Singly and doubly labeled base-discriminating fluorescent oligonucleotide probes containing oxo-pyrene chromophore

Article abstract of DOI:10.1016/j.bmcl.2010.04.063

We have developed new oxo-pyrene labeled fluorescent nucleoside, ^Oxo-PyU which showed a strong fluorescence dependency on solvent polarity at long wavelength. The designed singly and doubly ^Oxo-PyU labeled fluorescent oligonucleotide probes were found highly efficient for the discrimination of A and consecutive AA bases of target DNA opposite to the labeled base via generation of enhanced fluorescence signal.

Full text of DOI:10.1016/j.bmcl.2010.04.063

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3227–3230
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Singly and doubly labeled base-discriminating fluorescent oligonucleotide
probes containing oxo-pyrene chromophore
a
b
b
b
Subhendu Sekhar Bag a, , Rajen Kundu , Katsuhiko Matsumoto , Yoshio Saito , Isao Saito
*
a Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology, Guwahati 781039, Assam, India
b Department of Chemical Biology and Applied Chemistry, NEWCAT Institute, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
a r t i c l e i n f o
a b s t r a c t
We have developed new oxo-pyrene labeled fluorescent nucleoside, Oxo-PyU which showed a strong fluo-
rescence dependency on solvent polarity at long wavelength. The designed singly and doubly Oxo-PyU
labeled fluorescent oligonucleotide probes were found highly efficient for the discrimination of A and
consecutive AA bases of target DNA opposite to the labeled base via generation of enhanced fluorescence
signal.
Article history:
Received 10 March 2010
Revised 13 April 2010
Accepted 16 April 2010
Available online 19 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Oxo-pyrene chromophore
Oligonucleotides
Fluorescence
Solvatochromic
DNA detection
Fluorescent oligonucleotide probes are powerful tools for struc-
tural studies of nucleic acids, sequencing, molecular diagnostics,
and other applications relating genomics.1 Oligonucleotide probes
labeled with solvatochromic fluorophores are particularly useful
for detecting the change in microenvironment around the nucleic
acids and thus can be used for DNA detection and other applica-
tions like single nucleotide polymorphisms (SNPs) typing.1 The
concept of the labeled probe is based on the fluorescence change
of the labeled base itself in response to the opposite bases on a
complementary strand. As a part of our ongoing project in search-
ing for long wavelength emissive and highly solvatochromic
fluorescent nucleosides, we demonstrated a novel method for
homogeneous fluorescence assay that allows a clear distinction
of the base opposite to the labeled base by a drastic fluorescence
change.2 Such fluorescent nucleosides named as base-discriminat-
ing fluorescence (BDF) nucleosides have been used extensively for
gene detection and SNP genotyping.1c,f,2,3
Previously reported fluorescent probes containing BDF base
MPyU- and AMPyU with oxo-pyrene chromophore were found to
be useful for the discrimination of perfectly matched base A oppo-
site to the BDF base in a complementary strand.3a However, the
preparation of nucleosides MPyU- and AMPyU needed rather compli-
cated synthetic steps of low yield and the synthesis in large quan-
tity was very difficult.3a Therefore, the assay using these BDF bases
are not practical. In contrast, a new fluorescent nucleoside Oxo-PyU
described here was very easily synthesized from readily available
-oxo-pyrene butyric acid and the fluorescence emission was more
c
solvent polarity sensitive with stronger fluorescence intensity and
higher quantum yield (Fig. 1). Therefore, Oxo-PyU is more conve-
nient and practically more useful for the preparation of oxo-pyrene
labeled BDF probes as compared with previous MPyU- and AMPyU
probes.3a
Aiming a practical application of the BDF base, we thought that
it would be worthwhile to tether oxo-pyrene chromophore to the
oligonucleotide sequence and follow up the fluorescence behavior
in the presence of its complementary target sequences. We also
thought that there is a possibility of detecting two consecutive
opposite bases from the target strand if we could incorporate
two consecutive Oxo-PyU in the probe, either by an enhanced mono-
mer emission or via excimer emission, depending on the geomet-
rical disposition of the two pyrene rings. With this view in mind,
we have synthesized oxo-pyrene labeled 20-deoxyuridine, Oxo-PyU,
which was incorporated into short oligonucleotides as one and
two consecutive BDF bases.
