ΦF) were
calculated according to ref 6.
Conclusions
We have devised novel pyrene-labeled BDF nucleosides, Py
and PyC. BDF probes containing these fluorescent nucleosides
U
selectively emit fluorescence when the complementary bases
are A and G, respectively. Furthermore, the fluorescence of Py
U
N-Propargyl-1-pyrenecarboxamide (5a). A solution of 1-pyrene-
carboxylic acid (500 mg, 2.03 mmol) and EDCI (388 mg, 2.03 mmol)
and PyC is not quenched by the flanking base pairs, which is
9
J. AM. CHEM. SOC. VOL. 126, NO. 15, 2004 4825

A R T I C L E S
Okamoto et al.
in anhydrous DMF (10 mL) was stirred at room temperature. After 15
min, propargylamine (0.139 mL, 2.03 mmol) was added, and the
mixture was stirred at room temperature for 2.5 h. The resulting mixture
was concentrated in vacuo and diluted with ethyl acetate. This solution
was washed with sat. NH4Cl solution and brine, dried over MgSO4,
filtered and evaporated. The crude product was purified by silica gel
column chromatography (chloroform-methanol ) 30:1) to yield 5a
(500 mg, 87%) as a yellow solid: 1H NMR (CDCl3, 400 MHz) δ )
8.62 (d, 1H, J ) 9.3 Hz), 8.25 (d, 1H, J ) 7.6 Hz), 8.26-8.04 (7H),
6.30 (br, 1H), 4.45 (dd, 2H, J ) 5.2, 2.6 Hz), 2.35 (t, 1H, J ) 2.6 Hz);
13C NMR (DMSO-d6, 125 MHz) δ ) 169.5, 132.8, 131.1, 130.6, 128.9,
128.84, 128.81, 127.0, 126.4, 125.9, 125.8, 124.7, 124.5, 124.30, 124.26,
124.2, 79.4, 72.1, 30.0; FABMS (NBA/CDCl3), m/z 283 (M+), HRMS
calcd. for C20H13NO (M+) 283.0997, found 283.0996.
mixture was stirred for 5 min at room temperature. The resulting mixture
was evaporated and purified by recycling preparative HPLC (JAIST
LC-908, JAIGEL GS-310, methanol) to yield 4a (12 mg, 27%): 1H
NMR (DMSO-d6, 500 MHz) δ ) 11.63 (br, 1H), 9.22 (t, 1H, J ) 5.6
Hz), 8.53 (d, 1H, J ) 9.6 Hz), 8.55-8.10 (7H), 8.14 (s, 1H), 8.12 (dd,
1H, J ) 9.6, 8.0 Hz), 6.13 (t, 1H, J ) 6.6 Hz), 5.23 (br, 1H), 5.10 (br,
1H), 4.44 (d, 2H, J ) 5.5 Hz), 4.26-4.21 (m, 1H), 3.64-3.56 (m,
2H), 2.19-2.12 (m, 2H); 13C NMR (DMSO-d6, 125 MHz) δ ) 168.5,
161.8, 149.5, 143.6, 131.7, 131.0, 130.7, 130.1, 128.3, 128.2, 127.9,
127.1, 126.5, 125.8, 125.6, 125.2, 124.6, 124.3, 123.7, 123.6, 98.2,
89.6, 87.6, 84.7, 74.5, 70.2, 61.0, 40.0, 29.4; FABMS (NBA/DMSO),
m/z 510 ([M+H]+), HRMS calcd. for C29H24N3O6 ([M+H]+) 510.1665,
found 510.1667.
