Nucleic acid probe containing fluorescent tricyclic base-linked acyclonucleoside for detection of single nucleotide polymorphisms

Article abstract of DOI:10.1016/j.bmc.2011.11.045

The development of a reliable and simple method for detecting single nucleotide polymorphisms (SNPs), common genetic variations in the human genome, is currently an important research area because SNPs are important for identifying disease-causing genes a

Full text of DOI:10.1016/j.bmc.2011.11.045

Bioorganic & Medicinal Chemistry 20 (2012) 16–24
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Nucleic acid probe containing fluorescent tricyclic base-linked
acyclonucleoside for detection of single nucleotide polymorphisms
Kinji Furukawa a, , Mayumi Hattori a, , Tokimitsu Ohki a, Yoshiaki Kitamura a, Yukio Kitade a,
Yoshihito Ueno b,
⇑
a Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of a reliable and simple method for detecting single nucleotide polymorphisms (SNPs),
common genetic variations in the human genome, is currently an important research area because SNPs
are important for identifying disease-causing genes and for pharmacogenetic studies. Here, we developed
a novel method for SNP detection. We designed and synthesized DNA probes containing a fluorescent tri-
cyclic base-linked acyclonucleoside P. The type of nucleobases involved in the SNP sites in the DNA and
RNA targets could be determined using four DNA probes containing P. Thus, this system would provide a
novel and simple method for detecting SNPs in DNA and RNA targets.
Received 22 October 2011
Revised 19 November 2011
Accepted 21 November 2011
Available online 1 December 2011
Keywords:
DNA
SNPs
Ó 2011 Elsevier Ltd. All rights reserved.
Detection
Acyclonucleoside
Tricyclic base
1. Introduction
It would be advantageous to directly detect the formation of
base pairing with a target nucleoside, for example, hydrogen bond-
The most frequent form of genetic variation in the human gen-
ome is a single nucleotide polymorphism (SNP), which occurs
roughly once every 1000 bases and is currently thought to be
linked to diseases and individual differences in drug response.1,2
Therefore, the development of a reliable and simple method for
detecting SNPs is very important for pharmacogenomics3 and the
realization of personalized medicine. To date, a number of SNP
genotyping technologies, such as microarrays,4,5 molecular inver-
sion probe genotyping,6 50 exonuclease fluorescence-based assay
(Taq-Man),7 pyrosequencing,8 single-base extension9 and matrix-
assisted laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF/MS),10,11 have been developed and applied. Most
discrimination principles in these methods are based on differ-
ences in the thermodynamic stabilities of the duplexes comprising
a probe strand and a complementary DNA target or DNA contain-
ing SNPs; the probe binds to the wild-type sequence but not to
DNA containing SNPs. However, because differences in duplex sta-
bilities between complementary sequences and sequences con-
taining one base mismatch are small, careful selection of the
oligonucleotide probe sequence and the annealing conditions,
especially temperature, is needed to prevent genotyping errors.
ing with a target nucleobase, as a fluorescence signal in duplex for-
mation. Saito and colleagues reported that oligodeoxynucleotides
(ODNs) containing base-discriminating fluorescent (BDF) nucleo-
sides that were modified with a pyrene or dimethylaminonaphtha-
lene residue acted as nucleic acid probes.12–17 Seitz and colleagues
also reported forced intercalation probes (FIT-probes), which con-
tained thiazole orange as a fluorescent base.18–22
In this paper, we report a novel method for SNP detection using
a
nucleic acid probe that contains a fluorescent tricyclic
base-linked acyclonucleoside, 8-amino-3-(2,3-dihydroxypropyl)
imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (P) (Fig. 1). The design
principle of SNP detection using the nucleoside surrogate P is
outlined in Figure 2. The fluorescence intensity of the tricyclic
base-linked P depended on solvent polarity; fluorescence intensity
NH
2
NH
2
N
N
N
N
HO
N
X
N
O
N
HO
N
N
HO
HO
2: X = CH
3: X = N
1 (P)
⇑
Corresponding authors. Tel./fax: +81 58 293 2919.
E-mail address: uenoy@gifu-u.ac.jp (Y. Ueno).
These authors contributed equally to this work.
Figure 1. Structures of tricyclic base-linked nucleoside surrogates.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.045

K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
17
moiety and the protons of the 1,3-dioxolanylmethyl residue, but
no cross-peak was detected between H7 of the base moiety and
the 1,3-dioxolanylmethyl portion. From these results, the product
was identified as an N3-alkyl derivative 6. Treatment of 6 with
CH(OEt)3 at 100 °C followed by treatment with methanolic ammo-
nia in a steel sealed tube at 110 °C produced an isopropylidene
derivative of P. The amino group of 7 was protected with a benzoyl
group. Deprotection of the isopropylidene group, protection of the
primary hydroxy group with 4,40-dimethoxytrityl (DMTr) group,
and phosphitylation produced phosphoramidite unit 12.
