Naphthalene-based environmentally sensitive fluorescent 8-substituted 2′-deoxyadenosines: Application to DNA detection

Article abstract of DOI:10.1016/j.bmcl.2012.11.029

We synthesized various C8-naphthylethynylated 2′-deoxyadenosine derivatives and investigated their photophysical properties. Among them, cyano- and N,N-dimethylamino-substituted 8-naphthylethynylated 2′-deoxyadenosine derivatives (^cnA and ^dnA) showed strong fluorescence with high quantum yields and a remarkable solvatofuorochromicity. In particular, fluorescence of N,N-dimethylamino-substituted ^{2, 6dn}A was not quenched by neighboring guanines (Gs) when incorporated in DNA duplexes, in contrast to ^cnA. We developed a new fluorescent probe containing ^2,6dnA that can be used for the detection of target DNA via a bulge formation regardless of the neighboring sequences.

Full text of DOI:10.1016/j.bmcl.2012.11.029

Bioorganic & Medicinal Chemistry Letters 23 (2013) 886–892
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Naphthalene-based environmentally sensitive fluorescent
8-substituted 20-deoxyadenosines: Application to DNA detection
a
a,
Azusa Suzuki a, Naoya Takahashi a, Yuji Okada a, Isao Saito b, , Nobukatsu Nemoto , Yoshio Saito
⇑
⇑
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
We synthesized various C8-naphthylethynylated 20-deoxyadenosine derivatives and investigated their
photophysical properties. Among them, cyano- and N,N-dimethylamino-substituted 8-naphthylethyny-
lated 20-deoxyadenosine derivatives (cnA and dnA) showed strong fluorescence with high quantum yields
and a remarkable solvatofuorochromicity. In particular, fluorescence of N,N-dimethylamino-substituted
2,6dnA was not quenched by neighboring guanines (Gs) when incorporated in DNA duplexes, in contrast
to cnA. We developed a new fluorescent probe containing 2,6dnA that can be used for the detection of tar-
get DNA via a bulge formation regardless of the neighboring sequences.
Article history:
Received 3 October 2012
Revised 7 November 2012
Accepted 8 November 2012
Available online 6 December 2012
Keywords:
Nucleoside
DNA
Ó 2012 Elsevier Ltd. All rights reserved.
Fluorescent probe
Fluorescent molecules are powerful research tools since they
are simple, inexpensive, highly sensitive, and non-invasive. They
are used for bioimaging,1 drug delivery,2 sensing,3 monitoring of
chemical interactions of biomolecules,4 gene detection,5 diagnosis6
and targeted therapeutics.7 Thus, there is still a growing interest in
the development of newly designed fluorescent molecules, partic-
ularly, environmentally sensitive fluorophores of high quantum
yield. The design of such fluorophores that changes their emission
wavelength and quantum yield in response to the change of local
environments such as dielectric properties, structural variation,
surrounding pH and viscosity is of special interest owing to their
wide range of applications. Although numerous environmentally
sensitive fluorescent molecules are known, only limited number
of environmentally sensitive fluorescent (ESF) nucleosides have
been reported recently.8 Incorporation of fluorescent nucleosides
that are sensitive to their local environment into oligonucleotide
can provide useful tools for structural study of nucleic acids as well
as for the detection of target gene and for single nucleotide poly-
morphism (SNP) typing.
In our research program directed toward the design of environ-
mentally sensitive fluorescent (ESF) nucleoside,9 we have recently
reported various C8-arylethynylated 20-deoxyguanosines.9a,b,e
Among them, acetyl-substituted 8-arylethynylated deoxyguano-
sine acG, which contains an electron donor–acceptor system
within a molecule, exhibited a remarkable solvent-dependent fluo-
rescence property.9a,b While acetyl-substituted acG derivatives
exhibited interesting solvatofluorochromic properties, their emis-
sion wavelengths and quantum yields are not satisfactory and re-
mained to be improved. Further development of more sensitive
solvatofluorochromic nucleosides of high quantum yields is
apparently necessary. We report herein that C8-naphthylethyny-
lated 20-deoxyadenosines cnA and dnA possessing an intramolecu-
lar donor–acceptor system can serve as an excellent ESF
nucleobase of remarkably high quantum yield. Owing to the differ-
ences in the redox potentials of adenine and guanine, adenine can
act as either as an electron donor or as an acceptor, depending on
the substituent on the naphthalene ring unlike C8-arylethynylated
guanine derivatives (Fig. 1). The newly synthesized ESF nucleosides
were incorporated into oligodeoxynucleotides (ODNs). Using these
ESF nucleosides, a novel fluorescent probe that can detect target
DNA via a bulge formation was developed.
