Synthesis and photophysical properties of novel push-pull-type solvatochromic 7-deaza-2′-deoxypurine nucleosides

Article abstract of DOI:10.1016/j.tetlet.2011.06.089

We have synthesized two novel push-pull-type fluorescent 7-deazapurine nucleosides, ^CNZA and ^CNZG, and investigated their photophysical properties. In particular, ^CNZA was found to exhibit a remarkable solvatofluorochromic

Full text of DOI:10.1016/j.tetlet.2011.06.089

Tetrahedron Letters 52 (2011) 4726–4729
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and photophysical properties of novel push–pull-type
solvatochromic 7-deaza-20-deoxypurine nucleosides
⇑
⇑
Yoshio Saito , Azusa Suzuki, Shinya Ishioroshi, Isao Saito
Department of Chemical Biology and Applied Chemistry, 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 synthesized two novel push–pull-type fluorescent 7-deazapurine nucleosides, CNZA and CNZG,
and investigated their photophysical properties. In particular, CNZA was found to exhibit a remarkable sol-
Article history:
Received 30 May 2011
Revised 16 June 2011
Accepted 23 June 2011
Available online 30 June 2011
vatofluorochromicity (D A
kfl.max = 60 nm). We incorporated CNZA into oligonucleotides and found that CNZ
can form a stable base pair with both thymine and cytosine. Such environmentally sensitive fluorescent
nucleosides have a potential as a fluorescence sensor for structural studies of nucleic acids.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Nucleoside
DNA
Fluorescent probe
Numerous fluorescent molecules have been reported for fluo-
rescence labeling of biomolecules and widely used in bioscience.1
However, environmentally sensitive fluorescence nucleosides in
which emission spectra and quantum yields change sensitively
according to solvent polarity are of greatest interest owing to their
wide range of applications.2,3 Such environmentally sensitive fluo-
rescent nucleosides are used as fluorescence sensors in various
fields, as for example, incorporation of such solvatofluorochromic
nucleosides into oligonucleotides provides powerful tools for the
detection of target DNA, SNP genotyping,2 and for studying nucleic
acid–protein interactions.3
Recently, we reported C8-substituted push–pull-type fluores-
cent guanosines, AcG and CNG, which contain a covalently linked
electron donor–acceptor system consisting of guanosine as elec-
tron donor and pyrene fluorophore as acceptor (Fig. 1a).4 Although
these guanosine derivatives exhibited interesting solvatofluoro-
chromic properties, the steric bulk of the C8-substituents in both
AcG and CNG causes considerable destabilization of the DNA duplex
Figure 1. (a) Reported push–pull-type fluroscent nucleosides. (b) Novel push–pull-type fluroscent nucleosides.
⇑
Corresponding authors.
E-mail address: saitoy@chem.ce.nihon-u.ac.jp (Y. Saito).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.06.089