D = dA, dG, T, dC
D
Probe
3’
5’
3’
=
Nucleoside
Surrogate P
Target Sequence
5’
Y
Y = dA, dG, T, dC
rA, rG, U, rC
Matched
Base Pair
Mismatched
Base Pair
Fluoresce
Quenched
2.2. Photophysical properties of the nucleoside surrogate P
D
D
3’
5’
3’
5’
5’
3’
5’
3’
Typical emission spectra of the tricyclic base-linked nucleoside
surrogate P are shown in Fig. 3. The nucleoside surrogate P showed
emission maxima at ꢀ390 nm under 332-nm excitation. The fluo-
rescence intensity of P depended on solvent polarity; fluorescence
intensity of P was greater in more polar solvents such as methanol
and water compared to less polar solvents such as chloroform. The
photophysical data of the tricyclic base-linked nucleoside surro-
gate P are summarized in Table 1. The emission wavelength of P
was affected by solvent polarity. The emission maximum shifted
from 388 nm for chloroform to 399 nm for methanol. The fluores-
Y
Y
Hydrogen Bonding
Intercalation
Figure 2. Design principle of SNP detection using the nucleoside surrogate P. Ds
denotes discriminating bases. Ys indicates SNP sites.
of P was greater in more polar solvents such as methanol and
water compared to less polar solvents such as chloroform. Based
on this result, we designed nucleic acid probes containing P at
the 50 (50-P-probes) or 30 sides (30-P-probes) of discriminating bases
Ds to produce bulges. It is believed that the inside of the grooves of
the DNA helix is more hydrophobic than bulk water.23 Thus, we
presumed that when the Ds match the sequences of opposite
bases, Ds form base pairs with complementary bases, causing the
nucleoside surrogate P to flip outside the DNA helix and strength-
ening the fluorescence intensity of P. On the other hand, when the
mismatched bases have sites opposite of Ds, P intercalates into the
DNA helix because the tricyclic base-linked nucleoside surrogate P
is more intercalative than natural mono or bicyclic natural nucleo-
bases. This weakens the fluorescence intensity of P. We anticipated
that we could determine the type of nucleobase involved in the
SNP site by comparing the fluorescence intensities of duplexes
composed of each probe containing P.
cence quantum yields (
U
s) of P increased with increasing solvent
polarity. The
water.
U value of P was 0.13 in chloroform and 0.97 in
2.3. Synthesis of ODNs containing the nucleoside surrogate P
The sequences of ODNs containing the nucleoside surrogate P
are given in Table 2. CYP2C9 is one of the predominant cytochrome
P450 (CYP) enzymes expressed constitutively in the human li-
ver.29,30 It metabolizes a variety of therapeutically important drugs
such as phenytoin, warfarin, and a number of anti-inflammatory
drugs, including indomethacin, mefenamic acid, flurbiprofen, ibu-
profen, diclofenac, and the oxicams.29,30 In the present study, we
chose the sequence containing an A1075(C) mutation in the
CYP2C9 gene as a model sequence. The probe ODNs were composed
of sequences complementary to the CYP2C9 gene. The probes (50-P-
probes) termed PA, PG, PC, and PT contained P at the 50 sides of Ds,
whereas the probes (30-P-probes) termed AP, GP, CP, and TP pos-
sessed P at the 30 sides of Ds. Target sequences are abbreviated
as S.
2. Results
2.1. Design and synthesis of a fluorescent nucleoside surrogate
Size-expanded nucleoside surrogates 2 and 3 were reported
fluoresce under ultraviolet light, emitting fluorescence at
ꢀ400 nm.24–27 Thus, we chose 8-amino-3-(2,3-dihydroxypro-
pyl)imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (P) as a nucleoside
surrogate for this method. We expected that the nucleoside surro-
gate P could be accommodated into a DNA/DNA or DNA/RNA helix
without disturbing the local helical structure because P is com-
posed of an acyclic three-carbon propylene glycol instead of
All the ODNs containing P were synthesized using a DNA/RNA
synthesizer. Fully protected ODNs (1.0 lmol each) linked to solid
supports were treated with concentrated NH4OH at 55 °C for
12 h. The ODNs released after treatment were purified by 20%
polyacrylamide gel electrophoresis (PAGE) to afford deprotected
ODNs. These ODNs were analyzed by MALDI-TOF/MS, and the ob-
served molecular weights were in agreement with their structures.
deoxy-
D
-ribose, although it carries a size-expanded tricyclic base.
2.4. Thermal denaturation study and fluorescence experiments
of duplexes containing P
We also expected that P would flip outside of the helix more easily
than natural nucleosides because P has a flexible acyclic structure.
The synthesis of the tricyclic base-linked nucleoside surrogate P
is shown in Scheme 1. 5-Amino-6-cyanoimidazo[4,5-b]pyridine
(4),28 which was synthesized by a method reported previously,
was coupled with (R)-2,2-dimethyl-4-(p-toluenesulfonyloxymeth-
yl)-1,3-dioxolane (5) in the presence of K2CO3 in dimethylformam-
ide (DMF) at 60 °C. Only one product was observed on thin-layer
chromatography (TLC). After purification on a silica gel column,
the product was analyzed by 1H–1H correlation spectroscopy
(COSY), heteronuclear multiple quantum coherence (HMQC), het-
eronuclear multiple bond correlation (HMBC) and nuclear overha-
user enhancement spectroscopy (NOESY) spectra. In the NOESY
experiments, cross-peaks were observed between H2 of the base
First, we examined the ability of the probes to detect SNPs in
the DNA targets. The Tm values of the duplexes between the probes
and the DNA targets are shown in Table 3 (left column). The high-
est Tm values were from the duplexes comprising complementary
sequences, for example, when the discriminating bases and the tar-
get base sequences matched.
DTm variation between the matched
and mismatched sequences was 3–7 °C. Thus, the discriminating
bases possessed sufficient base-selectivity, although they con-
tained the tricyclic nucleoside surrogate P at the 50 or 30 sides of
the discriminating bases.