Various substituted C8-naphthylethynylated 20-deoxyadeno-
sines 1a–c were prepared via the palladium(0)-mediated Sono-
gashira cross-coupling reaction10 (Scheme 1). 8-Ethynyl-20-
deoxyadenosine 3 prepared by a previous method11 was coupled
with 2-bromo-6-cyanonaphthalene 4a and 2-bromonaphthalene
4c to afford the corresponding C8-substituted 20-deoxyadenosines
2,6cnA (1a) and 2nA (1c), respectively.12 Because of the low reactiv-
ity of bromonaphthalene containing electron-donor substituents
toward cross-coupling reactions, bromonaphthalene 4b was con-
verted to the corresponding iodide 5b, which was then used in
the Pd(0)-mediated cross-coupling reaction to afford 2,6dnA (1b).12
C8-substituted-20-deoxyadenosines 2a–c with 1,4-disubsti-
tuted naphthalenes were also synthesized by a similar route.
8-Ethynyl-20-deoxyadenosine 3 was coupled with 1-bromo-4-cya-
nonaphthalene 6a and commercially available 1-iodonaphthalene
7c to afford 1,4cnA (2a) and 1nA (2c), respectively. Iodonaphthalene
⇑
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.2012.11.029

A. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 886–892
887
cn
sumed that such an intramolecular donor–acceptor system is the
origin for the solvatofluorochromic effects.
dn
2
The photophysical properties of C8-substituted-20-deoxyadeno-
sines with 1,4-disubstituted 1,4cnA (2a) and 1,4dnA (2b) and non-
substituted 1nA (2c)-naphthalenes were also investigated. As
shown in Figure 3, very similar results to those observed for 2,6-
disubstituted naphthalene derivatives were obtained. The fluores-
cence wavelengths of non-substituted 1nA (2c) were not changed
remarkably by changing solvent polarity, showing almost no solva-
tochromocity. In contrast, 1,4cnA (2a) and 1,4dnA (2b) exhibited very
strong fluorescence emissions in non-polar solvents but only weak
red-shifted fluorescence emission in polar solvents, indicating that
both 1,4cnA (2a) and 1,4dnA (2b) are effective ESF nucleosides of a
2
2
cn
ac
dn
Figure 1. Structures of solvatofluorochromic nucleosides with donor–acceptor
system.
7b prepared from 6b was coupled with 3 using Pd(PPh3)4 to afford
1,4dnA (2b).
considerable solvatofluorochromicity (2a:
Dk = 71 nm; 2b:
The photophysical properties of newly synthesized 20-deoxya-
denosine derivatives 1a–c and 2a–c were examined. Initially, we
measured the fluorescence spectra of 2,6-disubstituted naphtha-
lene derivative 2,6cnA (1a) containing an electron-withdrawing cy-
ano substituent in various solvents of different polarities. By
excitation of 2,6cnA (1a) at 340 nm in 1,4-dioxane, strong fluores-
cence emission was observed at 424 nm with a high quantum yield
(0.82) as shown in Figure 2a. Fluorescence emission of 2,6cnA (1a)
was observed at 454 nm in acetonitrile. In contrast, in polar sol-
vents such as methanol (462 nm), DMF (478 nm), and DMSO
(490 nm), weaker fluorescence emissions were observed for
2,6cnA (1a) at longer wavelengths. Thus, 2,6cnA (1a) exhibited a
typical solvatofluorochromocic behavior with a considerable large
D
k = 36 nm). Interestingly, each of 2,6- and 1,4-disubstituted
naphthalene derivatives 2,6cnA (1a), 2,6dnA (1b), 1,4cnA (2a), and
1,4dnA (2b) exhibited high solvatofluorochromic properties. Among
them, 2,6dnA (1b) bearing an electron donating N,N-dimethylamino
substituent is a very attractive ESF nucleoside that has a high quan-
tum yield (0.88 in dioxane) and may probably be usable regardless
of the flanking sequences, because unlike cyano-substituted 2,6cnA
(1a), electron donor substituted naphthalene 2,6dnA (1b) may not
be a quenchable fluorophore by neighboring Gs when incorporated
in duplex DNA. It should be reminded here that N,N-dimethyl-
amino substituted 2,6dnA (1b) exhibit strong fluorescence at nonpo-
lar environments but only weak fluorescence in polar aqueous
solvents at longer wavelength (Fig. 2b).