Thus, we synthesized nucleoside 1 bearing a rigid acetylene lin-
ker via Sonogashira cross-coupling4 of 5-iodo-20-deoxyuridine
with
c-oxo-pyrene carboxamide 2 (Scheme 1). The synthesis of
c-oxo-pyrene butyric acid (3) was accomplished from commer-
cially available pyrene and succinic anhydride via Friedel–Craft
acylation. Nucleoside 1 was ultimately incorporated into three
oligonucleotide sequences ODN 1, 2 and 3 via standard DNA
synthesis protocol and the ODNs were analyzed by MALDI-TOF
mass spectrometry (Table 1). While 13-mer ODN 1 and 2 contained
* Corresponding author. Tel.: +91 361 258 2324; fax: +91 361 258 2349.
E-mail address: ssbag75@iitg.ernet.in (S.S. Bag).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.063

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S. S. Bag et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3227–3230
O
1.1
0.55
0
70 nm shift
O
DMSO
Isopropanol
EtOH
O
N
N
H
HN
MeOH
O
Py
20% W
50% W
70% W
TFE
HO
O
1(Oxo-PyU)
OH
375
475
575
Wavelength (nm)
O
O
O
O
N
HN
HN
O
N
Py
Py
O
HO
O
HO
O
AMPyU
MPyU
OH
OH
Figure 1. BDF nucleoside 1 (Oxo-PyU), its solvatochromicity and the structures of AMPyU and MPyU.
O
N
O
O
I
Py
Py
O
N
O
N
HN
N
H
N
H
O
O
HN
HN
O
(d)
(b)
HO
(c)
HO
O
O
O
DMTrO
O
O
4
1 [(Oxo-PyU), 60%)]
5 (70%)
OH
OH
OH
O
Py
O
N
N
H
O
HN
O
DNA Synthesis
ODN 1, 2, 3
DMTrO
O
N
O
O
6
H
N
O
P
(a)
OH
Py
Py
NC
O
O
O
3
2 (85%)
Scheme 1. Synthesis of BDF nucleoside 1 and ODNs.
Photophysical properties of nucleoside 1 were then examined.
We observed that with increasing solvent polarity a decreased
and a red shifted patterns were observed in the UV absorption
spectra of 1 (kmax = 345 in DMSO; kmax = 355 nm on TFE). Most
interestingly, both the fluorescence intensity and the wavelength
were markedly affected by solvent polarity (Fig. 1). In DMSO nucle-
oside 1 displayed a very weak emission with a maximum at
Table 1
Oligonucleotide sequences used in this study
ODNs
Sequences
1
2
3
4
5
6
7
8
9
50-d(CGCAACOxo-PyUCAACGC)-30
50-d(CGCAATOxo-PyUTAACGC)-30
50-d(CGCAATOxo-PyUOxo-PyUTAACGC)-30
50-d(CGCAAC T CAACGC)-30
50-d(CGCAAT T TAACGC)-30
50-d(CGCAAT TT TAACGC)-30
50-d(GCGTTG N GTTGCG)-30 [N = A, G]
50-d(GCGTTA N ATTGCG)-30 [N = A, G,]
50-d(GCGTTA NN ATTGCG)-30 [NN = AA, AG, GG]
396 nm (
tested, 1 showed a strong emission (
and decayed deeply into the visible region (>565 nm). We have
also calculated fluorescence brightness factor (F.B. = ) which
U
= 0.015), while in TFE, the most polar organic solvent
U
= 0.22) at around 467 nm
eU
showed an 11-fold increase in TFE in comparison with DMSO (Sup-
plementary data, Fig. S1, Table S1). The fluorescence titration of a
solution of 1 in EtOH with water revealed a similar observation.
singly labeled Oxo-PyU and differed only by flanking bases near the
labeled nucleoside (ODN 1 contained C, while ODN 2 contained
T as the flanking bases). 14-mer ODN 3 labeled with two consecu-
tive Oxo-PyU was also prepared.