5-[3-(1-Pyrenylmethylamino)propynyl]-5′-O-(4,4′-dimethoxy-
trityl)-2′-deoxyuridine (2b). To a solution of 1 (523 mg, 0.961 mmol),
5b (333 mg, 1.24 mmol), and triethylamine (0.530 mL, 3.78 mmol) in
10 mL of anhydrous DMF was added tetrakis(triphenylphosphine)-
palladium(0) (492 mg, 0.426 mmol) and copper(I) iodide (162 mg,
0.852 mmol) under nitrogen. The mixture was stirred at room
temperature for 12 h. The resulting mixture was concentrated in vacuo
and diluted in ethyl acetate. This solution was washed with 5% (w/V)
EDTA solution and 5% (w/V) sodium bisulfite solution, dried over Na2-
SO4, filtered and evaporated. The crude product was purified by silica
gel column chromatography (chloroform-methanol ) 50:1) yield 2b
(465 mg, 60%) as a white solid: 1H NMR (DMSO-d6, 400 MHz) δ )
11.70 (s, 1H), 8.45-7.96 (11H), 7.35-7.09 (9H), 6.89-6.75 (4H), 6.13
(t, 1H, J ) 6.7 Hz), 5.35 (d, 1H, J ) 4.4 Hz), 4.38 (d, 2H, J ) 5.7
Hz), 4.31-4.28 (m, 1H), 3.94-3.89 (m, 1H), 3.63 (s, 3H), 3.60 (s,
3H), 3.40 (d, 2H, J ) 3.1 Hz), 3.23 (dd, 1H, J ) 10.2, 5.0 Hz), 3.07
(dd, 1H, J ) 10.2, 2.4 Hz), 2.28 (dt, 1H, J ) 13.4, 6.8 Hz), 2.20 (ddd,
1H, J ) 13.4, 6.4, 3.6 Hz); 13C NMR (DMSO-d6, 125 MHz) δ ) 161.7,
158.0, 157.9, 149.3, 144.6, 142.5, 135.5, 130.7, 130.3, 130.0, 129.7,
129.6, 129.5, 128.7, 128.6, 127.8, 127.5, 127.4, 127.3, 127.0, 126.8,
126.5, 126.0, 125.0, 124.9, 124.5, 124.1, 123.92, 123.85, 113.2, 113.13,
113.09, 98.8, 86.0, 85.81, 85.80, 85.79, 84.9, 79.1, 75.3, 70.4, 63.6,
54.93, 54.87, 54.86, 49.2, 39.9, 38.0, 37.8; FABMS (NBA/DMSO),
m/z 798 ([M+H]+), HRMS calcd. for C50H43N3O7 ([M+H]+) 798.3179,
found 325.1473.
N-(1-Pyrenylmethyl)propargylamine (5b). To a solution of pyren-
ecarboxaldehyde (1.00 g, 4.34 mmol) and propargylamine (0.365 mL,
5.21 mmol) in methanol (10 mL) was added sodium cyanoborohydride
(327 mg, 5.21 mmol). The mixture was stirred at room temperature
for 1 h. The resulting mixture was concentrated and diluted in ethyl
acetate. This solution was washed with sat. NH4Cl and sat. NaHCO3,
dried over Na2SO4, filtered and evaporated. The crude product was
purified by silica gel column chromatography (chloroform-methanol
) 50:1) to yield 5b (901 mg, 77%) as a white solid: 1H NMR (CDCl3,
400 MHz) δ ) 8.42-7.97 (9H), 4.58 (s, 2H), 3.56 (s, 2H), 2.39 (t,
1H, J ) 2.2 Hz); 13C NMR (DMSO-d6, 125 MHz) δ ) 133.8, 130.7,
130.3, 129.9, 128.7, 127.3, 127.2, 127.0, 126.7, 126.0, 124.94, 124.90,
124.5, 124.1, 123.9, 123.7, 82.9, 73.9, 49.2, 37.1; FABMS (NBA/
CDCl3), m/z 269 (M+), HRMS calcd. for C20H15N (M+) 269.1204, found
269.1204.
5-[3-(1-Pyrenecarboxamido)propynyl]-5′-O-(4,4′-dimethoxytrityl)-
2′-deoxyuridine (2a). To a solution of 5-iodo-5′-O-(4,4′-dimethox-
ytrityl)-2′-deoxyuridine5 (1, 523 mg, 0.961 mmol), 5a (323 mg, 1.24
mmol), and triethylamine (0.53 mL, 3.79 mmol) in 10 mL of anhydrous
DMF was added tetrakis(triphenylphosphine)palladium(0) (492 mg,
0.426 mmol) and copper(I) iodide (162 mg, 0.852 mmol) under
nitrogen. The mixture was stirred at room temperature for 12 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 (chloroform-methanol ) 50:1) to yield 2a (465 mg,
60%) as a yellow solid: 1H NMR (DMSO-d6, 400 MHz) δ ) 11.70
(br, 1H), 9.13 (t, 1H, J ) 5.5 Hz), 8.51 (d, 1H, J ) 5.6 Hz), 8.37-
8.08 (8H), 7.96 (s, 1H), 7.42-7.20 (9H), 6.88 (dd, 4H, J ) 9.2, 3.2
Hz), 6.11 (t, 1H, J ) 6.8 Hz), 5.33 (d, 1H, J ) 4.4 Hz), 4.29 (d, 2H,
J ) 4.8 Hz), 3.93-3.90 (m, 1H), 3.73 (s, 1H), 3.69 (s, 3H), 3.68 (s,
3H), 3.28-3.24 (m, 1H), 3.07 (dd, 1H, J ) 10.3, 2.7 Hz), 2.29 (dt,
1H, J ) 13.2, 6.4 Hz), 2.19 (ddd, 1H, J ) 13.6, 6.4, 3.5 Hz); 13C
NMR (DMSO-d6, 125 MHz) δ ) 168.4, 161.6, 158.1, 158.0, 149.3,
144.8, 143.2, 135.6, 135.2, 131.7, 131.0, 130.7, 130.1, 129.71, 129.65,
129.59, 128.3, 128.1, 127.90, 127.85, 127.5, 127.1, 126.6, 126.5, 125.8,
125.6, 125.2, 124.6, 124.3, 123.74, 123.56, 113.23, 113.19, 98.3, 89.6,
85.9, 85.8, 85.0, 79.1, 74.1, 70.5, 63.7, 55.0, 54.9, 29.4; FABMS (NBA/
DMSO), m/z 811 (M+), HRMS calcd. for C50H41N3O8 (M+) 811.2894,
found 811.2883.
