Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine

Article abstract of DOI:10.1016/j.bmcl.2012.04.011

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described

Full text of DOI:10.1016/j.bmcl.2012.04.011

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3723–3726
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorometric detection of adenine in target DNA by exciplex formation
with fluorescent 8-arylethynylated deoxyguanosine
Yoshio Saito a, , Kenji Kugenuma a, Makiko Tanaka b, Azusa Suzuki a, Isao Saito b,
⇑
⇑
a Department of Chemical Biology and Applied Chemistry, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
b 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
Article history:
We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense
new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent
8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donat-
ing fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for
the structural study and the fluorometric analysis of nucleic acids.
Received 5 March 2012
Revised 30 March 2012
Accepted 3 April 2012
Available online 10 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Nucleoside
Fluorescence
Probe
Fluorescent nucleosides and oligonucleotides are powerful tools
for structural studies on nucleic acids, sequencing, molecular diag-
nostics, and other applications relating to genomics. Numerous ef-
forts to impart useful photophysical features upon non-emissive
native nucleobases have been reported.1 Most of the previous ap-
proaches involve the linking of nucleobases to fluorescent aromatic
or heteroaromatic chromophores via an ethynyl linker2 or the direct
attachment of fluorescent chromophores on the native nucleo-
bases.1,3 Particularly, the fluorescent nucleosides possessing
fluorescence ‘off-on’ property are of special importance for the
application to fluorometric assays of nucleic acids and in fluores-
cence imaging. The concepts of previous fluorescence ‘off-on’ assay
of nucleic acids usually rely either on the attachment of fluorophore
and quencher at both ends of a oligodeoxynucleotide (ODN) probe
as exemplified by molecular beacon4 or on the PET-based fluores-
cent probe.5 We previously reported a different type of fluorescent
nucleosides, base-discriminating fluorescent (BDF) nucleosides,6
which when incorporated in a probe ODN showed an enhancement
or quenching of emission intensities at a fixed wavelength depend-
ing on a base in a target DNA opposite to the BDF of the probe ODN.
Recently, we reported environmentally sensitive fluorescent
nucleosides such as 8-substituted deoxyadenosine and deoxygua-
nosine derivatives which indicate the difference in the local envi-
ronment by a significant change of the emission wavelength as
well as its intensity.7 We now wish to report herein a unique ap-
proach to discriminate specific base such as adenine in a target
DNA by incident appearance of a strong new emission at a long
wavelength via exciplex formation with newly designed fluorescent
electron donating 8-arylethynylated deoxyguanosine derivatives.8
Guanine is the most electron donating base among DNA bases
but non-emissive. The attachment of aromatic chromophores such
as phenyl or naphthyl group to the 8-position of non-emissive gua-
nosine through a triple bond induced a strong fluorescence to
provide easily accessible fluorescent guanine dreivatives.2,7 Meth-
oxy-substituted fluorescent deoxyguanosine derivatives 1b–d have
an intriguing property, that is, they possess lower oxidation poten-
tials than native guanosine and strong fluorescence at around
400 nm. Oxidation potentials Eox, (V vs SCE) obtained from cyclic
voltammetry in acetonitrile were 1.17 (1b), 1.07 (1c) and 1.14
(1d) that are lower than that of 20-deoxyguanosine (1.23 V). We ex-
pected that the stacking interaction between highly electron
donating fluorescent deoxyguanosines 1b–d and adenine base hav-
ing a moderately electron accepting property in a DNA duplex may
form an exciplex upon photoexcitation of the fluorescent deoxy-
guanosine. We report herein the synthesis of such electron donat-
ing fluorescent deoxyguanosine derivatives and their incorporation
into DNA oligomers which were used as a BDF probe for the detec-
tion of adenine base fluorometrically.
8-Arylethynylated 20-deoxyguanosine derivatives were pre-
pared by Pd(0)-mediated cross-coupling as reported previously
(Scheme 1).7a 8-Ethynyl-20-deoxyguanosine 27a was coupled with
various aryl iodides 3a–d under Pd(0)-mediated cross-coupling
conditions to provide corresponding fluorescent deoxyguanosine
derivatives (1a–d). Deoxyguanosine analog 1a was then incorpo-
rated into ODN via automated DNA synthesizer. After protection
of amino group with N,N-dimethylformamide diethylacetal, 4a
was reacted with DMTrCl in the presence of catalytic amount of
⇑
Corresponding authors.
E-mail address: saitoy@chem.ce.nihon-u.ac.jp (Y. Saito).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.011