Fluorescence emission spectra of the duplexes consisting of
50-P-probes, PA, PG, PC, and PT are shown in Fig. 4. Fluorescence

18
K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
CN
NH
N
NH
7
2
1
6
5
CN
NH
N
N
N
H
N
N
N
2
N
2
4
N
4
3
a
2
b
N
N
+
O
O
O
OTos
6
7
O
O
O
5
O
N
H
N
NH
2
N
N
N
N
c
N
e
N
N
N
N
RO
RO
OR
OR
1: R = H
9: R = TBDMS
10: R = H
d
f
8: R = TBDMS
H
Bz
N
N
H
Bz
N
N
N
N
N
N
N
N
g
h
N
N
DMTrO
O
O
DMTrO
P
N
CN
OH
11
12
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 60 °C, 70 h, 54%; (b) HC(OEt)3, 100 °C, 54 h; (2) NH3 in MeOH, 110 °C, 18 h, 65%; (c) CH3CO2H, 60 °C, 9 h, 98%; (d)
TBDMSCl, imidazole, DMF, rt, 18 h, 81%; (e) BzCl, pyridine, rt, 10 h, 67%; (f) TBAF, THF, rt, 2 h, 75%; (g) DMTrCl, pyridine, rt, 2 h, 56%; (h) chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine, i-Pr2NEt, THF, rt, 30 min, 67%.
Table 1
1000
800
600
400
200
0
Photophysical properties of P. The experimental conditions were described in the
Section 4
H2O
MeOH
CHCl3
Solvent
kex (nm)
kem (nm)
e
U
H2O
333.4
335.4
334.8
336.6
338.8
335.0
335.0
391.6
398.6
386.8
386.4
389.8
388.2
385.0
78.5
32.6
36.2
0.97
0.82
0.70
0.43
0.19
0.13
0.12
MeOH
CH3CN
EtOAc
THF
CHCl3
CH2Cl2
6.19
7.32
4.70
8.90
330
380
430
480
530
U = fluorescence quantum yield.
= dielectric constant.
e
Wavelength (nm)
Figure 3. Fluorescence emission spectra of P in various solvents. Spectra were
obtained under 332-nm excitation on a spectrofluorophotometer in quartz cuvettes
with a path length of 1.0 cm at a P concentration of 30 lM in an appropriate solvent
at 20 °C. Spectra were recorded using an excitation slit of 1.5 nm and emission slit
of 1.5 nm.
ble-type base pairing between dG and dT bases.31 The fluorescence
intensities of the single-stranded P-probes were almost equal to
those of the mismatched duplexes (data not shown).
The results for probes containing the surrogate P at the 30 side
of the discriminating bases (30-P-probes) are shown in Fig. 6 (left
column). Fluorescence intensities of the duplexes were greatest
when the discriminating bases were complementary to the target
bases (except for the CP probe): AP probe for the SdT target, TP
probe for the SdA target, and GP probe for the SdC target. No fluo-
rescence was observed with the CP probe for the target sequences.
From these results, we could determine the type of nucleobases in-
volved in the SNP sites in the DNA targets using the PA or AP probe
for the SdT target, the PC probe for the SdG target, the TP probe for
the SdA target, and the PG probe for the SdC target.
spectra intensity of the duplexes correlated with the Tm values of
the duplexes. In all sequences, the fluorescence intensities were
greatest when the discriminating bases were complementary to
the target bases. Next, we compared the fluorescence intensities
of the duplexes at 390 nm. As shown in Fig. 5 (left column), the
fluorescence intensities were greatest when the duplexes were
composed of matched sequences: PA probe for the SdT target, PC
probe for the SdG target, PT probe for the SdA target, and PG probe
for the SdC target. However, with the dG target base, fluorescence
was also observed for the PT probe. This was attributed to a wob-

K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
19
Table 2
Oligonucleotide sequences. The underlined letters indicate discriminating bases. The
italic letters represent target bases
Abbreviation
Sequence
50-d(GAA GGT CAA PAG TAT CTC T)-30
50-d(GAA GGT CAA PGG TAT CTC T)-30
50-d(GAA GGT CAA PCG TAT CTC T)-30
50-d(GAA GGT CAA PTG TAT CTC T)-30
50-d(GAA GGT CAA PsTG TAT CTC T)-30
50-d(GAA GGT CAA APG TAT CTC T)-30
50-d(GAA GGT CAA GPG TAT CTC T)-30
50-d(GAA GGT CAA CPG TAT CTC T)-30
50-d(GAA GGT CAA TPG TAT CTC T)-30
30-d(CTT CCA GTT ACA TAG AGA)-50
30-d(CTT CCA GTT GCA TAG AGA)-50
30-d(CTT CCA GTT CCA TAG AGA)-50
30-d(CTT CCA GTT TCA TAG AGA)-50
30-r(CUU CCA GUU ACA UAG AGA)-50
30-r(CUU CCA GUU GCA UAG AGA)-50
30-r(CUU CCA GUU CCA UAG AGA)-50
30-r(CUU CCA GUU UCA UAG AGA)-50
PA
PG
PC
PT
PsT
AP
GP
CP
TP
SdA
SdG
SdC
SdT
SrA
SrG
SrC
SrU
Table 3
Tm Values.