5-[3-(1-Pyrenylmethylamino)propynyl]-5′-O-(4,4′-dimethoxy-
trityl)-2′-deoxyuridine 3′-O-(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite (3b). To a solution of 2b (48 mg, 60 µmol) and tetrazole
(4.9 mg, 71 µmol) in anhydrous acetonitrile (0.6 mL) was added
2-cyanoethyl tetraisopropylphosphorodiamidite (22 µL, 70 µmol) under
nitrogen. The mixture was stirred at room temperature for 1 h. The
mixture was filtered and used for oligodeoxynucleotide synthesis
without further purification.
5-[3-(1-Pyrenylmethylamino)propynyl]-2′-deoxyuridine (4b). To
a solution of 2b (75 mg, 94.1 µmol) in 1 mL of dichloromethane was
added 3% trichloroacetic acid in dichloromethane (3 mL) and the
mixture was stirred for 5 min at room temperature. The resulting mixture
was evaporated and purified by silica gel column chromatography
(chloroform-methanol ) 50:1) yield 4b (35 mg, 75%): 1H NMR (CD3-
OD, 400 MHz) δ ) 8.51-8.48 (2H), 8.33-8.30 (4H), 8.22-8.08 (4H),
6.23 (t, 1H, J ) 6.4 Hz), 5.12 (s, 2H), 4.39 (dt, 1H, J ) 6.4, 3.6 Hz),
4.27 (s, 2H), 3.96 (q, 1H, J ) 3.6 Hz), 3.80 (dd, 1H, J ) 12.0 Hz),
3.72 (dd, 1H, J ) 12.0, 3.2 Hz), 2.36 (ddd, 1H, J ) 13.6, 6.4, 3.6 Hz),
2.22 (dt, 1H, J ) 13.6, 6.4 Hz); 13C NMR (CD3OD, 125 MHz) δ )
164.5, 151.0, 146.6, 134.0, 132.6, 132.0, 131.2, 130.3, 130.0, 129.8,
127.7, 127.2, 127.0, 126.2, 126.1, 125.6, 123.2, 98.4, 89.4, 87.5, 72.0,
62.5, 58.3, 49.9, 49.5, 49.3, 42.0, 38.3; FABMS (DTT-TG/CH3OH),
m/z 496 ([M+H]+), HRMS calcd. for C29H25N3O5 ([M+H]+) 496.1868,
found 496.1872.
5-[3-(1-Pyrenecarboxamido)propynyl]-5′-O-(4,4′-dimethoxytrityl)-
2′-deoxyuridine
3′-O-(2-cyanoethyl)-N,N-diisopropylphosphor-
amidite (3a). To a solution of 2a (57 mg, 70 µmol) and tetrazole (5.4
mg, 77 µmol) in anhydrous acetonitrile (0.8 mL) was added 2-cyano-
ethyl tetraisopropylphosphorodiamidite (25 µL, 77 µmol) under nitro-
gen. The mixture was stirred at room temperature for 1h. The mixture
was filtered and used for oligodeoxynucleotide synthesis without further
purification.