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Y. Saito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3723–3726
Scheme 1. Reagents and conditions: (a) Compounds 3a–d, Pd(PPh3)4, CuI, Et3N, DMF, 50 °C, 3 h; (b) N,N-dimethylformamide diethylacetal, DMF, rt, 3 h; (c) DMTrCl, DMAP,
pyridine, rt, 17 h.
DMAP in dry pyridine to give 5a. Phosphoramidite obtained from
5a was used to prepare modified ODNs via automated DNA synthe-
sizer. Other 8-arylethynylated guanosine derivatives 1b–d were
also incorporated into ODNs in a similar way. ODNs thus obtained
were purified by HPLC and characterized by MALDI-TOF mass
spectrometry.
After getting all the nucleosides in hand, we first examine their
photophysical properties in organic solvents of varying polarity.
The fluorescence spectra of monomer 8-arylethylated deoxyguano-
sine derivatives 1a and 1b in different solvents were shown in
Figure 1. These deoxyguanosine derivatives exhibited strong fluo-
rescence at around 390 nm but did not show solvatochromic
behavior. Next, UV–visible absorption and fluorescence spectra of
ODNs containing 1a–d were examined. Fluorescence emission of
single stranded ODN I (X = 1b) containing 1b in the middle of the
strand was found to be relatively weak and appeared at ca.
390 nm. When the opposite base of a complementary strand is ade-
nine (N = A) in duplex ODN I (X = 1b)/ODN II (N = A), very strong
new emission was observed at longer wavelength around 435 nm
(Fig. 2).10 The emission at 435 nm was not observed when the oppo-
site bases are G, C and T, suggesting that the new emission is an
exciplex emission from a complex formed between 1b and adenine
(A). In support of this hypothesis, a weak new absorption band in a
longer wavelength region (ca. 360 nm) was observed in the UV–
visible spectra of duplex ODN I (X = 1b)/ODN II (N = A), indicating
the formation of ground state complex between 1b and A in the
(a)
(b)
Figure 1. Fluorescence spectra of (a) 1a (2.5 lM) and (b) 1b (2.5 lM) in various solvents. Excitation wavelength was 325 nm.

Y. Saito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3723–3726
3725
Figure 2. Fluorescence spectra of (a) ODN I (X = 1a), (b) ODN I (X = 1b), (c) ODN I (X = 1c) and (d) ODN I (X = 1d) together with those of duplexes ODN I/ODN II formed by
hybridization with complementary ODN II (N = A, T, G, C), respectively, (2.5 M ODNs, 50 mM sodium phosphate, 0.1 M sodium chloride, pH 7.0, rt).
l
duplex form (Supplementary data, Fig. S1-b). When ODN I (X = 1a)
containing unsubstituted phenyl group instead of 1b was used,
such exciplex emission has not been observed in hybridization with
ODN II (N = A) (Fig. 2a), indicating a requirement of the electron do-
nor substituent for the exciplex formation. A similar strong exciplex
emission was also observed at ca. 450 nm with duplex ODN I
(X = 1c)/ODN II (N = A) containing trimethoxy-substituted 1c
(Fig. 2c).
A more prominent example is the emission from
methoxy-substituted naphthalene derivative 1d. A characteristic
fluorescence from the naphthalene chromophore appeared at
390–440 nm for single stranded ODN I (X = 1d). However, in a du-
plex form [ODN I (X = 1d)/ODN II (N = A)], a new strong exciplex
emission at 470 nm emerged together with the appearance of
original naphthalene fluorescence, suggesting a partial formation
of excited state complex (Fig. 2d). The formation of ground state
complex between the naphthalene chromophore and A was also
indicated by the appearance of a new absorption band in the
UV–visible spectrum of the duplex ODN I (X = 1d)/ODN II (N = A)
(Supplementary data, Fig. S1-d). The fluorescence color change
depending upon the opposite base was readily observable by naked
eye under illumination with 365 nm transilluminator (Fig. 3).
To evaluate the formation of exciplex in detail, we measured
Figure 3. Comparison of the fluorescence colour change upon the bases opposite to
1b in duplex ODN I (X = 1b)/ODN II (50 mM sodium phosphate, 0.1 M sodium
chloride, pH 7.0, room temperature). ‘ss’ denotes as
a single-stranded ODN I
(X = 1b). The sample solutions were illuminated with a 365 nm transilluminator.
(X = 1d) and ODN I (X = 1d)/ODN II (N = A), respectively. As shown
in Table 1, longer lifetime component ( = 2.2 ns) was ascribable to
the formation of an exciplex between 1d and A, whereas the shorter
s
fluorescence lifetimes of single and double stranded ODN
I