D
Tms [Tm (a duplex between a probe and a complementary target) ꢁ Tm (a
duplex between a probe and a target containing a mismatched base)] are indicated in
parentheses. The experimental conditions are as described in the Section 4
SdA
SdG
47.8
SdC
SdT
SrA
SrG
SrC
SrU
PA 45.6
44.9
50.1
—
46.8
45.0
47.1
44.6
48.1
—
46.9
(ꢁ5.4)
45.7
(ꢁ4.5) (ꢁ2.3) (ꢁ5.1)
(ꢁ3.1) (ꢁ1.0) (ꢁ3.5)
PG 45.8
45.8
51.6
—
44.6
46.8
47.8
52.3
—
44.9
(ꢁ5.8) (ꢁ5.8)
(ꢁ3.8) (ꢁ5.5) (ꢁ4.5)
PC
PT
44.5
(ꢁ5.6)
50.5
—
52.1
—
47.1
46.1
45.5
51.5
—
48.0
(ꢁ5.5) (ꢁ6.0) (ꢁ7.7)
(ꢁ6.6) (ꢁ5.5)
45.7
47.3
49.2
—
46.5
44.8 47.0
(ꢁ3.4) (ꢁ4.8) (ꢁ3.2)
(ꢁ1.2) (ꢁ4.4) (ꢁ2.2)
AP 47.8
46.8
46.4
50.3
—
48.3
49.6
46.1
(ꢁ1.8)
52.8
—
47.9
—
47.4
(ꢁ5.4)
49.3
(ꢁ2.5) (ꢁ3.5) (ꢁ3.9)
(ꢁ1.4) (+1.7)
GP 49.1
47.2
52.6
—
48.0
48.1 48.7
(ꢁ3.5) (ꢁ5.4)
(ꢁ4.3) (ꢁ4.7) (ꢁ4.1)
CP
TP
49.8
(ꢁ5.1)
52.4
—
54.9
—
48.9
49.1
47.2
56.1
—
51.7
(+1.7)
48.0
(ꢁ6.9) (ꢁ5.8) (ꢁ8.9)
(ꢁ8.1) (ꢁ6.8)
48.2 48.9
(ꢁ2.1) (ꢁ1.1)
47.7
49.4
50.0
—
(ꢁ3.5) (ꢁ4.7) (ꢁ3.0)
The results for the RNA targets are shown in Table 3 (right col-
umn), Fig. 5 (right column), and Fig. 6 (right column). When the
discriminating bases and the target bases matched, the Tm values
of the duplexes were greatest in all sequences, except for the AP
and TP probes, whose
DTm values for duplexes comprising
matched and mismatched sequences varied from 1 to 9 °C. Thus,
the discriminating bases seemed to have base selectivities in the
DNA/RNA duplexes containing the tricyclic nucleoside surrogate
P. The Tm values of the AP/SrG (Tm = 49.6 °C) and TP/SrG
(Tm = 51.7 °C) duplexes were slightly greater than those of the
AP/SrU (Tm = 47.9 °C) and TP/SrA (Tm = 50.0 °C) duplexes, which
were complementary. These differences in the Tm values were
attributed to sheared G:A base pairing32,33 and a wobble-type base
pairing between dG and dT bases.31
The results of the fluorescence experiment for the RNA targets
were similar to those for the DNA targets. The fluorescence inten-
sities of the duplexes containing the 50-P-probes were greatest
when the discriminating bases were complementary to the target
bases, except for the PT/SrG duplex (Fig. 5, right column). The fluo-
rescence of the mismatched PT/SrG duplex was attributed to the
wobble-type base pairing between dT and rG bases. Results for
the 30-P-probes are shown in Figure 6 (right column). The fluores-
cence intensities were greatest when the duplexes were comple-
Figure 4. Fluorescence emission spectra of the duplexes. Spectra were obtained
under 341-nm excitation on a spectrofluorophotometer in quartz cuvettes with a
path length of 1.0 cm and a duplex concentration of 3.0 lM in a Tm buffer at 20 °C.
Spectra were recorded with using an excitation slit of 1.5 nm and emission slit of
1.5 nm.
mentary, although the fluorescence intensities of the duplexes
composed of the CP probe were weak in all target sequences. How-
ever, in principle, we could determine the type of nucleobases in-
volved in the SNP sites in the RNA target using four 50-P-probes or
four 30-P-probes.
To improve sensitivity and solve the problem related to wobble-
type base pairing, we designed and synthesized a probe containing
2-thiothymidine (sT) as a discriminating base. Sekine et al. re-
ported that the formation of wobble-type base pairing between
dG and dT can be suppressed by introducing a thiocarbonyl group
into the 2-position of dT instead of a 2-carbonyl oxygen (Fig. 7).34

20
K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
Figure 5. Fluorescence intensities of duplexes at 390 nm. Left column: 50-P-probe/DNA target. Right column: 50-P-probe/RNA target. Spectra were measured at 20 °C.
Therefore, we expected that fluorescence due to the formation of
wobble-type base pairing could be suppressed by introducing sT
instead of dT (Table 2). The results of fluorescence experiments
are shown in Fig. 8. The left graph shows the result of the DNA tar-
get, while the right graph represents the result of the RNA target.
Fluorescence due to the formation of wobble-type base pairing
was reduced by introducing sT instead of dT without affecting
other base pairings.
the probe containing P hybridized with a complementary se-
quence, the probe emitted fluorescence. However, when the probe
hybridized with a target containing a mismatched base, the probe
did not fluoresce in the almost sequences. Thus, in principle, we
can identify the kind of nucleotide involved in an SNP site using
4 probes containing only 1 nucleoside surrogate P.
The fluorescence intensity values varied significantly even
when the duplex types were the same. For example, the values ob-
tained for the PA/SdT and PC/SdG duplexes, both of which are suc-
cessful probes, were 75 and 200, respectively. Similarly, the values
obtained for the PA/SrU and PC/SrG duplexes were 75 and 160.
