Design and synthesis of highly solvatochromic fluorescent 2′-deoxyguanosine and 2′-deoxyadenosine analogs

Article abstract of DOI:10.1016/j.bmcl.2010.11.129

We synthesized various substituted 8-styryl-2′-deoxyguanosine and 8-styryl-2′-deoxyadenosine derivatives. Among them only acetyl substituted 8-styryl-2′-deoxyguanosine analog 5b showed a remarkable solvatochromicity (Δλ_max^em=91nm),th

Full text of DOI:10.1016/j.bmcl.2010.11.129

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1275–1278
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of highly solvatochromic fluorescent 20-deoxyguanosine
and 20-deoxyadenosine analogs
Katsuhiko Matsumoto b, Naoya Takahashi a, Azusa Suzuki a, Takashi Morii b, Yoshio Saito a, , Isao Saito a,
⇑
⇑
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 synthesized various substituted 8-styryl-20-deoxyguanosine and 8-styryl-20-deoxyadenosine deriva-
Article history:
tives. Among them only acetyl substituted 8-styryl-20-deoxyguanosine analog 5b showed a remarkable
Received 9 November 2010
Revised 26 November 2010
Accepted 30 November 2010
Available online 13 December 2010
em
solvatochromicity (
Dk
¼ 91 nm),that is, strong fluorescence at 477 nm in 1,4-dioxane, but in metha-
max
nol the fluorescence was red shifted to 558 nm with very low intensity. Such environmentally sensitive
solvatochromic fluorescent guanosine analogs may be useful as a sensor for investigating interactions of
DNA with DNA binding proteins.
Keyword:
Nucleoside DNA fluorescence
Ó 2010 Elsevier Ltd. All rights reserved.
Numerous efforts to impart useful photophysical features
(e.g., long emission wavelength and high quantum efficiency) upon
non-emissive natural nucleobases have been reported.1 Most of the
previous approaches involve the linking of natural nucleobases to
fluorescent aromatic or heteroaromatic chromophores via an
ethynyl linker1 or the direct attachment of fluorescent chromoph-
ores on the natural nucleobases.2 Although numerous 8-arylethyny-
lated adenine3 and guanine4 derivatives have been reported,
tuted 8-styryl-20-deoxyguanosine (5b) showed a highly solvent
polarity sensitive fluorescence emission with a large solvatochrom-
em
max
icity (Dk
¼ 91 nm) that may be useful for investigating nucleic
acid structure, dynamics and recognition (Fig. 1). In contrast to our
previously reported base-discriminating fluorescent (BDF) nucleo-
sides,1c,6 which show increasing or decreasing emission intensity
at fixed wavelength, solvatochromic fluorescent 20-deoxyguanosine
and 20-deoxyadenosine analogs described here may indicate the
change in their local environments by a drastic change of emission
wavelength together with intensity change.
guanine derivatives that are
p-conjugated with aromatics at the
8-positionvia a double bond are few.5 Inour researchprogram direc-
ted toward the design of environmentally sensitive fluorescent
nucleosides, we have successfully designed for the first time highly
solvatochromic fluorescent 20-deoxyguanosine and 20-deoxyadeno-
We synthesized 20-deoxyguanosine analog 5b according to the
synthetic route shown in Scheme 1. Amino group of 8-bromo-
20-deoxyguanosine 1 was protected by N,N-dimethylformamide
diethylacetal to yield 2. Protected 2 was reacted with tetravinyltin
(IV) under palladium (0) mediated Stille coupling conditions to
sine analogs that are
p-conjugated with aromatic chromophores
through a double bond. We now report herein that acetyl substi-
O
O
N
NH2
N
O
O
N
N
N
N
N
NH
NH
NH2
N
N
NH2
N
HO
HO
HO
O
O
O
OH
OH
OH
5a
5b
8
Figure 1. Chemical structures of aromatic fluorophore substituted purine nucleosides.
⇑
Corresponding authors.
E-mail address: saitoy@chem.ce.nihon-u.ac.jp (Y. Saito).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.129

