Design of environmentally sensitive fluorescent 2′-deoxyguanosine containing arylethynyl moieties: Distinction of thymine base by base-discriminating fluorescent (BDF) probe

Article abstract of DOI:10.1016/j.bmcl.2010.03.055

We have synthesized various substituted 8-arylethynylated 2′-deoxyguanosine derivatives. Among them, acetyl substituted deoxyguanosine analogue 4c showed a remarkable solvent dependent fluorescence property, that is, an intense fluorescence in non-polar solvents but extremely weak fluorescence in polar solvents like methanol. By using solvatofluorochromic deoxyguanosine analogue 4c, we have developed highly thymine (T) selective fluorescent DNA probes that can sense T opposite 4c in a target DNA regardless of the flanking sequences. We were able to demonstrate that 4c can be used as a T specific base-discriminating fluorescent (BDF) nucleoside in homogeneous fluorescence assay.

Full text of DOI:10.1016/j.bmcl.2010.03.055

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2817–2820
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of environmentally sensitive fluorescent 20-deoxyguanosine containing
arylethynyl moieties: Distinction of thymine base by base-discriminating
fluorescent (BDF) probe
a,
Yuta Shinohara a, Katsuhiko Matsumoto b, Kenji Kugenuma a, Takashi Morii b, Yoshio Saito a, , Isao Saito
*
*
a Department of Chemical Biology and Applied Chemistry, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
b Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan
a r t i c l e i n f o
a b s t r a c t
We have synthesized various substituted 8-arylethynylated 20-deoxyguanosine derivatives. Among them,
acetyl substituted deoxyguanosine analogue 4c showed a remarkable solvent dependent fluorescence
property, that is, an intense fluorescence in non-polar solvents but extremely weak fluorescence in polar
solvents like methanol. By using solvatofluorochromic deoxyguanosine analogue 4c, we have developed
highly thymine (T) selective fluorescent DNA probes that can sense T opposite 4c in a target DNA regard-
less of the flanking sequences. We were able to demonstrate that 4c can be used as a T specific base-dis-
criminating fluorescent (BDF) nucleoside in homogeneous fluorescence assay.
Article history:
Received 5 February 2010
Revised 8 March 2010
Accepted 11 March 2010
Available online 16 March 2010
Keywords:
BDF probe
DNA
Ó 2010 Elsevier Ltd. All rights reserved.
Nucleoside
SNPs
Fluorescent nucleosides and oligonucleotides are powerful tools
for structural studies of nucleic acids, sequencing, molecular diag-
nostics, and other applications relating genomics.1 Particularly, the
development of fluorescent nucleosides that can monitor local
microenvironmental change around nucleic acids is extremely
important for understanding biological events accompanying
interbiomolecular interactions such as replication, transcription,
expression activation, and inactivation. The number of reported
fluorescent nucleosides has increased greatly in recent years, and
numerous efforts to impart useful photophysical features upon
natural nucleobases have been reported.1,2
In our continuous studies directed toward the development of
practically useful fluorescent oligonucleotide probes, we reported
a new concept for the design of important fluorescent nucleosides
such as base-discriminating fluorescent (BDF) nucleosides3,4 and
microenvironment sensitive fluorescent nucleosides.5
In order to devise sensitive fluorescent nucleosides to their local
environment, molecular design of highly solvent dependent fluo-
rescent nucleobase is critically important. While 8-arylethynylated
adenosine6 and guanosine7 are known, solvatochromic nature of
such fluorescent ribonucleosides has not been reported. Through
extensive studies of the substituent effects on 8-arylethynylated
20-deoxyguanisine derivatives, we found that acetyl group on the
aryl moiety induced a remarkable solvatofluorochromic property
of the deoxyguanosine analogues. Unlike unsubstituted 8-aryleth-
ynylated deoxyguanosine, acetyl substituted deoxyguanosine ana-
logue 4c showed a remarkable solvent dependent fluorescence
property. We report herein unique fluorescent 20-deoxyguanosine
analogues that are highly sensitive to their local environment. By
using such fluorescent deoxyguanosine analogues, we have devel-
oped highly thymine (T) selective BDF probes that can sense T
opposite BDF base in a target DNA regardless of the flanking se-
quences (Fig. 1).
We already reported pyrene labeled BDF nucleosides pyU and
pyC that can distinguish A and G, respectively, opposite the BDF
bases.4 In contrast, adenosine type T specific BDF base previously
reported3d was only effective in AT rich sequences due to the
quenching by neighboring Gs. Environment sensitive fluorescent
deoxyguanosine derivatives usable as
a fluorescence sensor
regardless of the flanking sequences are highly desirable for struc-
tural studies of nucleic acids and molecular diagnostics such as
SNP genotyping.
* Corresponding authors.
E-mail address: saitoy@chem.ce.nihon-u.ac.jp (Y. Saito).
Figure 1. Schematic illustration of DNA detection using BDF nucleoside.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.055