Y. Saito et al. / Tetrahedron Letters 52 (2011) 4726–4729
4727
Br
CN
a)
Br
Br
3
4
CN
b), c)
5
NC
NH2
NH2
N
I
N
d)
N
N
N
N
HO
O
HO
O
OH
CNZA (1)
6
OH
Scheme 1. Reagents and conditions: (a) 4-ethynylbenzonitrile, Pd(PPh3)4, Cul, Et3N, DMF, 80 °C, overnight, 52%; (b) trimethylsilylacetylene, Pd(PPh3)4, Cul, Et3N, toluene,
90 °C, overnight; (c) K2Co3, MeOH/THF, rt, 2 h, 66% in two steps; (d) 5, Pd(PPh3)4, Cul, Et3N, DMF, 80 °C, 2 h, 73%.
are directly attached to pyrene chromophore via triple bonds in or-
O
N
der to construct an intramolecular donor–acceptor system. We re-
port herein the synthesis and photophysical properties of novel
push–pull-type 7-deaza-20-deoxyadenosine (CNZ A) and 7-deaza-
20-deoxyguanosine (CNZ G) containing a 1,6-disubstituted pyrene
chromophore (Fig. 1b).
O
N
I
I
NH
N
NH
NH2
a)
N
N
N
HO
HO
O
O
The synthetic route of pyrene-labeled push–pull-type 7-deaza-
20-deoxyadenosine (CNZA) is outlined in Scheme 1. 1,6-dibromopy-
rene 37 was coupled with 4-ethynylbenzonitrile using Pd(PPh3)4 to
afford compound 4. Sonogashira cross-coupling reaction8 of 4 with
TMS-acetylene followed by deprotection with K2CO3 yielded 5.
Compound 5 was then coupled with 7-iodo-7-deaza-20-deoxya-
denosine 6 prepared according to the protocol of Seela et al.9 to
form CNZA.10
OH
7
OH
8
NC
O
N
NH
N
b)
N
N
HO
O
7-Deaza-20-deoxyguanosine derivative (CNZG) was synthesized
according to Scheme 2 via a similar route. The amino group of 7-
iodo-7-deaza-20-deoxyguanosine 711 was reacted with N,N-
dimethylformamide diethyl acetal to give protected amine 8 in
70% yield. Compound 8 was then coupled with 5 under Sonogash-
ira conditions using Pd(PPh3)4 to afford 9. After deprotection of
DMF group with 28% aq NH4OH-methanol, 7-deaza-20-deoxygua-
nosine derivative (CNZG) containing an electron-withdrawing sub-
stituent was obtained.10
9
OH
NC
O
NH
NH2
c)
N
N
HO
O
The photophysical properties of newly synthesized push–pull-
type 7-deazapurine derivatives, CNZA and CNZG, were examined. Ini-
tially, we measured the fluorescence spectra of CNZA in various sol-
vents of different polarity. Upon excitation of CNZA at 416 nm in
chloroform, strong fluorescence emission was observed at
470 nm as shown in Figure 2a (Ufl = 0.448).12 Upon excitation of
CNZA in THF, we observed moderate emission at 510 nm
OH
CNZG (2)
Scheme 2. Reagents and conditions: (a) N,N-dimethylformamide diethyl acetal,
DMF, 60 °C, 2 h, 70%; (b) 5, Pd(PPh3)4, Cul, Et3N, DMF, 80 °C, 2 h, 52%; (c) NH4OH,
MeOH, 60 °C, overnight, 41%.
structure due to their syn-conformation, and thus, these molecules
may not be suitable for the use as fluorescent DNA probes. We have
thus designed novel push–pull-type 7-deazapurine nucleosides.
While 8-substituents on purines cause steric repulsion that drives
the molecule into the syn-conformation, 7-substituents on 7-dea-
zapurine do not sterically interfere with the sugar-phosphate back-
bone and should fit into the major groove of the B-form of DNA.5 In
addition, the electron-donating ability of 7-deazapurine seems to
be similar to that of natural guanine bases, because the oxidation
potentials of 7-deazaadenine and 7-deazaguanine are closer to
and lower than those of natural guanine bases, respectively.6 On
the basis of these concepts, an electron-withdrawing 4-cyano-
phenyl group (acceptor) and 7-deazapurine nucleoside (donor)
(Ufl = 0.305). In contrast, very weak fluorescence emission was ob-
served at 530 nm in a polar solvent such as DMF (Ufl = 0.089). As
expected, push–pull-type 7-deaza-20-deoxyadenosine derivative
CNZA
exhibited
a
considerable
solvatofluorochromicity
(Dkfl.max = 60 nm), as also demonstrated by the fluorescence color
image depicted in Figure 2c.
The photophysical properties of 7-deaza-20-deoxyguanosine
derivative CNZG were also examined. While the fluorescence inten-
sity of CNZG is strong in low-polarity solvents such as chloroform,
weak fluorescence was observed in polar solvents as is presented
in Figure 2b. In the case of CNZG, no remarkable redshift of fluores-
cence emission was observed by changing solvent polarity, unlike

