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M. Hattori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 253–257
NH
2
NH
2
CN
N
N
N
N
N
N
N
N
NH
2
a
N
b
N
N
NH
2
N
N
NH
2
O
O
O
HO
3
4
1
O
OH
H
N
H
N
N
N
N(
n-Bu)
2
N
N
N(
n
-Bu)
2
N
N
N
N
H
H
c
e
N
N
N(n-Bu)
2
N
N
N(n
-Bu)
2
DMTrO
RO
d
O
O
OH
P
N
CN
5: R = H
6: R = DMTr
7
Scheme 1. Reagents and conditions: (a) NH@C(NH2)2, EtOH, reflux, 23 h, 76%; (b) 80% CH3CO2H, 60 °C, 8 h, 97%; (c) (MeO)2CHN(n-Bu)2, DMF, 60 °C, 24 h; (d) DMTrCl,
pyridine, rt, 3 h, 23% (two steps); and (e) chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, i-Pr2NEt, THF, rt, 3 h, 93%.
SNP diagnosis rather than as DNA after RT-PCR. Furthermore, in
relation to discovery of a large number of noncoding RNAs, a meth-
600
od to detect intracellular RNAs in real-time is required. Thus, in
this study, we focused on detection of SNPs on RNA strands.
The synthesis of the tricyclic base-linked nucleoside surrogate N
is shown in Scheme 1. 5-Amino-6-cyano-3-[(2,2-dimethyl-1,
3-dioxolan-4-yl)methyl]imidazo[4,5-b]pyridine (3), which was
synthesized by a method reported previously,8 was treated with
guanidine to produce an isopropylidene derivative of N in 76%
yield. Deprotection of the isopropylidene group, protection of the
amino groups with a di(n-butyl)formamidine group,9 and protec-
tion of the primary hydroxy group with a 4,40-dimethoxytrityl
(DMTr) group afforded the properly protected nucleoside surrogate
6. Phosphitylation of the secondary hydroxy group of 6 gave
phosphoramidite unit 7 in 93% yield.
Typical emission spectra of the tricyclic base-linked nucleoside
surrogate N are shown in Figure 3. The nucleoside surrogate N
showed emission maxima at 427 nm under 334-nm excitation in
H2O, which was a wavelength approximately 35 nm longer than
that of Q. The fluorescence intensity of N was dependent on solvent
polarity; that is, the fluorescence intensity of N was greater in
more polar solvents such as methanol and water than in less polar
solvents such as chloroform (Table S1).
The sequences of oligodeoxynucleotides (ODNs) containing N
are listed in Table 1. CYP2C9 is one of the predominant cytochrome
P450 (CYP) enzymes expressed constitutively in the human li-
ver.10,11 It metabolizes a variety of therapeutically important
drugs.10,11 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-N-probes) termed NA, NG, NC,
and NT contained N at the 50 sides of Ds, whereas the probes
(30-N-probes) termed AN, GN, CN, and TN possessed N at the 30
sides of Ds. Target sequences are abbreviated as S. All the ODNs
containing N were synthesized using a DNA synthesizer. The ob-
tained ODNs were analyzed by MALDI-TOF/MS, and the observed
molecular weights were in agreement with their structures.
The Tm values of the duplexes between the probes and the RNA
targets are listed in Table S2. When the discriminating bases and
the target bases matched, the Tm values of the duplexes were the
greatest in all sequences except for the TN probe for the SrU target.
Thus, the discriminating bases seemed to have base selectivities in
the DNA/RNA duplexes containing the tricyclic nucleoside surro-
gate N.
H2O
450
300
150
0
MeOH
CHCl3
300
400
500
Wavelength (nm)
600
Figure 3. Fluorescence emission spectra of N in various solvents. Spectra were
obtained on a spectrofluorophotometer in quartz cuvettes with a path length of
1.0 cm and a 30 lM N concentration in an appropriate solvent. Spectra were
recorded by using an excitation slit of 1.5 nm and an emission slit of 1.5 nm.
detect the SNPs in the targets by visible colors. Here, we report SNP
detection by a visible color using DNAs that contain a fluorescent
tricyclic base-linked acyclonucleoside, 6,8-diamino-3-(2,3-dihydr-
oxypropyl)imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (N).
In general, amplified DNAs are used for the SNP typing.
However, the first product of gene expression is RNA, which is a
transcription product. Therefore, it is advantageous to use RNA in
Table 1
Sequences of oligonucleotides
Abbreviation
Sequence
50-d(GAA GGT CAA NAG TAT CTC T)-30
50-d(GAA GGT CAA NGG TAT CTC T)-30
50-d(GAA GGT CAA NCG TAT CTC T)-30
50-d(GAA GGT CAA NTG TAT CTC T)-30
50-d(GAA GGT CAA ANG TAT CTC T)-30
50-d(GAA GGT CAA GNG TAT CTC T)-30
50-d(GAA GGT CAA CNG TAT CTC T)-30
50-d(GAA GGT CAA TNG TAT CTC T)-30
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
NA
NG
NC
NT
AN
GN
CN
TN
SrA
SrG
SrC
SrU
The underlined letters indicate discriminating bases. The small italic letters rep-
resent target bases.