2818
Y. Shinohara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2817–2820
O
N
O
N
N
N
N
N
NH
NH2
NH
NH2
Br
TMS
HO
(a)
HO
O
O
OH
OH
1
2 (73 %)
O
N
O
N
N
N
NH
NH2
NH
NH2
R
(b)
(c)
N
N
HO
HO
O
O
OH
OH
3 (80 %)
4 a(66 %) R = H
b(50 %)
c(61 %)
R = NMe2
R= COCH3
Scheme 1. Reagents and conditions: (a) trimethylsilyl acetylene, Pd(PPh3)4, TEA, DMF, 50 °C, 3 h; (b) TBAF, THF, rt, 2 h; (c) 4-iodobenzene, 4-dimethylaminoiodobenzene or
4-bromoacetophenone, Pd(PPh3)4, TEA, DMF, rt, 3 h.
8-Arylethynylated 20-deoxyguanosine derivatives 4 were pre-
pared via palladium (0) mediated Sonogashira cross-coupling
(Scheme 1).8 8-Bromo-20-deoxyguanosine 1 was coupled with tri-
methylsilyl acetylene to yield compound 2. Silyl group of 2 was
deprotected by tetrabutylammonium fluoride to give 8-ethynyl-
20-deoxyguanosine 3. Compound 3 was coupled with substituted
aryl halides to afford corresponding nucleosides 4a, 4b, and 4c in
high yields.
We first examined the photophysical properties of 8-arylethy-
nylated deoxyguanosine derivatives in various solvents. All these
deoxyguanosines (4a, 4b, 4c) showed strong fluorescence at
around 475 nm in non-polar solvents like ethyl acetate but rela-
tively weak fluorescence in polar solvents like methanol and aceto-
nitrile. Interestingly, fluorescence intensity of acetyl analogue 4c
showed a strong solvent dependency, that is, an intense fluores-
cence in non-polar solvent at 475 nm but extremely weak fluores-
cence in methanol, showing that introduction of acetyl group on
the aromatic moiety induces a solvatochromic fluorescence prop-
erty (Fig. 2c). Thus, we thought that solvatofluorochromic deoxy-
guanosine 4c may be used as a fluorescence sensor for SNP
genotyping.
of amino group with N,N-dimethylformamide diethylacetal, 4c
was reacted with DMTrCl in the presence of catalytic amount of
DMAP in dry pyridine to give 6. Protected 6 was converted to phos-
phoramidite 7, which was used for ODN synthesis by automated
DNA/RNA synthesizer without further purification (Scheme 2).
Two ODNs containing 4c in AT rich site (ODN 1) and GC rich site
(ODN 2) were prepared (Table 1).
Fluorescence spectra of ODN 1 and ODN 2 were measured in the
absence and presence of complimentary strands. Fluorescence
intensities of single stranded ODN 1 and ODN 2 and of the duplexes
with complimentary strands ODN 3 and ODN 4 (N = A, G, C),
respectively, were very weak. In contrast, the fluorescence spec-
trum of duplex ODN 1/ODN 3 (N = T) showed an intense fluores-
cence at 510 nm in AT rich sequence. In GC rich sequence a
similar T specific fluorescence emission was observed for duplex
ODN 2/ODN 4 (N = T), although the fluorescence intensity in GC
rich sequence was negatively low due to the quench by neighbor-
ing Gs (Fig. 3a, b). The fluorescence emission was visible with
naked eye only for T opposite BDF base 4c under illumination with
365 nm transilluminator (Fig. 3c). In contrast, such T specific rec-
ognition opposite BDF base was not observed for ODNs containing
4a and 4b. Thus, acetyl substituted fluorescent deoxyguanosine 4c
is exceptionally useful as a T specific BDF base regardless of the
Deoxyguanosine 4c was then incorporated into oligodeoxynu-
cleotides (ODNs) via automated DNA synthesis. After protection
a
b
c
1
0.8
0.6
0.4
0.2
0
AcOEt
AcOEt
1
0.8
0.6
0.4
0.2
0
AcOEt
1
0.8
0.6
0.4
0.2
0
CH CN
CH CN
3
3
CH CN
3
MeOH
MeOH
MeOH
350
450
550
650
350 400 450 500 550 600
wavelength (nm)
350 400 450 500 550 600
wavelength (nm)
wavelength (nm)
Figure 2. Fluorescence spectra of 4a (a), 4b (b) and 4c (c) in various solvent (each 10 lM conc.). Excitation wavelength was 355 nm (a), 345 nm (b), and 350 nm (c).