4728
Y. Saito et al. / Tetrahedron Letters 52 (2011) 4726–4729
(a)
(b)
100
25
Chloroform
20
Chloroform
AcOEt
80
60
40
AcOEt
15
10
5
THF
THF
DMF
DMF
Acetonitrile
Methanol
Acetonitrile
Methanol
20
0
0
400 450 500 550 600 650 700
400 450 500 550 600 650 700
Wavelength (nm)
Wavelength (nm)
(c)
chloroform
MeOH
acetonitrile
THF
DMF
EtOAc
Figure 2. Fluorescence spectra of (a) CNZA (1, 2.5
l
M) and (b) CNZG (2, 2.5
l
M) in various solvents, (c) Fluorescence color image of CNZA (1) in different solvents. The sample
solutions were illuminated with 365 nm transilluminator.
7-deazapurine derivative, CNZA which exhibited a redshift with
increasing solvent polarity. As shown inFigure 2a and b, the shapes
of the fluorescence spectra of CNZA and CNZG were very different.
CNZA showed broad emission bands and solvent polarity-depen-
dent fluorescence emissions at longer wavelengths, whereas the
emission bands of CNZG are similar to those of the vibrational struc-
tures of the parent pyrene chromophore.
To study the thermal stability and photophysical properties of
the synthesized solvatofluorochromic nucleoside in DNA, CNZA
was incorporated into oligodeoxynucleotide (ODN) via automated
DNA synthesis. The synthetic route of the corresponding phospho-
ramidite is indicated in Scheme 3. After protection of the amino
group with N,N-dimethylformamide diethylacetal, 10 was reacted
with DMTrCl in the presence of a catalytic amount of DMAP in
dry pyridine to give 11. Protected 11 was then converted to phos-
phoramidite 12. Phosphoramidite of CNZA was used for ODN syn-
thesis by using an automated DNA/RNA synthesizer without
further purification (Table 1).
Table 1
Thermal melting properties and photophysical properties ODN 1: 50-CGCAAT
X
TAACGC-30 (X = CNZA or A) ODN 2: 30-GCGTTA N ATTGCG-50 (N = T, C, A or G)
Abs
max
fl
max
Duplexes
Tm (°C)
/
fl
k
k
ODN 1 (CNZA)
—
424
422
435
434
421
—
484
484
483
484
485
—
0.372
0.454
0.466
0.387
0.428
—
ODN 1 (CNZA)/ODN 2 (T)
ODN 1 (CNZA)/ODN 2 (C)
ODN 1 (CNZA)/ODN 2 (A)
ODN 1 (CNZA)/ODN 2 (G)
ODN 1 (A)/ODN 2 (T)
45.5
46.7
43.4
43.4
52.6
Synthesized single-stranded oligonucleotide (ODN 1) contain-
ing push–pull-type fluorescent nucleoside CNZA was hybridized
with complimentary DNA strands (ODN 2) and resulting duplexes
were tested for thermal stability. Seela and co-workers reported
that 7-deaza-20-deoxyadenosine (ZA) generates a strong base pair,
not only with T but also with C and G. In particular, replacement of
the purine base by a pyrrolo[2,3-d]pyrimidine system causes
Scheme 3. Reagents and conditions: (a) N,N-dimethylformamide diethyl acetal, DMF, 60 °C, l h, 73%; (b) DMTrCl, DMAP, pyridine, r.t, 2 h, 88%; (c) 2-cyanoethyl N,N-
diissopropylchlorophosphoramidite, Et3N, acetonitrile, rt, l h.