These phenomena may partly reflect the influence of the number
of hydrogen bonding between the discriminating bases Ds and
the target bases. The dA:dT base pairing is formed by 2 hydrogen
bonds, whereas the base pairing between dG and dC is composed
of 3 hydrogen bonds. Thus, when the dG:dC base pair is formed be-
tween the discriminating base D and the target base, the nucleo-
side surrogate P may flip outside of the DNA helix more largely
3. Discussion and conclusions
A reliable and simple method for detecting nucleotide muta-
tions is very important clinically because sequence variations in
human DNA cause genetic diseases and genetically influenced
traits. The majority of sequence variations are attributed to SNPs.1,2
In this study, we developed a novel method for SNP detection. The
fluorescence intensity of the nucleoside surrogate P,
a key
compound in this system, depended on solvent polarity. When

K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
21
Figure 6. Fluorescence intensities of duplexes at 390 nm. Left column: 30-P-probe/DNA target. Right column: 30-P-probe/RNA target. Spectra were measured at 20 °C.
contrast, the position of the nucleoside surrogate P within the
probes affected the fluorescence intensity of the duplexes. For
example, the PC probe (50-P-probe) worked well for detecting SNPs
for the DNA and RNA targets, whereas the CP probe (30-P-probe)
did not emit fluorescence even when it hybridized with comple-
mentary targets. It is known that a guanine base can form not only
a Watson–Crick base pairing with a cytosine base but also a
sheared-type base pairing with an adenine base.32,33 The base moi-
ety of the nucleoside surrogate P has a proton donor–acceptor pat-
tern same as adenosine. Thus, the nucleoside surrogate P might
form a base pairing with the target dG or rG base like as the
sheared-type base pairing in the CP/SdG or CP/SrG duplex. In this
study, we tested only one sequence. In the future, we will system-
atically examine a large number of target sequences to confirm the
general applicability of this system.
Wobble Type
O
H N
O
Me
O
H N
S
Me
O
N
O
N
N
N
N
N
N H
N H
N
N H
H
N
T
sT
N H
H
dG
dG
Figure 7. Base pairing between dG and T and between dG and 2-thiothymidine
(sT).
as compared with the case of the dA:dT base pair: this may allow
the nucleoside surrogate P to emit more fluorescence than the case
of the dA:dT base pair.
Fluorescence signal patterns of the DNA and RNA target du-
plexes were not significantly different. Thus, global conformation
differences between the duplexes, that is, B-type and A-type
conformations, did not influence probe fluorescence intensity. In
Fluorescence attributed to wobble-type T:G base pairing was
observed for the PT probe (50-P-probe), while the TP probe (30-P-
probe) worked well for detecting SNPs for both DNA and RNA
targets. These results imply that the geometry of the nucleoside
surrogate P in the duplexes was different in the 30-P-probe and

22
K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
Figure 8. Fluorescence intensities of duplexes containing 2-thiothymidine (sT) at 390 nm. Spectra were measured at 20 °C.
the 50-P-probe. Furthermore, intensity differences between the
fluorescence signals of the matched and mismatched sequences
were dependent on the kind of probe and the target sequences.
The AP, PC, and TP probes worked well as SNP-detecting probes.
When they hybridized with complementary targets, the fluores-
cence intensities of the duplexes increased 5- to 20-fold as com-
pared to those with mismatched sequences.
As mentioned-above, it is known that a guanine base can form
not only a Watson–Crick base pairing with a cytosine base but also
a wobble-type base pairing with a thymine base and a sheared-
type base pairing with an adenine base.31–33 In this study, we ob-
served fluorescence signals attributed to wobble-type G:T base
pairing. We succeeded in reducing these fluorescence signals by
introducing sT instead of dT in the probe. Furthermore, when the
GP probe hybridized with the SdA target, fluorescence signal inten-
sity increased slightly in comparison to other mismatched targets.
This was attributed to sheared-type G:A base pairing. Introducing a
chemically modified guanine base might solve the problem and
improve the sensitivity of the probe.
In conclusion, we demonstrated the synthesis of DNA contain-
ing a fluorescent tricyclic base-linked acyclonucleoside P. We
examined the properties of the DNA containing P as an SNP-detect-
ing probe. We found that, in principle, we can determine the types
of nucleobases involved in the SNP sites in DNA and RNA targets
with high sensitivities using 4 probes containing P. Thus, this sys-
tem would provide a novel and simple method for detecting SNPs
in DNA and RNA targets although we must optimize the linker
length and the structure of the base moiety. Synthesis of other
fluorescent base-linked acyclonucleosides with distinct emission
wavelengths are under investigation in our laboratory.
in DMF (80 mL) was stirred at 60 °C for 70 h. The mixture was par-
titioned between EtOAc and H2O. The organic layer was washed
with brine, dried (Na2SO4), and concentrated. The residue was
purified by column chromatography (SiO2, 1–2% MeOH in CHCl3)
to give 6 (0.70 g, 2.56 mmol) in 54% yield: 1H NMR (400 MHz,
DMSO-d6) d 1.33 (s, 3H, 2-CH3), 1.38 (s, 3H, 2-CH3), 3.71 (dd, 1H,
J = 6.0 and 8.7, 5-CHaHb), 4.11 (dd, 1H, J = 6.5 and 8.7, 5-CHaHb),
4.19 (dd, 1H, J = 6.0 and 14.5, NCHaHb), 4.32 (dd, 1H, J = 3.6 and
14.5, NCHaHb), 4.46 (m, 1H, 4-CH), 5.11 (s, 2H, 5-NH2), 7.97 (s,
1H, 2-H), 8.07 (s, 1H, 7-H); 13C NMR (100 MHz, DMSO-d6) d 24.9,
26.4, 45.0, 65.8, 72.9, 85.3, 108.5, 117.3, 125.9, 133.3, 144.1,
148.6, 156.9; HRMS (FAB) calcd for C13H15N5O2: 273.1226, found;
273.1229. Anal. Calcd for C13H16N6O2ꢂ11/50H2O: C, 56.32; H,
5.61; N, 25.26. Found: C, 56.58; H, 5.47; N, 24.96.