4-N-(N,N-Dimethylaminomethylidenyl)-5-iodo-5′-O-(4,4′-dimeth-
oxytrityl)-2′-deoxycytidine (7). To a solution of the 5-iodo-2′-
deoxycytidine (6, 500 mg, 1.42 mmol) in dry DMF (5 mL) was added
N,N-dimethylformamide dimethylacetal (5.3 mL, 30.0 mmol), and the
5-[3-(1-Pyrenecarboxamido)propynyl]-2′-deoxyuridine (4a). To
a solution of 2a (70 mg, 86.2 µmol) in 1 mL of dichloromethane was
added 3% trichloroacetic acid in dichloromethane (2 mL) and the
9
4826 J. AM. CHEM. SOC. VOL. 126, NO. 15, 2004

Base-Discriminating Fluorescent DNA Probes
A R T I C L E S
solution was stirred at 55 °C for 2 h and concentrated in vacuo. The
residue was solved in dry pyridine (5 mL), and then 4,4′-dimethoxytrityl
chloride (577 mg, 1.70 mmol) was added. The mixture was stirred at
room temperature for 5 h. The mixture was concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(chloroform-methanol ) 20:1) to yield 7 (710 mg, 50%) as a white
solid: 1H NMR (CDCl3, 500 MHz) δ ) 8.71 (s, 1H), 8.24 (s, 1H),
7.43-7.13 (9H), 6.85-6.80 (4H), 6.30 (t, 1H, J ) 6.5 Hz), 4.45 (dt,
1H, J ) 6.0, 3.1 Hz), 4.11 (q, 1H, J ) 3.4 Hz), 3.77 (s, 6H), 3.38 (dd,
1H, J ) 10.4, 3.6 Hz), 3.33 (dd, 1H, J ) 10.4, 3.6 Hz), 3.19 (s, 3H),
3.16 (s, 3H), 2.65 (ddd, 1H, J ) 13.7, 5.7, 3.1 Hz), 2.19 (dt, 1H, J )
13.6, 6.2 Hz); 13C NMR (CDCl3, 125 MHz) δ ) 158.9, 158.63, 158.57,
146.5, 144.5, 139.3, 135.7, 130.09, 139,06, 129.1, 128.1, 128.0, 127.82,
127.75, 127.71, 127.1, 126.9, 113.3, 113.2, 113.0, 87.5, 86.8, 86.2,
81.4, 77.2, 72.3, 63.5, 55.2, 41.4, 35.5; FABMS (DTT-TG/CHCl3),
m/z 711 ([M+H]+), HRMS calcd. for C33H36N4O6 ([M+H]+) 711.1680,
found 711.1675.
with calf intestine alkaline phosphatase (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 CHEMCO-
BOND 5-ODS-H column (4.6 × 150 mm), elution with a solvent
mixture of 0.1 M triethylamine acetate (TEAA), pH 7.0, flow rate 1.0
mL/min). Concentration of each ODN was determined by comparing
peak areas with standard solution containing dA, dC, dG, and dT at a
concentration of 0.1 mM. ODN1(PyU) 5′-d(CGCAATPyUTAACGC)-
3′: ESI-TOF [(M-3H)3-] calcd 1394.24, found 1394.17. ODN1-
(
Py(amino)U) 5′-d(CGCAATPy(amino)UTAACGC)-3′: MALDI-TOF [(M-
H)-] calcd 4170.88, found 4170.79. ODN2(PyU) 5′-d(CGCAACPyUC-
AACGC)-3′: ESI-TOF [(M-3H)3-] calcd 1384.13, found 1384.28.
ODN2(PyC) 5′-d(CGCAACPyCCAACGC)-3′: ESI-TOF [(M-5H)5-
]
calcd 829.96, found 827.57. ODNras(PyU) 5′-d(GGCGCCGPyUCGGT-
GTG)-3′: ESI-TOF [(M-3H)3-] calcd 1635.09, found 1636.59.
Py
ODN
( C) 5′-d(GCAGCCTPyCAGGCAGC)-3′: ESI-TOF [(M-
BRCA2
4H)4-] calcd 806.37, found 804.01.