Y. Shinohara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2817–2820
2819
O
O
N
N
N
N
NH
NH
R
R
N
N
N
N
N
N
(a)
(c)
(b)
HO
DMTrO
4c
O
O
OH
OH
5 (73 %)
6 (88 %)
O
N
NH
R
N
N
N
N
DMTrO
O
ODNs
O
O
P
N
CN
7 R= COCH3
Scheme 2. Reagents and conditions: (a) N,N-dimethylformamide diethylacetal, DMF, rt, 3 h; (b) DMTrCl, DMAP, pyridine, rt, 17 h; (c) N,N-diisopropylchlorophosphoramidite,
TEA, CH2Cl2, rt, 15 min.
Table 1
Sequences of oligonucleotides
flanking sequences in a fluorescence hybridization assay. The rea-
son for the T specific fluorescence emission is not clear at present.
However, it seems likely that in base pairing of 4c with T the chro-
mophore of 4c is located at a hydrophobic site inside the groove as
suggested by the fact that intense fluorescence of 4c appeared only
in non-polar environment. Thus the microenvironment around 4c
when base paired with T is slightly different from those for other
ODNs
Sequence
1
2
3
4
50-d(CGCAATXTAACGC)-30 (X = 4c)
50-d(CGCAACXCAACGC)-30 (X = 4c)
50-d(GCGTTANATTGCG)-30 (N = A, G, C, T)
50-d(GCGTTGNGTTGCG)-30 (N = A, G, C, T)
4
b
18
a
A
T
G
C
ss
A
T
15
12
9
G
3
2
1
0
C
ss
6
3
0
430
530
630
430
530
630
wavelength (nm)
wavelength (nm)
c
ss
A
T
G
C
Figure 3. Fluorescence spectra of ODN 1 (a) and ODN 2 (b) together with those of the duplexes formed by hybridization with ODN 3 and 4 (N = A, T, G, C), respectively (2.5 lM
ODNs, 50 mM sodium phosphate, 0.1 M sodium chloride, pH 7.0, rt). (c) Fluorescence image of ODN 1 (ss) and the duplexes formed with ODN 3 (N = A, T, G, C) under
illumination with transilluminator (365 nm). ‘ss’ denotes single stranded ODN.

2820
Y. Shinohara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2817–2820
Table 2
Supplementary data
Thermal melting behavior of ODNsa
X/N
ODN 1/ODN 3 (°C)
ODN 2/ODN 4 (°C)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.055.
4c/A
4c/G
4c/T
4c/C
G/C
49.6
51.5
48.7
52.5
58.7
58.0
58.1
55.4
59.3
63.8
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.
J. 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.
a
All Tms of ODNs (2.5 lM conc.) were measured in 50 mM sodium phosphate
buffers (pH 7.0) containing 100 mM sodium chloride.
2. Tor, Y. Ed., Tetrahedron symposium in print, 2007, 63.
3. (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.; Tainaka, 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) Dohno, C.; Saito, I. ChemBioChem 2005, 6, 1075; (f) Saito, Y.; Bag, S.
S.; Kusakabe, Y.; Nagai, C.; Matsumoto, K.; Kodate, S.; Suzuka, I.; Saito, I. Chem.
Commun. 2007, 2133; (g) Saito, Y.; Motegi, K.; Bag, S. S.; Saito, I. Bioorg. Med.
Chem. 2008, 16, 107.
4. Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820.
5. (a) Tainaka, K.; Tanaka, K.; Ikeda, S.; Nishiza, K.-i.; Unzai, T.; Fujiwara, Y.; Saito,
I.; Okamoto, A. J. Am. Chem. Soc. 2007, 129, 4776; (b) Saito, Y.; Miyauchi, Y.;
Okamoto, A.; Saito, I. Chem. Commun. 2004, 1704.
6. (a) Volpini, S.; Costanzi, S.; Lambertucci, C.; Vittori, S.; Klotz, K.-N.; Lorenzen, A.;
Cristalli, G. Bioorg. Med. Chem. Lett. 2001, 11, 1931; (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) Seo, Y. J.; Rhee, H.; Joo, T.; Kim, B. H. J. Am. Chem. Soc. 2007, 129, 5244; (e)
O’Mahony, G.; Ehrman, E.; Grøtli, M. Tetrahedron 2008, 64, 7151; (f) Abou-
Elkhair, R. A. I.; Dixon, D. W.; Netzel, T. L. J. Org. Chem. 2009, 74, 4712.
7. (a) Sessler, J. L.; Sathiosatham, M.; Brown, C. T.; Rhodes, T. A.; Wiederrecht, G. J.
Am. Chem. Soc. 2001, 123, 3655; (b) Mayer, E.; Valis, L.; Wagner, C.; Rist, M.;
Amann, N.; Wagenknecht, H. A. ChemBioChem 2004, 5, 865; (c) Okamoto, A.;
Ochi, Y.; Saito, I. Chem. Commun. 2005, 1128; (d) Firth, A. G.; Fairlamb, I. J. S.;
Darley, K.; Baumann, C. G. Tetrahedron Lett. 2006, 47, 3529.
bases (A, G) on the opposite site, not inconsistent with melting
temperature experiments (Table 2).
In conclusion, we have succeeded in the molecular design of a
solvatofluorochromic 20-deoxyguanosine derivative for the first
time. We found that acetyl substitution on the aromatic moiety
of 8-arylethynylated 20-deoxyguanosine induces a remarkable sol-
vatofluorochromic property. We also demonstrated that fluores-
cent deoxyguanosine 4c can be used as a thymine specific base-
discriminating fluorescent (BDF) nucleoside in homogenous fluo-
rescence assay. Fluorescence DNA probes containing BDF base 4c
are very useful for genetic analysis to detect point mutations and
SNPs and may provide multiplexing capability.
Acknowledgment
This work was supported by a Grant-in-Aid of MEXT, Japanese
Government.
8. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467.