4.1.2. (S)-8-Amino-3-[(2,2-dimethyl-1,3-dioxolan-4-yl)
methyl]imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (7)
A solution of 6 (1.20 g, 4.39 mmol) in triethyl orthofomate
(40 mL, 240 mmol) was stirred at 100 °C for 54 h, cooled to room
temperature, and concentrated. The residue was dissolved in sat-
urated NH3/MeOH (50 mL). The tube was sealed and the solution
was stirred at 110 °C for 18 h. After cooling the mixture, the tube
was opened and the excess NH3 was allowed to escape slowly.
The mixture was concentrated. The residue was purified by col-
umn chromatography (SiO2, 6–10% MeOH in CHCl3) to give 7
(0.86 g, 2.86 mmol) in 65% yield: 1H NMR (400 MHz, DMSO-d6)
d 1.22 (s, 3H), 1.27 (s, 3H), 3.82 (dd, 1H, J = 5.1 and 8.7), 4.06
(dd, 1H, J = 6.5 and 8.7), 4.39 (dd, 1H, J = 6.5 and 14.0), 4.47
(dd, 1H, J = 4.2 and 14.0), 4.56 (m, 1H), 7.99 (br s, 2H, D2O
exchangeable), 8.47 (s, 1H), 8.64 (s, 1H), 9.03 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d 25.0, 26.5, 45.4, 66.1, 73.3, 91.2, 106.0,
106.9, 109.0, 123.2, 150.3, 151.6, 157.4, 163.9; HRMS (FAB) calcd
4. Experimental section
4.1. General remarks
for
C14H17N6O2: 301.1422, found; 301.1413. Anal. Calcd for
C
14H17N6O2ꢂ1/10H2O: C, 55.66; H, 5.44; N, 27.82. Found: C,
55.79; H, 5.42; N, 27.50.
Thin-layer chromatography was carried out on Merck coated
plates 60F254. Silica gel column chromatography was carried out
on Wakogel C-300. 1H-, 13C-, and 31P NMR spectra were obtained
with a JEOL JNM AL-400 spectrometer. CDCl3 (CIL) or DMSO-d6
(CIL) was used as a solvent for obtaining NMR spectra. Chemical
shifts (d) are given in parts per million (ppm) downfield from
(CH3)4Si (d 0.00 for 1H NMR in CDCl3), 80% H3PO4 (d 0.00 for 31P
NMR), or a solvent (for 13C NMR and 1H NMR in DMSO-d6) as an
internal reference with coupling constants (J) in Hz. The abbrevia-
tions s, d, and q signify singlet, doublet, and quartet, respectively.
4.1.3. (S)-8-Amino-3-[2,3-dihydroxypropyl]imidazo[40,50:5,6]
pyrido[2,3-d]pyrimidine (1)
A solution of 7 (0.86 g, 2.86 mmol) in 80% CH3CO2H (30 mL)
was stirred at 60 °C for 9 h. The solvent was evaporated in vacuo
to give 1 (0.74 g, 2.83 mmol) in 98% yield: UV kmax (H2O) 260 nm
(e = 14700), 333 nm (e
= 18300); 1H NMR (400 MHz, DMSO-d6) d
3.39 (m, 2H), 3.93 (m, 1H), 4.14 (dd, 1H, J = 8.7 and 13.9), 4.48
(dd, 1H, J = 3.4 and 14.0), 4.90 (t, 1H, J = 5.5), 5.14 (d, 1H,
J = 5.9), 7.97 (br s, 2H), 8.47 (s, 1H), 8.59 (s, 1H), 9.01 (s, 1H);
13C NMR (100 MHz, DMSO-d6) d 46.6, 63.7, 69.3, 105.8, 122.9,
134.0, 150.7, 151.6, 155.1, 157.3, 163.9; HRMS (FAB) calcd for
4.1.1. (S)-5-Amino-6-cyano-3-[(2,2-dimethyl-1,3-dioxolan-4-yl)
methyl]imidazo[4,5-b]pyridine (6)
C
11H13N6O2: 261.1111, found; 261.1100. Anal. Calcd for
A mixture of (R)-2,2-dimethyl-4-(p-toluenesulfonyloxymethyl)-
1,3-dioxolane (1.70 g, 5.94 mmol), 5-amino-6-cyanoimdazo[4,5-
b]pyridine (0.76 g, 4.77 mmol),28 and K2CO3 (0.80 g, 5.79 mmol)
C11H13N6O2ꢂ1/5 H2O: C, 50.29; H, 4.74; N, 30.87. Found: C,
50.37; H, 4.87; N, 30.89.

K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
23
4.1.4. (S)-8-Amino-3-[2,3-bis(tert-butyldimethylsilyloxy)
propyl]imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (8)
(s, 1H); 13C NMR (100 MHz, CDCl3) d 47.6, 55.3, 64.9, 69.0, 86.6,
113.3, 126.4, 127.1, 127.3, 128.0, 128.1, 128.4, 130.0, 130.1,
132.7, 135.4, 135.6, 135.7, 144.6, 150.5, 152.3, 158.7. Anal. Calcd
for C39H34N6O5ꢂ9/5H2O: C, 67.00; H, 5.42; N, 12.02. Found: C,
67.34; H, 5.08; N, 11.63.