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Y. Saito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3723–3726
Table 1
Saito, I. Photochem. Photobiol. C: Photochem. Rev. 2005, 6, 108; (d) Ranasinghe, R.
Fluorescence lifetimes of single stranded ODN I (X = 1d) and duplex ODN I/ODN IIa
T.; Brown, T. Chem. Commun. 2005, 5487; (e) Tor, Y. Ed.; Tetrahedron
Symposium in print. 2007, 63.; (f) Venkatesan, N. Y.; Seo, J.; Kim, B. H. Chem.
Soc. Rev. 2008, 37, 648; (g) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev.
2010, 110, 2579.
Sample
sf (ns)
ODN I (X = 1d)
0.90 (420 nm)
2. (a) Volpini, S.; Costanzi, S.; Lambertucci, C.; Vittori, S.; Klotz, K.-N.; Lorenzen,
A.; Cristalli, G. Bioorg. Med. Chem. Lett. 1931, 2001, 11; (b) Costanzi, S.;
Lambertucci, C.; Vittori, S.; Volpini, R.; Cristalli, G. J. Mol. Graphics Modell. 2003,
21, 253; (c) Abou-Elkhair, R. A. I.; Netzel, T. L. Nucleosides Nucleotides Nucleic
Acids 2005, 24, 85; (d) Okamoto, A.; Ochi, Y.; Saito, I. Chem. Commun. 2005,
1128; (e) Firth, A. G.; Fairlamb, I. J. S.; Darley, K.; Baumann, C. G. Tetrahedron
Lett. 2006, 47(3529), 5; (f) Seo, Y. J.; Rhee, H.; Joo, T.; Byeang, H. Kim. J. Am.
Chem. Soc. 2007, 129, 5244; (g) O’Mahony, G.; Ehrman, E.; Grøtli, M. Tetrahedron
2008, 64, 7151; (h) Abou-Elkhair, R. A. I.; Dixon, D. W.; Netzel, T. L. J. Org. Chem.
2009, 74, 4712.
3. For example, see: Greco, N. J.; Tor, Y. J. Am. Chem. Soc. 2005, 127, 10784. and
references therein.
4. Kostrikis, L. G.; Tyagi, S.; Mhlanga, M. M.; Ho, D. D.; Kramer, F. R. Science 1998,
279, 1228.
5. (a) Czarnic, A. W. Acc. Chem. Res. 1994, 27, 302; (b) Spaarano, B. A.; Shahi, S. P.;
Koide, K. Org. Lett. 1947, 2004, 6; (c) Sparano, B. A.; Koide, K. J. Am. Chem. Soc.
2005, 127, 14954.
ODN I (X = 1d)/ODN II (X = A)
1.1 (420 nm), 2.2 (470 nm)
a
Experimental conditions: [ssODN or duplex ODN] = 3.0
sodium phosphate buffer, pH 7.0, 100 mM NaCl, kex = 365 nm.
lM in deaerated 50 mM
lifetime component (s = 1.1 ns) was resulted from the naphthalene
chromophore of 1d. The study of fluorescence lifetime clearly indi-
cated the existence of two excited species that are responsible for
fluorescence emission and strongly suggests that the longer life-
time component with a strong emission at 470 nm is from the exci-
plex between 1d and A. Actually, molecular modeling study of
duplex ODN I (X = 1d)/ODN II (N = A) by using MacroModel ver.
9.0 indicated a strong
p-stacking between methoxynaphthalene
6. (a) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125, 4972; (b)
Okamoto, A.; Tanaka, K.; Fukuda, T.; Saito, I. J. Am. Chem. Soc. 2003, 125, 4972;