4-N-(N,N-Dimethylaminomethylidenyl)-5-[3-(1-pyrenecarbox-
amido)propynyl]-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine (8). To
a solution of 7 (500 mg, 0.71 mmol), 5a (191 mg, 0.71 mmol), and
triethylamine (0.12 mL, 0.86 mmol) in 7 mL of anhydrous DMF was
added tetrakis(triphenylphosphine)palladium(0) (164 mg, 0.142 mmol)
and copper(I) iodide (54.1 mg, 0.284 mmol) under nitrogen. The mixture
was stirred at room temperature for 12 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 (chloroform-
methanol ) 25:1) to yield 8 (291 mg, 47%): 1H NMR (DMSO-d6,
400 MHz) δ ) 9.10 (t, 1H, J ) 5.6 Hz), 8.62 (s, 1H), 8.49-8.07 (9H),
7.42-7.19 (9H), 6.90-6.87 (4H), 6.14 (t, 1H, J ) 6.4 Hz), 5.38 (d,
1H, J ) 4.0 Hz), 4.35-4.31 (m, 1H), 4.27 (d, 2H, J ) 5.2 Hz), 3.98-
3.95 (m, 1H), 3.693 (s, 3H), 3.685 (s, 3H), 3.29 (dd, 1H, J ) 12.0, 4.8
Hz), 3.14 (s, 3H), 3.12-3.09 (m, 1H), 3.04 (s, 3H), 2.30 (ddd, 1H, J
) 13.6, 6.4, 3.6 Hz), 2.15 (dt, 1H, J ) 13.6, 6.4 Hz); 13C NMR (DMSO-
d6, 125 MHz) δ ) 169.9, 168.4, 158.1, 158.0, 157.8, 153.3, 144.7,
144.6, 135.7, 135.2, 131.7, 131.1, 130.7, 130.1, 129.7, 129.6, 128.3,
128.0, 127.9, 127.5, 127.1, 126.62, 126.56, 125.8, 125.6, 125.1, 124.5,
124.3, 123.8, 123.6, 113.24, 113.21, 97.7, 88.9, 85.91, 85.87, 79.1,
75.9, 70.6, 63.7, 55.0, 40.9, 40.8, 34.8, 29.5; FABMS (NBA/DMSO),
m/z 866 ([M+H]+), HRMS calcd. for C53H47N5O7 ([M+H]+) 866.3554,
found 866.3550.
4-N-(N,N-Dimethylaminomethylidenyl)-5-[3-(1-pyrenecarbox-
amido)propynyl]-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine 3′-O-
(2-cyanoethyl)-N,N-diisopropylphosphoramidite (9). To a solution
of 8 (52 mg, 64 µmol) and tetrazole (4.9 mg, 70 µmol) in anhydrous
acetonitrile (0.8 mL) was added 2-cyanoethyl tetraisopropylphospho-
rodiamidite (22 µL, 70 µmol) under nitrogen. The mixture was stirred
at room temperature for 1 h. The mixture was filtered and used for
oligodeoxynucleotide synthesis without further purification.
Oligodeoxynucleotide (ODN) Synthesis and Characterization.
ODNs were synthesized by a conventional phosphoramidite method
by using an Applied Biosystems 392 DNA/RNA synthesizer. ODNs
were purified by reverse phase HPLC on a 5-ODS-H column (10 ×
150 mm, elution with 0.1 M triethylamine acetate (TEAA), pH 7.0,
linear gradient over 50 min from 0% to 50% acetonitrile at a flow rate
3.0 mL/min). ODNs containing modified nucleotides were fully digested
Melting Temperature (Tm) Measurements. All Tms of the ODNs
(2.5 µM, final duplex concentration) were taken 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 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 Tm values.
UV Absorption Measurements. ODN solutions were prepared as
described in Tm measurement experiment. Absorption spectra were
obtained using an Ultraspec 3000pro UV-vis spectrophotometer
(Amarsham Pharmacia Biotech) at room temperature using 1 cm path
length cell.
Fluorescence Experiments. ODN solutions were prepared as
described in Tm measurement experiment. Fluorescence spectra were
obtained using a Shimadzu RF-5300PC spectrofluorophotometer at 25
°C using 1 cm path length cell. The excitation bandwidth was 1.5 nm.
The emission bandwidth was 1.5 nm.
The fluorescence quantum yields (ΦF) were determined using 9,-
10-diphenylanthracene as a reference with a known ΦF of 0.95 in
ethanol.6 The area of the emission spectrum was integrated using the
software available in the instrument, and the quantum yield is 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 yield of the sample
and the reference, respectively. A(S) and A(R) are the area under the
fluorescence spectra of the sample and the reference, respectively,
(Abs)(S) and (Abs)(R) are the respective optical densities of the sample
and the reference solution at the wavelength of excitation, and n(S) and
n(R) are the values of refractive index for the respective solvents used
for the sample (1.333) and the reference (1.383).
Fluorescence imaging was performed using Bio-Rad VersaDoc 3000.
The sample solution was illuminated with a 290-365 nm transillumi-
nator. Image was taken through a 380-nm long pass emission
filter.
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J. AM. CHEM. SOC. VOL. 126, NO. 15, 2004 4827