solvatofluorochromicity (
much larger solvatofluorochromicity (
D
k = 66 nm). Interestingly, a similar but
k = 71 nm) was observed
Next, to study the photophysical properties of these ESF nucleo-
sides in duplex DNA, 2,6cnA (1a), 2,6dnA (1b), 1,4cnA (2a), and 1,4dnA
(2b) were incorporated into oligodeoxynucleotides (ODNs) via
automated DNA synthesis. The synthetic route to the correspond-
ing phosphoramidites of the 2,6-disubstituted naphthylethynylat-
ed 20-deoxyadenosines is indicated in Scheme 2. After protection
of the amino group with N,N-dimethylformamide diethylacetal,
compounds of general formula 8 were reacted with DMTrCl in
the presence of catalytic amount of DMAP in dry pyridine to give
9. The protected compounds 9 were then converted to phospho-
ramidites 10. Using a similar protocol, phosphoramidites of 1,4-
disubstituted naphthylethynylated 20-deoxyadenosines were also
prepared (Supplementary data, Scheme S1). Each phosphoramidite
D
with 2,6dnA (1b) bearing an electron donor substituent (Fig. 2b).
While C8-naphthylethynylated 20-deoxyadenosine derivatives
2,6cnA (1a) and 2,6dnA (1b) exhibited highly solvatofluorochromic
properties, non-substituted naphthalene derivative 2nA (1c) did
not show a clear solvatofluorochromic behavior, indicating the
requirement of an intramolecular donor–acceptor system within
a molecule for the solvatofluorochromicity (Fig. 2c, Table 1). In case
of electron-withdrawing cyano-substituted naphthalene 2,6cnA
(1a), adenine moiety acts as an electron donor. In contrast, adenine
moiety can act as an electron acceptor in 2,6dnA (1b), which con-
tains an electron-donating N,N-dimethylamino group. It is as-
NH2
R6
N
N
N
N
NH2
N
HO
HO
2,6cnA(1a):
2,6dnA(1b):
2nA(1c):
R
R
R
6 = CN (22%)
6 = NMe2 (69%)
6 = H (39%)
O
(a)
N
N
N
OH
NH2
R4 or R6
HO
O
X
N
N
N
OH
3
R4
X = I or Br
N
R = CN, NMe2 or H
O
1,4cnA(2a):
1,4dnA(2b):
1nA(2c):
R
R
R
4 = CN (46%)
4 = NMe2 (74%)
4 = H (61%)
OH
R6
R6
(b)
(b)
Br
R4
I
R4
I
Br
4a
6a: R4 = CN
5b: R6 = NMe2
7b: R4 = NMe2 (40%)
: R6 = CN
b: R4 = NMe2
b: R6 = NMe2
c: R6 = H
: R4 = H
c
(67%)
Scheme 1. Reagents and conditions: (a) 4a,c, 5b, 6a, 7b, or 7c, Pd(PPh3)4 CuI, Et3N, DMF, 50–80 °C, 30 min–3 h; (b) n-BuLi, THF, ꢀ78 °C, 30 min and then I2, THF, rt, 30 min.

888
A. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 886–892
a
b
c
Figure 2. Fluorescence spectra of (a) 2,6cnA (1a, 10 m), (b) 2,6dnA (1b,10 m) and (c) 2nA (1c, 10
l l lm) in various solvents. The excitation was conducted at (a) 340 nm, (b)
382 nm and (c) 326 nm. (slit width: 1.5).
was used for ODN synthesis using an automated DNA synthesizer.
Two 13 mer fluorescent ODN probes containing ESF nucleoside X at
AT rich site (ODN 1) and at GC rich site (ODN 2) were synthesized
(Table 2).