(c) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820; (d)
Saito, Y.; Hanawa, K.; Motegi, K.; Omoto, K.; Okamoto, A.; Saito, I. Tetrahedron
Lett. 2005, 46, 7605; (e) Saito, Y.; Bag, S. S.; Kusakabe, Y.; Nagai, C.; Matsumoto,
K.; Kodate, S.; Suzuka, I.; Saito, I. Chem. Commun. 2007, 2133; (f) Saito, Y.;
Motegi, K.; Bag, S. S.; Saito, I. Bioorg. Med. Chem. 2008, 16, 107.
7. (a) Shinohara, Y.; Matsumoto, K.; Kugenuma, K.; Morii, T.; Saito, Y.; Saito, I.
Bioorg. Med. Chem. Lett. 2010, 20, 2817; (b) Matsumoto, K.; Takahashi, N.;
Suzuki, A.; Morii, T.; Saito, Y.; Saito, I. Bioorg. Med. Chem. Lett. 2011, 21, 1275; (c)
Saito, Y.; Shinohara, Y.; Ishioroshi, S.; Suzuki, A.; Tanaka, M.; Saito, I.
Tetrahedron Lett. 2011, 52, 2359.
moiety and adenine (Supplementary data, Fig. S2).
In conclusion, we have devised an unprecedented type of fluo-
rescent probe containing highly electron donating fluorescent
deoxyguanosine analogs which gave rise to the appearance of a
strong new emission at longer wavelength via exciplex formation
with adenine in hybridization with target DNA. Such electron
donating deoxyguanosine analogs 1b,c,d capable of forming ex-
cited state complexes specifically with adenine may be widely
used as a fluoresce probe for structural study of DNA and RNA
and for the fluorometric analysis of nucleic acids and fluorescent
imaging.
8. While many examples for fluorometric assay of DNA using pyrene eximer
emission are known,9 there has been no example, to our knowledge, for the
fluorometric analysis of nucleic acids using exciplex emission.
9. For examples, see: (a) Tong, G.; Lawlor, J. M.; Tregear, G. W.; Haralambidis, J. J.
Am. Chem. Soc. 1995, 117, 12151; (b) Yamana, K.; Iwai, T.; Ohtani, Y.; Sato, S.;
Nakamura, M.; Nakano, H. Bioconjugate Chem. 2002, 13, 1266; (c) Okamoto, A.;
Ichiba, T.; Saito, I. J. Am. Chem. Soc. 2004, 126, 8364; (d) Yamana, K.; Ohshita, Y.;
Fukunaga, Y.; Nakamura, M.; Maruyama, A. Bioorg. Med. Chem. 2008, 16, 78; (e)
Conlon, P.; Yang, C. J.; Wu, Y.; Chen, Y.; Martinez, K.; Kim, Y.; Stevens, N.; Marti,
A. A.; Jockusch, S.; Turro, N. J.; Tan, W. J. Am. Chem. Soc. 2008, 130, 336. and
references therein; (f) Matsumoto, K.; Shinohara, Y.; Bag, S. S.; Takeuchi, Y.;
Morii, T.; Saito, Y.; Saito, I. Bioorg. Med. Chem. Lett. 2009, 19, 6392.
10. It should be noted that such adenine specific exciplex formation is sequence
dependent. When the flanking base pair of fluorescent deoxyguanosine 1b was
G–C base pair, such A specific exciplex emission was not observed.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.
011.
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.;