Y. Saito et al. / Tetrahedron Letters 52 (2011) 4726–4729
4729
strong deazapurine (ZA)–C interaction.13 As shown in Table 1, the
References and notes
melting temperatures of ODN 1(CNZA)/ODN 2(T) and ODN
1(CNZA)/ODN 2(C) were considerably higher than those observed
in other mismatched duplexes in a sodium phosphate buffer (pH
7.0), suggesting that CNZA can also form a stable base pair with both
natural T and C, although incorporation of bulky CNZA into duplexes
1. (a) Greco, N. J.; Sinkeldam, R. W.; Tor, Y. Org. Lett. 2009, 11, 1115; (b) Srivatsan,
S. G.; Tor, Y. J. Am. Chem. Soc. 2007, 129, 2044; (c) Printz, M.; Richert, C.
Chemistry 2009, 15, 3390; (d) Cekan, P.; Sigurdsson, S. T. Chem. Commun. 2008,
3393.
2. (a) Saito, Y.; Miyauchi, Y.; Okamoto, A.; Saito, I. Tetrahedron Lett. 2004, 45, 7827;
(b) Matsumoto, K.; Takahashi, N.; Suzuki, A.; Morii, T.; Saito, Y.; Saito, I. Bioorg.
Med. Chem. Lett. 2011, 21, 1275.
3. Tainaka, K.; Tanaka, K.; Ikeda, S.; Nishiza, K.; Unzai, T.; Fujiwara, Y.; Saito, I.;
Okamoto, A. J. Am. Chem. Soc. 2007, 129, 4776.
4. Saito, Y.; Suzuki, A.; Imai, K.; Nemoto, N.; Saito, I. Tetrahedron Lett. 2010, 51,
2606.
5. Seela, F.; Shaikh, K. I. Tetrahedron 2005, 61, 2675.
6. Baik, M.-H.; Silverman, J. S.; Yang, I. V.; Ropp, P. A.; Szalai, V. A.; Yang, W.;
Thorp, H. H. J. Phys. Chem. B 2001, 105, 6437.
7. Grimshaw, J.; Trocha-Grimshaw, J. J. Chem. Soc., Perkin Trans. 1 1972, 1622.
8. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467.
9. Seela, F.; Zulauf, M. Synthesis 1996, 726.
resulted in
Tm = 7.1 °C) as compared with unmodified matched ODN 1(A)/
ODN 2(T).
a
considerable destabilization of the duplex
(D
Next, we measured the fluorescence spectra of ODN 1 in the
presence or absence of complementary strands. Strong fluores-
cence emissions of single-stranded ODN 1 and the duplexes with
complementary strands ODN 2 (T, C, A, G) were observed at around
485 nm. However, the fluorescence wavelength and intensity were
not changed remarkably by changing the nucleosides opposite to
CNZA (Table 1, Fig. S1), indicating that CNZA cannot be used as a
base-discriminating fluorescent nucleoside.14
10. Spectroscopic data for CNZA (1): 1H NMR (DMSO-d6, 400 MHz) d 2.31 (ddd,
J = 2.9, 6.0, 13.2 Hz, 1H), 2.57 (m, 1H), 3.60–3.71 (complex, 2H), 3.91 (m, 1H),
4.44 (m, 1H), 4.97 (dd, J = 5.5, 5.5 Hz, 1H), 5.20 (d, J = 4.2 Hz, 1H), 6.60 (m, 1H),
6.77 (br, 2H), 7.96–8.01 (complex, 4H), 8.11 (s, 1H), 8.28 (s, 1H), 8.36–8.45
(complex, 6H), 8.69 (d, J = 9.0 Hz, 1H), 8.70 (d, J = 9.0 Hz, 1H); 13C NMR (DMSO-
d6, 100 MHz) d 61.7, 70.7, 78.9(ꢀ2), 83.2, 87.5, 89.5, 89.6, 91.9, 93.9, 110.9,
116.4, 118.1, 118.2, 123.1, 123.2, 125.3, 125.4, 125.7, 125.9, 127.0, 127.3, 128.3,
128.7, 129.8, 130.2, 130.3, 130.7, 131.2, 131.4, 132.1(ꢀ2), 132.4(ꢀ2), 149.5,
152.6, 157.5. C(20) overlapped with DMSO; HRMS (ESI) m/z 622.1855 calcd for
In summary, we have synthesized novel push–pull-type fluo-
rescent 7-deazapurine nucleosides CNZA and CNZG, the first highly
fluorescent pyrene containing 7-deaza-20-deoxypurine nucleo-
sides. CNZA exhibited strong fluorescence at long wavelength and
a remarkable solvatofluorochromicity (D
kfl.max = 60 nm). CNZA was
C
38H25N5O3Na [M+Na]+, found 622.1858.
incorporated into oligonucleotides and the thermal stability of
the resulting duplex was evaluated. The thermal melting data indi-
cated that CNZA can form a stable base pair with natural T and C,
similar to that reported for 7-deaza-20-deoxyadenosine (ZA). Such
types of environmentally sensitive and strongly fluorescent
nucleosides may be used as a fluorescence sensor for structural
studies of nucleic acids and as a building block for constructing a
fluorescent DNA nanostructure.
Spectroscopic data for CNZG (2): 1H NMR (DMSO-d6, 400 MHz) d 2.21 (ddd,
J = 2.8, 5.9, 13.1 Hz, 1H), 2.45 (ddd, J = 5.9, 8.1, 13.1 Hz, 1H), 3.57–3.65
(complex, 2H), 3.85 (m, 1H), 4.38 (m, 1H), 4.86 (m, 1H), 5.16 (d, J = 2.9 Hz,
1H), 6.37–6.40 (complex, 3H), 7.56 (s, 1H), 7.96–8.01 (complex, 4H), 8.22 (d,
J = 8.0 Hz, 1H), 8.32–8.41 (complex, 5H), 8.66 (d, J = 9.1 Hz, 1H), 9.07 (d,
J = 9.1 Hz, 1H), 10.40 (br, 1H); 13C NMR (DMSO-d6, 100 MHz) d 61.8, 70.7, 82.4,
87.2, 88.7, 91.2, 92.1, 93.8, 98.4, 99.6, 110.9, 116.0, 118.2, 119.4, 122.1, 123.2,
123.2, 124.9, 125.1, 125.7, 127.0, 127.1, 127.8, 128.8, 128.9, 129.8, 130.1, 131.0,
131.4, 131.5, 132.1(ꢀ2), 132.4(ꢀ2), 150.5, 153.3, 158.8. C(20) overlapped with
DMSO; HRMS (ESI) m/z 638.1804 calcd for C38H25N5O4Na [M+Na]+, found
638.1818.
11. Ramzaeva, N.; Seela, F. Helv. Chim. Acta 1995, 78, 1083.
12. Quantum yields (U) were calculated according to the following reference:
Acknowledgment
Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976, 80, 969.
13. Peng, X.; Li, H.; Seela, F. Nucleic Acids Res. 2006, 34, 5987.
14. (a) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820; (b)
Saito, Y.; Miyauchi, Y.; Okamoto, A.; Saito, I. Chem. Commun. 2004, 1704.
This work was supported by a Grant-in-Aid for Scientific re-
search of MEXT, from Japanese Government.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.06.089.