Table 1
Photophysical properties of fluorescent 2’-deoxyadenosine derivatives
abs
max
fl
max
Compound
Solvent
Ufl
k
(nm)
k
(nm)
As predicted by molecular modeling, adenine base containing
large substituents such as naphthylethynyl group at C8 position
cannot form Watson–Crick AT base pair when hybridized with
complementary strand, because the steric bulkiness of the naphth-
ylethynyl group is repulsive to sugar backbone and the syn-confor-
mation of the adenine glycoside bond is generally preferable for
adenines with large substituents at C8 position. In fact, melting
temperature of full matched 13 mer duplex ODN 1(X = 2,6cnA)/30-
GCGTTATATTGCG-50 (13 mer) is 9.3 °C lower than that of the native
duplex (52.6 °C) containing unmodified ODN1(X = A), suggesting a
lack of base pairing between 2,6cnA and T to result in the observed
large destabilization of the duplex (Supplementary data, Table S1).
Thus, 8-naphthylethynylated adenosines such as 2,6cnA (1a) and
2,6dnA (1b) cannot form AT base pair with opposite T in hybridiza-
tion with a complementary strand and such ESF nucleosides are
not usable as a modified adenine base opposite to T in a fluores-
cence hybridization assay. We, therefore, selected 12 mer blODN
1 as a target ODN that may form a bulge structure at X in hybrid-
ization with ODN1 (X). For GC rich sequence, ODN2 (X) and 12 mer
blODN 2 were selected. Examples of such ODNs are summarized in
Table 2.
2,6cnA (1a)
1,4-Dioxane
Acetonitrile
DMF
347
338
348
338
424
454
478
462
0.82
0.58
0.33
0.36
Methanol
2,6dnA (1b)
1,4-Dioxane
Acetonitrile
DMF
360
365
385
385
435
488
480
506
0.88
0.51
0.62
0.05
Methanol
2nA (1c)
1,4-Dioxane
Acetonitrile
DMF
330
325
333
328
392
398
414
392
0.66
0.57
0.51
0.20
Methanol
1,4cnA (2a)
1,4dnA (2b)
1nA (2c)
1,4-Dioxane
Acetonitrile
DMF
362
357
365
359
450
482
503
494
0.51
0.14
0.04
0.04
Methanol
1,4-Dioxane
Acetonitrile
DMF
371
371
375
374
442
465
464
478
0.41
0.40
0.47
0.05
Methanol
1,4-Dioxane
Acetonitrile
DMF
339
335
340
336
397
401
424
391
0.52
0.40
0.36
0.20
First, the fluorescence of cyano-substituted ESF nucleoside
Methanol
(
2,6cnA)-containing 13 mer ODN was examined. The fluorescence

A. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 886–892
889
a
b
c
1,
Figure 3. Fluorescence spectra of (a) 1,4cnA (2a, 10
l
m), (b) 4dnA (2b, 10
l
m) and (c) 1nA (2c, 10
l
m) in various solvents. The excitation was conducted at (a) 360 nm, (b)
375 nm and (c) 337 nm. (slit width: 1.5).
N
NH2
N
R6
N
R6
N
N
N
N
(a)
N
N
N
HO
O
2,6cnA(1a):
2,6dnA(1b):
6 = CN
6 = NMe2
R
6 = CN (93%)
HO
R
R
8a:
O
b: R6 = NMe2 (97%)
OH
OH
N
N
N
N
N
R6
N
N
R6
N
N
(b)
N
N
N
(c)
DMTrO
O
DMTrO
O
10a: R6 = CN
O
R
6 = CN (37%)
b: R6 = NMe2 (80%)
OH
9a:
b:
R
6 = NMe2
P
CN
N
O
DNA synthesis
Scheme 2. Reagents and conditions: (a) DMF acetal, DMF, 80 °C, 30 min; (b) DMTrCl, DMAP, pyridine, rt, 3 h; (c) 2-cyanoethyldiisopropylchlorophosphoramidite, Et3N,
acetonitrile, rt, 30 min.
spectra of single-stranded ODN1 (2,6cnA) exhibited relatively weak
fluorescence at 446 nm. Then, the fluorescence of ODN1 (2,6cnA)
when hybridized with a complementary 12 mer blODN1 forming
a bulge structure at 2,6cnA was examined. Strongly enhanced fluo-
rescence emission appeared with a maximum at 446 nm (Fig. 4a),
suggesting that the naphthalene fluorophore is located at the

890
A. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 886–892
formed in duplex ODN2 (2,6cnA)/blODN2 (Fig. 4c). This result
Table 2
showed that the fluorescence emission of 2,6cnA in DNA is com-
pletely quenched by neighboring guanines (Gs) in a bulged struc-
ture, indicating that there is a strict sequence limitation for the
use of cyano substituted ESF nucleoside fluorescence 2,6cnA.