A
solution of 1 (0.75 g, 2.88 mmol), imidazole (1.75 g,
25.7 mmol), TBDMSCl (1.58 g, 10.5 mmol) in DMF (30 mL) was stir-
red at room temperature for 18 h. EtOH (1 mL) was added to the
mixture, and the whole was stirred for 10 min. The mixture was
partitioned between EtOAc and H2O. The organic layer was washed
with aqueous NaHCO3 (saturated) and brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography
(SiO2, 3% MeOH in CHCl3) to give 8 (1.14 g, 2.33 mmol) in 81%
yield: 1H NMR (400 MHz, DMSO-d6) d ꢁ0.63 (s, 3H), ꢁ0.15 (s,
3H), 0.08 (s, 6H), 0.65 (s, 9H), 0.90 (s, 9H), 3.66 (m, 2H), 4.26 (m,
2H), 4.47 (m, 1H), 7.98 (br s, 2H), 8.47 (s, 1H), 8.58 (s, 1H), 9.01
(s, 1H); 13C NMR (100 MHz, DMSO-d6) d ꢁ5.9, ꢁ5.5, ꢁ5.4, ꢁ4.9,
17.4, 18.1, 25.5, 25.9, 46.7, 65.7, 70.4, 105.8, 123.0, 134.1, 150.6,
151.6, 155.2, 157.3, 163.9; HRMS (FAB) calcd for C23H40N6O2Si2:
488.2751, found; 488.2760. Anal. Calcd for C23H40N6O2Si2: C,
56.52; H, 8.25; N, 17.19. Found: C, 56.37; H, 8.10; N, 17.24.
4.1.8. N8-Benzoylamino-3-[(R)-3-(4,40-dimethoxytrityloxy)
-2-[[(2-cyanoethoxy)(N,N-diisopropylamino)phosphanyl]oxy]
-propyl]imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (12)
A solution of 11 (0.50 g, 0.75 mmol), N,N-diisopropylethylamine
(0.65 mL, 3.74 mmol), and chloro(2-cyanoethoxy)(N,N-diisopro-
pylamino)phosphine (0.33 mL, 1.48 mmol) in THF (4 mL) was stir-
red at room temperature. After 30 min, the mixture was
partitioned between CHCl3 and H2O. The organic layer was washed
with aqueous NaHCO3 (saturated) and brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography
(SiO2, 50% EtOAc in hexane) to give 12 (0.43 g, 0.50 mmol) in 67%
yield: 31P NMR (162 MHz, DMSO-d6) d 149.1, 150.2.
4.1.5. (S)-N8-Benzoylamino-3-[2,3-bis(tert-butyldimethylsilyl
oxy)propyl]imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (9)
4.2. Oligonucleotide synthesis
A
solution of
8
(1.82 g, 3.72 mmol) and BzCl (0.64 mL,
The synthesis was carried out with a DNA/RNA synthesizer (Ap-
plied Biosystems Model 3400) by the phosphoramidite method. In
the case of the coupling of the amidite 12, a 0.12 M solution of the
amidite 12 in CH3CN and a coupling time of 15 min were used.
Deprotection of the bases and phosphates was performed in con-
centrated NH4OH at 55 °C for 16 h. The oligonucleotides were puri-
fied by 20% PAGE containing 7 M urea to give the highly purified
oligonucleotides, PA (14), PG (40), PC (20), PT (8), AP (15), GP
(15), CP (14), TP (29), and PsT (15). The yields are indicated in
5.51 mmol) in pyridine (20 mL) was stirred at room temperature.
After 10 h, the mixture was partitioned between CHCl3 and H2O.
The organic layer was washed with aqueous NaHCO3 (saturated)
and brine, dried (Na2SO4), and concentrated. The residue was puri-
fied by column chromatography (SiO2, 0–10% MeOH in CHCl3) to
give 9 (1.48 g, 2.50 mmol) in 67% yield: 1H NMR (400 MHz, CDCl3)
d ꢁ0.47 (s, 3H), ꢁ0.08 (s, 3H), 0.07 (s, 6H), 0.78 (s, 9H), 0.92 (s, 9H),
3.61 (m, 1H), 3.70 (m, 1H), 4.23 (m, 1H), 4.37 (m, 1H), 4.67 (m, 1H),
7.51–7.57 (m, 3H), 8.37–8.48 (m, 4H), 9.53 (s, 1H); 13C NMR
(100 MHz, CDCl3) d ꢁ5.4, ꢁ5.3, ꢁ4.7, 17.8, 18.4, 25.7, 26.0, 47.4,
65.4, 70.7, 113.6, 126.9, 128.3, 130.0, 132.7, 135.9, 137.2, 143.6,
150.6, 152.6, 154.2, 159.0, 180.0. Anal. Calcd for C23H44N6O3Si2:
C, 60.77; H, 7.48; N, 14.09. Found: C, 60.51.; H, 7.40; N, 14.11.
parentheses as OD units at 260 nm starting from 1.0 lmol scale.