In pure EtOH, the emission was around 438 nm (
was shifted to 452 nm and decayed deeply into visible region, 580–
600 nm, when the ratio of water and ethanol was 70:30 ( = 0.12).
As expected, the fluorescence of oxo-pyrene-labeled nucleoside
U = 0.023) which
U

S. S. Bag et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3227–3230
3229
20
10
0
16
8
a
0.7
0.35
0
b
c
c
b
0.07
0.035
0
ss
AA
AG
GG
ss
ss
ss
A
G
310
380 450
AA
AG
GG
AA
AG
GG
Wavelength (nm)
0
220
350
Wavelength (nm)
480
370
510
650
370
510
650
Wavelength (nm)
Wavelength (nm)
Figure 2. (a) Fluorescence of single strand ODN 2, (b) UV–vis and (c) fluorescence spectra of ODN 3 (2.5
lM) and the different duplexes formed by hybridization with ODN 8
[N = A, G] and 9 [NN = AA, AG, GG], (2.5 M, 50 mM sodium phosphate, 0.1 M sodium chloride, pH 7.0, rt; kex = 369 nm.).
l
showed a high sensitivity to the solvent polarity. It is noteworthy
that the emission wavelength in 30% water is about 55 nm longer
than that reported for previous pyrenecarboxamide (PyU)-labeled
emission or via an excimer emission depending on the geometrical
disposition of the two oxo-pyrene rings. Therefore, we prepared a
doubly Oxo-PyU-labeled probe, ODN 3 [50-d(CGCAAT Oxo-PyU Oxo-Py
U
probes (397 nm in water)2c and the solvatochromicity (
D
70 nm)
TAACGC)-30] and measured the fluorescence of the duplexes
formed via hybridization with complementary strands ODN 9
[{50-d(GCGTTA NN ATTGCG)-30}; NN = AA, AG, GG]. Unfortunately,
in any case we could not observe an excimer fluorescence. How-
ever, the detection of –AA– bases was perfectly accomplished by
was 20 nm larger than those of MPyU and AMPyU (
D
50 nm).3a
Thus, the solvent polarity dependent fluorescence property of
Oxo-PyU would be usable for monitoring the change in DNA micro-
environment. Therefore, we examined the photophysical proper-
ties of single stranded ODN 1 (50-CGCAAC Oxo-PyU CAACGC-30)
and 2 (50-CGCAAT Oxo-PyU TAACGC-30) in the presence and absence
of their complementary strands. As revealed from the fluorescence
spectra (Supplementary data, Fig. S2), it was observed that the
fluorescence of ODN 1 with flanking GC base pair was completely
quenched when hybridized to perfectly matched or single base
mismatched complementary sequences ODN 7 [N = A, G]. This is
so obvious and a positive result of our expectation that the flanking
GC base pair is responsible for the quenching of Oxo-PyU fluores-
cence. Thus, Oxo-PyU could potentially be useful in designing
G-quenched molecular beacon as well.5
a strong monomer emission at 467 nm (U = 0.15) with a long tail
in the visible region (>567 nm) together with a high signal to noise
ratio and a large fluorescence brightness factor (F.B. = 3284). In
contrast, the emission of the single stranded ODN 3 and the du-
plexes ODN 3/9 [NN = AG] and ODN 3/9 [NN = GG] was strongly
suppressed (Fig. 2b and Table 2). Therefore, ODN 3 can act as a use-
ful probe which emits strong fluorescence with a high AA selectiv-
ity. To understand the fact of no excimer emission by two
consecutive Oxo-PyU we have examined the molecular modeling
study. From the optimized structure it was found that the pyrene
rings are far apart and positioned parallel and in cofacial configu-
ration to each other, thus inhibiting an excimer formation (Supple-
mentary data, Fig. S5).8
In conclusion, we have investigated the fluorescence emission
properties of fluorescent DNA probes containing singly and dou-
bly labeled Oxo-PyU9 in the presence of complementary strands.