Then, the fluorescence spectrum of the electron-donating N,N-
dimethylamino-substituted 2,6dnA-containing oligodeoxynucleo-
tide ODN1(2,6dnA) was examined in the absence and presence of
bulge-forming 12 mer blODN1. As shown in Figure 4b, the fluores-
cence intensity of single-stranded ODN1(2,6dnA) was weak at
520 nm, probably because the naphthalene fluorophore is ex-
truded to a polar aqueous solvent. In contrast, the fluorescence of
the duplex ODN1(2,6dnA)/blODN1 (bulge) was enhanced 3.6-folds
with the fluorescence maximum being red-shifted to 537 nm
Oligonucleotides (ODNs) used in this study
ODNs
Sequences
50-d(CGCAATXTAACGC)-30 X = cnA or dnA (13 mer)
50-d(CGCAACXCAACGC)-30 X = cnA or dnA (13 mer)
30-d(GCGTTAATTGCG)-50 (12 mer, bulge)
ODN1 (x)
ODN2 (x)
b1ODN1
b1ODN2
30-d(GCGTTGGTTGCG)-50 (12 mer, bulge)
hydrophobic site in the bulge structure as illustrated in Figure 4e.
On the other hand, when the fluorescence probe ODN2 (2,6cnA) for
GC rich sequence was used, the fluorescence emission was com-
pletely quenched by the neighboring C/G base pairs in the bulge
2,6cn
2,6dn
b
a
2,6cn
2,6dn
d
c
e
Figure 4. Fluorescence spectra of ODN1 [X = 2,6cnA (a) and 2,6dnA (b)] hybridized with b1ODN1 and of ODN2 [X = 2,6cnA (c) and 2,6dnA (d)] hybridized with blODN2 (2.5
lm
ODNs, 50 mM sodium phosphate, 0.1 M sodium chloride, pH 7.0, rt). The excitation wavelength was (a) 349 nm, (b) 420 nm, (c) 342 nm, and (d) 420 nm; (e) schematic
illustration of the target DNA detection by 2,6dnA-containing ODN probe via a bulge formation. (slit width: 1.5).

A. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 886–892
891
a
b
c
d
Figure 5. Fluorescence spectra of ODN1 [X = 1,4cnA (a) and 1,4dnA (b)] hybridized with blODN1 and of ODN2 [X = 1,4cnA (c) and 1,4dnA (d)] hybridized with blODN2 (2.5
ODNs, 50 mM sodium phosphate, 0.1 M sodium chloride, pH 7.0, rt). The excitation wavelength was (a) 368 nm, (b) 380 nm, (c) 365 nm and (d) 380 nm. (slit width:1.5).
lm
(Fig. 4b), probably because the naphthalene fluorophore is located
at the hydrophobic site in the bulge structure as proposed in case
of 2,6cnA. Interestingly, when the fluorescence probe containing GC
rich sequence ODN2 (2,6dnA) was used, the quenching by flanking
C/G base pairs in the bulge was not observed as shown in Figure 4d.
The fluorescence intensity of duplex ODN2(2,6dnA)/blODN2 (bulge)
was enhanced 2.9-folds compared to single-stranded ODN2(2,6d-
nA). These results indicate that N,N-dimethylamino substituted
ESF nucleoside 2,6dnA is usable for the detection of target DNA via
the formation of a bulge structure regardless of the flanking bases,
in contrast to 2,6cnA whose fluorescence was strongly quenched by
neighboring Gs. Essentially the same results were obtained with
ODNs containing 1,4-disubstituted naphthylethynylated 20-deoxy-
adenosines, 1,4cnA and 1,4dnA as shown in Figure 5. Thus, ESF nucle-
oside 1,4dnA is also useful for the detection of target sequence
regardless of the flanking sequences via the bulge formation.