The extinction coefficients of the oligonucleotides were calculated
from those of mononucleotides and dinucleotides according to the
nearest-neighbor approximation method.35
4.3. MALDI-TOF/MS analyses of oligonucleotides
4.1.6. (S)-N8-Benzoylamino-3-[2,3-dihydroxypropyl]imidazo[40,
50:5,6]pyrido[2,3-d]pyrimidine (10)
Spectra were obtained with a SHIMADZU AXIMA-CFR plus time-
A
solution of
9
(1.48 g, 2.50 mmol) and TBAF (9.96 mL,
of-flight mass spectrometer equipped with a nitrogen laser
9.96 mmol, 1 M THF solution) in THF (50 mL) was stirred at room
temperature. After 2 h, the solvent was evaporated in vacuo. The
residue was purified by column chromatography (SiO2, 5–14%
MeOH in CHCl3) to give 10 (0.68 g, 1.87 mmol) in 75% yield: 1H
NMR (400 MHz, DMSO-d6) d 3.41 (ddd, 1H, J = 5.5, 5.5 and 11.0),
3.48 (ddd, 1H, J = 5.5, 5.5 and 11.0), 3.97 (m, 1H), 4.21 (dd, 1H,
J = 8.8 and 14.0), 4.55 (dd, 1H, J = 3.4 and 14.0), 4.92 (t, 1H,
J = 5.5), 5.18 (d, 1H, J = 5.5), 7.54–8.38 (m, 7H), 8.76 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) d 47.4, 64.3, 69.8, 125.6, 128.9, 129.7,
132.7 (multiple), 135.7 (multiple), 152.8; HRMS (FAB) calcd for
(337 nm, 3-ns pulse). A solution of 3-hydroxypicolinic acid (3-
HPA) and diammonium hydrogen citrate in H2O was used as the
matrix. PA: calculated mass, 5844.8; observed mass, 5846.2. PG:
calculated mass, 5860.8; observed mass, 5856.2. PC: calculated
mass, 5820.8; observed mass, 5823.8. PT: calculated mass,
5835.8; observed mass, 5833.0. AP: calculated mass, 5844.8; ob-
served mass, 5843.6. GP: calculated mass, 5860.8; observed mass,
5862.2. CP: calculated mass, 5820.8; observed mass, 5823.0. TP:
calculated mass, 5835.8; observed mass, 5837.1. PsT: calculated
mass, 5851.7; observed mass, 5851.1.
C18H16N6O3: 365.1362, found; 365.1361. Anal. Calcd for
C
18H67N6O3ꢂ7/10H2O: C, 57.35; H, 4.65; N, 22.29. Found: C,
4.4. Absorption experiments
57.45; H, 4.73; N, 22.08.
Absorption spectra (200–500 nm) were obtained on a SHIMAZU
UV-2450 UV–Vis spectrophotometer in quartz cuvettes with a path
4.1.7. (S)-N8-Benzoylamino-3-[3-(4,40-dimethoxytrityloxy)-2-
hydroxypropyl]imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (11)
A solution of 10 (0.60 g, 1.65 mmol) and DMTrCl (0.84 g,
2.48 mmol) in pyridine (6 mL) was stirred at room temperature.
After 2 h, the mixture was partitioned between CHCl3 and H2O.
The organic layer was washed with aqueous NaHCO3 (saturated)
and brine, dried (Na2SO4), and concentrated. The residue was puri-
fied by column chromatography (SiO2, 1% MeOH in CHCl3) to give
11 (0.62 g, 0.93 mmol) in 56% yield: 1H NMR (400 MHz, CDCl3) d
3.09 (m, 1H), 3.17 (dd, 1H, J = 6.3 and 9.9), 3.31 (dd, 1H, J = 4.4
and 9.9), 4.29 (m, 1H), 4.42 (dd, 1H, J = 7.5 and 14.4), 4.63 (dd,
1H, J = 3.2 and 14.4), 6.08–6.82 (m, 4H), 7.22–7.31 (m, 7H), 7.34–
7.41 (m, 2H), 7.51–7.60 (m, 3H), 8.40 (m, 2H), 8.48 (m, 2H), 9.52
length of 1.0 cm and a 30
solvent.
lM P concentration in an appropriate
4.5. Thermal denaturation study
The solution containing the duplex in a buffer comprising
10 mM sodium phosphate (pH 7.0) and 0.1 M NaCl was heated at
95 °C for 3 min, cooled gradually to an appropriate temperature,
and then used for the thermal denaturation study. The thermal-in-
duced transition of each mixture was monitored at 260 nm on a
SHIMAZU UV-2450 UV–Vis spectrophotometer fitted with a tem-
perature controller in quartz cuvettes with a path length of

24
K. Furukawa et al. / Bioorg. Med. Chem. 20 (2012) 16–24
2. Venter, J. C.; Adams, M. D.; Myers, E. W.; Li, P. W.; Mural, R. J.; Sutton, G. G.;
1.0 cm and a 3.0 lM duplex concentration in a buffer of 10 mM so-
dium phosphate (pH 7.0) and 0.1 M NaCl. The sample temperature
was increased by 0.5 °C/min.
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4.6. Fluorescence experiments
Steady-state fluorescence emission spectra (370–670 nm) were
obtained on a SHIMADZU RF-5300PC spectrofluorophotometer in
quartz cuvettes with a path length of 1.0 cm and a 30
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excitation slit of 1.5 nm and emission slit of 1.5 nm for P or excita-
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2
following equation: Uem(S)
/
Uem(R) = (I(S)/I(R)) ꢃ (A(S)/A(R)) ꢃ (n(S)
/
n(R)2). Here, Uem(S) and Uem(R) are the fluorescence quantum yields
of the sample and the reference, respectively, I(S) and I(R) are the
integrated fluorescence intensities of the sample and the reference,
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2
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This study was supported in part by a Grant-in-Aid from Precur-
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Japan Science and Technology (JST), a Grant-in-Aid from the
Koshiyama Research Grand and a Grant-in-Aid for Scientific Re-
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Supplementary data
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