We showed that the fluorescence of Oxo-PyU is strongly quenched
by neighboring G-base. We have also shown that the probes are
highly specific for the detection of matched base A. The doubly
labeled ODN which exhibit unique fluorescence properties
depending on the number of adenines on the complementary
strands would be also useful for the detection of consecutive A
sequence.
On the other hand, ODN 2 (50-CGCAAT Oxo-PyU TAACGC-30) upon
hybridization with perfectly matched sequence ODN 8 [N = A]
showed a very strong emission at 465 nm (Fig. 2a). A very weak
fluorescence was observed in case of the duplex ODN 2/8 [N = G]
where mismatched base G is opposite to the labeled nucleoside
in the complementary strand. This observation again indicated that
the fluorescence of Oxo-PyU is tremendously quenched by neighbor-
ing G-base and can allow us to detect matched base A with an in-
tense fluorescence signal.3,6
The strong fluorescence emission for perfectly matched duplex
ODN 2/8 [N = A] can be explained if we consider the more hydro-
philic microenvironment faced by oxo-pyrene chromophore outside
the groove. As the opposite base is matched base A, the fluorophore
has to extrude outside the groove and thus faced to more aqueous
microenvironment, resulting in an enhancement of fluorescence
(Fig. 2a). The thermal melting behaviors of the duplex ODNs also
support our explanation. The thermal melting behavior showed that
the matched duplex ODN 2/8 [N = A] is more stable (Tm = 64.1 °C)
than unmodified duplex ODN 5/8 [N = A] (Tm = 56.1 °C). Thus, the
fluorophore resides along the major groove and involved in stacking
type interaction and hence bringing more stability. Molecular
modeling study using AMBER* force field in water employing
MacroModel vs. 9.0 also supported this explanation.
Table 2
Thermal melting temperatures and photophysical properties of ODNs
Duplexes
Tm (°C)
emax (Vꢀ1 cmꢀ1
)
U7
F.D.
ODN 1
—
11,200
11,200
10,800
—
11,600
12,000
11,200
—
22,648
21,896
21,492
19,696
—
0.054
0.002
0.001
—
0.046
0.216
0.030
—
0.036
0.150
0.025
0.007
—
605
22
11
ODN 1/7 [N = A]
ODN 1/7 [N = G]
ODN 4/7 [N = A]
ODN 2
ODN 2/8 [N = A]
ODN 2/8 [N = G]
ODN 5/8 [N = A]
ODN 3
ODN 3/9 [NN = AA]
ODN 3/9 [NN = AG]
ODN 3/9 [NN = GG]
ODN 6/9 [NN = AA]
70.9
60.7
59.8
—
64.1
52.5
56.1
—
64.1
58.4
53.2
61.1
—
543
2640
336
—
815
3284
550
153
—
Recently, Yoshida et al., have reported a doubly labeled probe
for efficient SNP typing using PyU and AMPyU.3b Since ODN 2
showed a highly A-selective fluorescence emission, we thought
that there could be a possibility of detecting opposite two consec-
utive AA sequence from the target strand if we incorporate two
consecutive Oxo-PyU in the probe either by an enhanced monomer

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S. S. Bag et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3227–3230
3. (a) Saito, Y.; Miyauchi, Y.; Okamoto, A.; Saito, I. Tetrahedron Lett. 2004, 45, 7827;
Acknowledgments
(b) Yoshida, Y.; Niwa, K.; Yamada, K.; Tokeshi, M.; Baba, Y.; Saito, Y.; Okamoto,
A.; Saito, I. Chem. Lett. 2010, 39, 116.
Financial supports from the DST (SR/SI/OC-69/2008), Govt. of
India, and IIT Guwahati are gratefully acknowledged.
4. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467.
5. (a) Matsumoto, K.; Shinohara, Y.; Bag, S. S.; Takeuchi, Y.; Morii, T.; Saito, Y.;
Saito, I. Bioorg. Med. Chem. Lett. 2009, 19, 6392; (b) Saito, Y.; Shinohara, Y.; Bag, S.