1276
K. Matsumoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1275–1278
O
N
O
O
N
N
N
N
N
N
NH
NH2
NH
NH
N
Br
Br
(a)
(b)
N
N
N
N
N
HO
HO
HO
O
O
O
OH
OH
OH
1
2
3
O
N
O
O
O
N
N
N
N
(d)
(c)
NH
NH
N
N
N
NH2
HO
HO
O
O
OH
OH
4
5b
Scheme 1. Reagents and conditions: (a) N,N-dimethylformamide diethylacetal, MeOH, 60 °C, 12 h, (98%); (b) tetravinyltin (IV), Pd(PPh3)4, Et3N, DMF, 80 °C, 12 h, (62%);
(c) 4-bromoacetophenone, Pd(PPh3)4, AcONa, DMF, 100 °C, 6 h, (quant.); (d) NH4OH, MeOH, 55 °C, 8 h, (68%).
NH2
N
NH2
N
NH2
N
O
N
N
N
N
N
N
Br
N
N
N
(a)
(b)
HO
HO
HO
O
O
O
OH
OH
OH
6
7
8
Scheme 2. Reagents and conditions: (a) tetravinyltin (IV), Pd(PPh3)4, AcONa, DMF, 90 °C, 3 h, (82%); (b) 4-bromoacetophenone, Pd(PPh3)4, AcONa, DMF, 120 °C, 5 h, (36%).
120
100
80
1,4-dioxane
THF
acetonitrile
DMSO
methanol
1,4-dioxane
THF
acetonitrile
DMSO
methanol
a
b
140
120
100
80
60
60
40
40
20
0
20
0
350
400
450
500
550
600
350
450
550
650
wavelength (nm)
wavelength (nm)
c
d
160
120
THF
acetonitrile
DMSO
methanol
600
550
500
450
400
5a
5b
8
80
40
0
350
450
550
650
35
45
55
65
ET (30)
wavelength (nm)
e
1,4-dioxane acetonitrile DMSO MeOH
Figure 2. Fluorescence spectra of 5a (a), 5b (b) and 8 (c) in various solvents (each 10
lM concn). Excitation wavelength was 355 nm (a), 370 nm (b), and 350 nm (c). (d) Plots
of emission maximum of 5a ( ), 5b ( ) and 8 ( ) as a function of ET (30). (e) Fluorescence image of 5b under illumination with transilluminator (365 nm).

K. Matsumoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1275–1278
1277
Table 1
emission in polar solvents, indicating that 5b is an effective envi-
ronmentally sensitive nucleoside (Fig. 2b, e, Table 1). Interestingly,
5b showed almost no fluorescence in methanol. We thought that
such phenomenon may also occur with acetylated 20-deoxyadeno-
sine 8. In fact, 8 showed strong fluorescence in non-polar solvents
and the fluorescence in polar solvents was red shifted with low
intensity (Fig. 2c). These fluorescence emission maxima in various
solvents were plotted against ET (30) values, a solvent polarity
parameter.8 The slopes obtained from the linear plots indicated
that 5b is most solvatofluorochromic (Fig. 2d). It should be note-
worthy that during fluorescence measurement acetyl substituted
5b and 8 were not photoisomerized, whereas 5a gradually gave a
mixture of (E) and (Z) isomers during fluorescence measurement.
Solvatochromic fluorescent deoxyguanosine 5b was incorpo-
rated into 16mer oligodeoxynucleotide (ODN 1, 50-GTATCCA5bAG
ATTGAA-30). 50-Hydroxyl group of 4 was protected by dimethoxy-
trityl group to afford 9. The compound 9 was reacted with N,N-dii-
sopropylchlorophosphoramidite to give amidite 10, which was
incorporated into oligonucleotide by automated DNA/RNA synthe-
sizer without further purification (Scheme 3). The synthesized
ODN 1 was purified by reverse phase HPLC.
Fluorescence spectrum of ODN 1 was measured in the absence
and presence of complementary 15mer ODN 2 (50-TTCAATCTT
GGATAC-30) forming a bulge structure at 5b. Fluorescence intensity
of single stranded ODN 1 was very weak and the fluorescence max-
imum was observed at 550 nm. In contrast, the fluorescence inten-
sity of duplex ODN 1/ODN 2 containing bulge was enhanced and
the fluorescence maximum was blue shifted to 526 nm (Fig. 3a).
The fluorescence color change was readily observable by naked
eye under illumination with 365 nm transilluminator (Fig. 3b,
Tm = 50.9 °C). The fluorescence change between single stranded
and double stranded structures may be ascribable to the local envi-
ronmental change around 5b.
Photophysical properties of fluorescent 20-deoxypurine nucleosides 5a, 5b and 8
Solvent
ET (30)8
5a
5b
8
kem
UF
kem
UF
kem
UF
(nm)
(nm)
(nm)
1,4-Dioxane
Elhylacelale
THF
DMSO
Acelonitrile
Methanol
36.0
38.1
37.4
45.1
45.6
55.4
432
438
442
456
442
440
0.26
0.19
0.34
0.16
0.25
0.12
477
491
499
541
525
558
0.62
0.48
0.49
0.34
0.29
0.02
—
—
460
470
502
474
517
0.39
0.37
0.27
0.33
0.10
produce 3. Compound 3 was coupled with 4-bromoacetophenone
under Heck reaction conditions and the treatment with ammonia
afforded acetylated (E)-8-styryl-deoxyguanosine 5b.
Acetyl substituted 8-styryl-20-deoxyadenosine
8 was also
synthesized by a similar route shown in Scheme 2. 8-Bromo-20-
deoxyadenosine 6 was reacted with tetravinyltin (IV) in the
presence of catalytic amount of Pd(0) to yield 8-vinyl-20-deoxya-
denosine 7. Compound 7 was coupled with 4-bromoacetophenone
under Heck reaction conditions to give acetylated (E)-8-styryl
deoxyadenosine 8. Acetyl substituted purine nucleosides 5b and
8 were characterized by 1H NMR, 13C NMR and mass spectros-
copy.7 (E)-8-Styryl-20-deoxyguanosine 5a was also synthesized
according to the reported protocol.5
We first examined the fluorescence behavior of phenyl substi-
tuted 5a and acetophenone conjugated 5b in various solvents.
Fluorescence wavelength and intensity of non-substituted 5a were
not changed remarkably by changing solvent polarity (Fig. 2a). In
contrast, acetyl substituted 5b showed strong fluorescence emis-
sion in non-polar solvents but very weak red shifted fluorescence
O
N
O
O
N
O
N
O
O
N
N
NH
N
N
N
N
N
NH
NH
N
N
(b)
(a)
DMTrO
N
N
N
O
HO
DMTrO
O
O
O
O
P
N
CN
OH
OH
4
9
10
Scheme 3. Reagents and conditions: (a) DMTrCl, 4,4-dimethylaminopyridine, pyridine, rt, 17 h, (54%); (b) N,N-diisopropylchlorophosphoramidite, triethylamine, acetonitrile,
rt, 5 min.
a
b
80
60
40
ODN 1
ODN 1/ODN 2
20
0
380
430
480
530
580
630
680
wavelength (nm)
ODN1
ODN 1/ODN 2
Figure 3. (a) Fluorescence spectra of single stranded ODN 1 and duplex formed by hybridization with ODN 2. (2.5
lM ODNs, 50 mM sodium phosphate, 0.1 M sodium
chloride, pH 7.0, rt) Excitation wavelength was 380 nm. (b) Fluorescence image of ODN 1 and the duplexes formed with ODN 2 under illumination with transilluminator
(365 nm).