In summary, we have succeeded in the molecular design of sol-
vatofluorochromic ESF 20-deoxyadenosine derivatives. We found
that cyano- and N,N-dimethylamino substitution on the naphtha-
lene ring of 8-naphthylethynylated 20-deoxyadeosine derivatives
induces a remarkable solvatofluorochromic property. In particular,
N,N-dimethylamino containing ESF nucleosides 2,6dnA and 1,4dnA
exhibited a strong fluorescence emission with high quantum yield
without quenching by neighboring Gs contained in DNA duplexes.
We demonstrated that 2,6dnA- and 1,4dnA-containing ESF oligonu-
cleotides can be used as an effective fluorescent probe for the
detection of target DNA via a bulge formation in a homogeneous
fluorescence assay without DNA sequence limitations.
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.
11.029.
References and notes
1. (a) Abo, M.; Urano, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.; Nagano, T. J. Am.
Chem. Soc. 2011, 133, 10629; (b) Komatsu, T.; Oushiki, D.; Takeda, A.;
Miyamura, M.; Ueno, T.; Terai, T.; Hanaoka, K.; Urano, Y.; Mineno, T.;
Nagano, T. Chem. Commun. 2011, 47, 10055.
2. (a) Lee, M. H.; Kim, J. Y.; Han, J. H.; Bhuniya, S.; Sessler, J. L.; Kang, C.; Kim, J. S. J.
Am. Chem. Soc. 2012, 134, 12668; (b) Jana, A.; Devi, K. S.; Maiti, T. K.; Singh, N. D.
J. Am. Chem. Soc. 2012, 134, 7656.
3. Dai, N.; Kool, E. T. Chem. Soc. Rev. 2011, 40, 5756.
4. Weissleder, R.; Tung, C. H.; Mahmood, U.; Bogdanov, A., Jr. Nat. Biotechnol.
1999, 17, 375.
5. For reviews, see: (a) Okamoto, A.; Saito, Y.; Saito, I. Photochem. Photobiol. C:
Photochem. Rev. 2005, 6, 108; (b) Ranasinghe, R. T.; Brown, T. Chem. Commun.
2005, 5487; (c) Wilson, J. N.; Kool, E. T. Org. Biomol. Chem. 2006, 4, 4265; (d)
Venkatesan, N.; Seo, Y. J.; Kim, B. H. Chem. Soc. Rev. 2008, 37, 648; (e)
Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev. 2010, 110, 2579; (f) Østergaard,
M. E.; Hrdlicka, P. J. Chem. Soc. Rev. 2011, 40, 5771.
6. Eser, S.; Messer, M.; Eser, P.; von Werder, A.; Seidler, B.; Bajbouj, M.;
Vogelmann, R.; Meining, A.; von Burstin, J.; Algül, H.; Pagel, P.; Schnieke, A.
E.; Esposito, I.; Schmid, R. M.; Schneider, G.; Saur, D. Proc. Natl. Acad. Sci. U.S.A.
2011, 108, 9945.
7. He, X.; Na, M. H.; Kim, J. S.; Lee, G. Y.; Park, J. Y.; Hoffman, A. S.; Nam, J. O.; Han,
S. E.; Sim, G. Y.; Oh, Y. K.; Kim, I. S.; Lee, B. H. Mol. Pharm. 2011, 8, 430.
8. (a) Tainaka, K.; Tanaka, K.; Ikeda, S.; Nishiza, K.-I.; Unzai, T.; Fujiwara, Y. I.;
Saito, I.; Okamoto, A. J. Am. Chem. Soc. 2008, 129, 4776; (b) Sinkeldam, R. W.;
Greco, N. J.; Tor, Y. ChemBioChem 2008, 9, 706; (c) Spadafora, M.; Postupalenko,
V. Y.; Shvadchak, V. V.; Klymchenko, A. S.; Mély, Y.; Burger, A.; Benhida, R.

892
A. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 886–892
Tetrahedron 2009, 65, 7809; (d) Pawar, M. G.; Srivatsan, S. G. Org. Lett. 2011, 13,
7.6 Hz, 1H), 5.41 (d, J = 4.4 Hz, 1H), 6.60 (m, 1H), 7.71 (br, 2H), 7.88 (m, 1H),
7.91 (dd, J = 1.6, 8.5 Hz, 1H), 8.18–8.24 (complex, 3H), 8.49 (m, 1H), 8.68 (m,
1H); 13C NMR (DMSO-d6, 100 MHz) d 37.8, 62.2, 71.2, 80.4, 85.1, 88.4, 93.9,
110.0, 118.9, 119.8, 120.5, 127.6, 129.4, 129.4, 129.5, 131.9, 132.4, 132.5, 133.8,
134.3, 148.6, 153.6, 156.2; HRMS (ESI) m/z 449.1338 calcd for C23H18N6O3Na
[M+Na]+, found 449.1309.