S.; Takeuchi, Y.; Matsumoto, K.; Saito, I. Tetrahedron 2009, 65, 934; (c) Saito, Y.;
Mizuno, E.; Bag, S. S.; Suzuka, I.; Saito, I. Chem. Commun. 2007, 4492; (d) Hwang,
G. T.; Seo, Y. J.; Kim, B. H. J. Am. Chem. Soc. 2004, 126, 6528.
Supplementary data
6. (a) Saito, Y.; Hanawa, K.; Motegi, K.; Omoto, K.; Okamoto, A.; Saito, I. Tetrahedron
Lett. 2005, 46, 7605; (b) Huang, F.-Y.; Chang, T.-Y.; Chen, M.-R.; Lee, H.-C.; Chiu,
N.-C.; Chi, H.; Hsu, C.-H.; Lin, S.-P.; Liu, H.-Fu.; Chen, W.-F.; Chu, C.-C.; Lin, M.;
Lee, Y.-J. Hum. Immunol. 2008, 69, 52.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.063.
7. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976, 80, 969.
8. (a) Robertson, J. M.; White, J. G. J. Chem. Soc. 1947, 358; (b) Birks, J. B.; Lumb, M.
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References and notes
1. For reviews, see (a) Hawkins, M. E. Cell Biochem. Biophys. 2001, 34, 257; (b) Rist,
M. J.; Marino, J. P. Curr. Org. Chem. 2002, 6, 775; (c) Okamoto, A.; Saito, Y.; Saito, I.
Photochem. Photobiol. C: Photochem. Rev. 2005, 6, 108; (d) Ranasinghe, R. T.;
Brown, T. Chem. Commun. 2005, 5487; (e) Venkatesan, N. Y.; Seo, J.; Kim, B. H.
Chem. Soc. Rev. 2008, 37, 648; (f) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev.
in press. doi: 10.1021/cr900301e.
2. (a) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125, 4972; (b)
Okamoto, A.; Tainaka, K.; Fukuta, T.; Saito, I. J. Am. Chem. Soc. 2003, 125, 9296;
(c) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820; (d)
Dohno, C.; Saito, I. ChemBioChem 2005, 6, 1075; (e) Bag, S. S.; Saito, Y.; Hanawa,
K.; Kodate, S.; Suzuka, I.; Saito, I. Bioorg. Med. Chem. Lett. 2006, 16, 6338; (f) Saito,
Y.; Bag, S. S.; Kusakabe, Y.; Nagai, C.; Matsumoto, K.; Kodate, S.; Suzuka, I.; Saito,
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9. Spectroscopic data for compound 1: 1H NMR (DMSO-d6, 400 MHz) d = 11.68 (d,
1H, J = 8.0 Hz), 8.76 (d, 1H, J = 9.4 Hz), 8.58 (d, 1H, J = 8.1 Hz), 8.41–8.27 (m, 5H),
8.25–8.15 (m, 2H), 8.14 (s, 1H), 6.13 (t, 1H, J = 8.2 Hz), 5.26 (br, 1H), 5.16 (br,
1H), 4.23 (br, 1H,), 4.14 (br, 1H), 3.80 (br, 1H), 3.6 (m, 2H), 3.49 (m, 1H), 3.36 (br,
2H), 2.69 (broad t, 1H), 2.50 (t, 2H, J = 1.8 Hz), 2.12 (br m, 2H); 13C NMR (DMSO-
d6, 100 MHz) d = 13C NMR (DMSO-d6, 100 MHz) d = 203.5, 170.9, 161.6, 149.4,
143.6, 132.9, 132.7, 130.6, 129.9, 129.2, 129.0, 128.0, 127.1, 126.7, 126.4, 126.3,
125.9, 124.5, 124.4, 123.9, 123.4, 98.1, 89.6, 87.5, 84.6, 74.3, 70.2, 60.9, 40.0,
37.0, 29.5, 28.7; FABMS, m/z 566 ([M+H]+), HRMS calcd for C32H27N3O7 ([M+H]+)
566.1927, found 566.1929.