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K. Matsumoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1275–1278
C.; Vittori, S.; Volpini, R.; Cristalli, G. J. Mol. Graphics Modell. 2003, 21, 253; (c)
In conclusion, we have succeeded in the design and synthesis of
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solvatofluorochromic 20-deoxyguanosine and adenosine analogs.
Interestingly, acetyl substituted 20-deoxypurine derivatives
showed a remarkable high solvatofluorochromicity. Particularly,
acetyl substituted 20-deoxyguanosine 5b showed a large solvato-
em
max
chromicity (
Dk
¼ 91 nm). Highly fluorescent solvatochromic
guanosine analog 5b may be used as a fluorescence sensor in var-
ious fields.
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Acknowledgements
6. (a) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820; (b)
Shinohara, Y.; Matsumoto, K.; Kugenuma, K.; Morii, T.; Saito, Y.; Saito, I. Bioorg.
Med. Chem. Lett. 2010, 20, 2817.
This work was supported by a Grant-in-Aid for Scientific re-
search of MEXT, Japanese Government. The authors are grateful
to Professor Tatsuo Arai (Univ. of Tsukuba) and Professor Masaya
Imoto (Keio Univ.) for helpful discussions.
7. 8-(4-Acetylstyryl)-20-deoxyguanosine (5b). 1H NMR (DMSO-d6, 400 MHz) d;
2.12 (m, 1H), 2.51–2.59 (complex, 4H), 3.69–3.73 (complex, 2H), 3.85 (m, 1H),
4.47 (m, 1H), 5.27 (dd, 1H, J = 5.0, 5.2 Hz), 5.31 (d, 1H, J = 4.0 Hz), 6.40 (dd, 1H,
J = 6.2, 8.8 Hz), 6.56 (br s, 2H), 7.60 (d, 1H, J = 15.8 Hz), 7.69 (d, 1H, J = 15.8 Hz),
7.85 (d, 2H, J = 8.4 Hz), 7.95 (d, 2H, J = 8.4 Hz), 10.72 (br s, 1H); 13C NMR (DMSO-
d6, 100 MHz) d; 26.7, 44.0, 61.5, 70.6, 83.4, 87.7, 117.6, 119.1, 127.6 (2C), 128.6
(2C), 134.5, 136.4, 140.0, 147.3, 149.8, 152.1, 155.6, 197.2; HRMS (ESI) m/z
434.1440 calcd for C20H21N5O5Na [M+Na]+, found 434.1418. 8-(4-Acetylstyryl)-
20-deoxyadenosine (8). 1H NMR (DMSO-d6, 400 MHz) d; 2.21 (m, 1H), 2.61 (s,
3H), 2.91 (m, 1H), 3.62 (m, 1H), 3.72 (m, 1H), 3.91 (m, 1H), 4.53 (m, 1H), 5.35 (d,
1H, J = 4.3 Hz), 5.50 (dd, 1H, J = 4.3, 6.9 Hz), 6.65 (dd, 1H, J = 6.6, 8.2 Hz), 7.41 (s,
2H), 7.78 (d, 1H, J = 15.8 Hz), 7.82 (d, 1H, J = 15.8 Hz), 7.91 (d, 2H, J = 8.6 Hz),
7.99 (d, 2H, J = 8.6 Hz), 8.12 (s, 1H) 13C NMR (DMSO-d6, 100 MHz) d; 26.6, 39.6,
61.1, 70.1, 82.4, 87.2, 116.7, 118.5, 127.2(2C), 128.6(2C), 131.4, 135.8, 140.7,
143.7, 151.7, 153.4, 156.4, 197.1; HRMS (ESI) m/z 418.1491 calcd for
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