1114; (e) Sinkeldam, R. W.; Marcus, P.; Uchenik, D.; Tor, Y. ChemPhysChem
2011, 12, 2260; (f) Riedl, J.; Pohl, R.; Rulíšek, L.; Hocek, M. J. Org. Chem. 2012, 77,
1026.
9. (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; (d) Tanaka, M.; Kozakai, R.; Saito, Y.; Saito, I.
Bioorg. Med. Chem. Lett. 2011, 21, 7021; (e) Saito, Y.; Kugenuma, K.; Tanaka, M.;
Suzuki, A.; Saito, I. Bioorg. Med. Chem. Lett. 2012, 22, 3723.
Spectroscopic data for 2,6dnA (1b): 1H NMR (DMSO-d6, 400 MHz) d 2.30 (ddd,
J = 2.6, 6.5, 13.2 Hz, 1H), 3.07 (s, 6H), 3.19 (m, 1H), 3.54 (ddd, J = 4.9, 7.9,
11.8 Hz, 1H), 3.70 (ddd, J = 4.3, 4.4, 11.8 Hz, 1H), 3.95 (m, 1H), 4.54 (m, 1H),
5.39–5.42 (complex, 2H), 6.59 (m, 1H), 6.98 (m, 1H), 7.31 (dd, J = 2.5, 9.2 Hz,
1H), 7.51 (dd, J = 1.7, 8.5 Hz, 1H), 7.64 (br, 2H), 7.74 (d, J = 8.5 Hz, 1H), 7.84 (d,
J = 9.2 Hz, 1H), 8.12 (m, 1H), 8.18 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) d 37.8,
40.0 (ꢁ2), 62.3, 71.3, 77.8, 85.2, 88.4, 96.2, 105.1, 111.8, 116.9, 119.7, 125.2,
126.6, 127.9, 129.0, 132.3, 133.4, 135.2, 148.6, 149.6, 153.2, 156.0; HRMS (ESI)
m/z 467.1808 calcd for C24H24N6O3Na [M+Na]+, found 467.1782.
10. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 2267.
11. (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.
Spectroscopic data for 2nA (1c): 1H NMR (DMSO-d6, 400 MHz) d 2.32 (ddd,
J = 2.8, 6.6, 13.2 Hz, 1H), 3.20 (m, 1H), 3.54 (ddd, J = 4.9, 7.7, 11.8 Hz, 1H), 3.71
(ddd, J = 4.4, 4.5, 11.8 Hz, 1H), 3.95 (m, 1H), 4.55 (m, 1H), 5.36 (dd, J = 4.4,
7.7 Hz, 1H), 5.41 (d, J = 4.4 Hz, 1H), 6.60 (m, 1H), 7.62–7.73 (complex, 5H),
8.01–8.07 (complex, 3H), 8.20 (s, 1H), 8.37 (m, 1H); 13C NMR (DMSO-d6,
100 MHz) d 37.8, 62.2, 71.2, 78.9, 85.1, 88.4, 94.8, 117.2, 119.7, 127.3, 127.7,
127.9, 128.0, 128.1, 128.8, 132.4, 132.5, 132.9, 133.1, 148.6, 153.5, 156.1;
HRMS (ESI) m/z 424.1386 calcd for C22H19N5O3Na [M+Na]+, found 424.1409.
12. Spectroscopic data for 2,6cnA (1a): 1H NMR (DMSO-d6, 400 MHz) d 2.32 (ddd,
J = 2.9, 6.6, 13.2 Hz, 1H), 3.20 (m, 1H), 3.54 (ddd, J = 5.0, 7.6, 11.8 Hz, 1H), 3.71
(ddd, J = 4.4, 4.5, 11.8 Hz, 1H), 3.94 (m, 1H), 4.56 (m, 1H), 5.33 (dd, J = 4.5,