Ends free and self-quenched molecular beacon with pyrene labeled pyrrolocytidine in the middle of the stem

Article abstract of DOI:10.1016/j.tet.2008.10.093

Pyrene labeled pyrrolocytidine was incorporated into an oligonucleotide to construct ends free and self-quenched molecular beacon in which the fluorophore containing pyrrolocytidine was placed in the middle of the stem and used for the detection of a targ

Full text of DOI:10.1016/j.tet.2008.10.093

Tetrahedron 65 (2009) 934–939
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Ends free and self-quenched molecular beacon with pyrene labeled
pyrrolocytidine in the middle of the stem
*
Yoshio Saito , Yuta Shinohara, Subhendu Sekhar Bag, Yoshiki Takeuchi,
*
Katsuhiko Matsumoto, Isao Saito
Department of Materials Chemistry and Engineering and 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
Article history:
Pyrene labeled pyrrolocytidine was incorporated into an oligonucleotide to construct ends free and self-
quenched molecular beacon in which the fluorophore containing pyrrolocytidine was placed in the
middle of the stem and used for the detection of a target DNA with an excellent efficiency.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 October 2008
Accepted 27 October 2008
Available online 5 November 2008
1. Introduction
features of our method are; (a) the generic design, which could be
applicable irrespective of the position of the fluorophore in the
Molecular beacons (MBs) (Fig. 1a) are widely used in fluoro-
metric analysis of nucleic acids.1 After publication of the idea of
molecular beacon by Tyagi et al., numerous papers have appeared
along with its modifications to afford more excellent sensitivity and
selectivity in DNA detection. As a result of tremendous efforts,
several conceptually new MB probes were developed,2 including
a most recent design of quencher-free molecular beacon.3 However,
all the recent advances in the design of quencher-free molecular
beacon are based on the introduction of a fluorophore at the stem
terminus. For efficient quenching of the hairpin state, these MBs
require at least one guanosine (G) base as a quencher at the op-
posite stem terminus, thus limiting their generality of the design
and the use for multiplex detection.3 There has been no report so
far for the quencher-free MBs having no terminal G but with the
fluorophore in the middle of the stem and with two free ends (30-
and 50-end) for introducing other functionalities. Therefore, we
thought that it will be worthwhile and of greatest versatility if we
develop such a new type of MBs having two free ends for further
application and modification.
Our long term efforts in designing fluorescently labeled oligo-
nucleotide probes4 for high-throughput genetic analyses have led
to a rational design of a novel self-quenched MB with two free ends
(30- and 50-end) that require no terminal G for quenching at hairpin
state3b and with the fluorophore attached to pyrrolocytidine (pC)5
placed in the middle of the stem. We sought a new concept, in
which the generation of signal ‘off’ hairpin state originates from the
quenching of the fluorophore by the opposite base G of the pC-G
base pair due to their close proximity (Fig. 1b).3g The unique
stem and the sequence of the stem, (b) the full regeneration of
fluorescence signal upon opening of the stem with optimum dis-
crimination, and (c) the allowance of almost unrestricted access of
fluorophores. Therefore, with its simplicity, sensitivity, and speci-
ficity, this strategy holds a great promise in applications such as in
heterogeneous assay and high-throughput DNA analysis.
2. Results and discussion
To assess the sensitivity and specificity of our method, two
molecular beacons (MB2 and MB3) were designed in which labeled
pyrrolocytidine (pC) was placed three base pairs apart from the 30-
terminus. We have first synthesized alkylamino substituted pyr-
rolocytidine (Fig. 1c) according to the reported synthetic protocol.6
The synthesis of pyrrolo-dC derivative 5 and its cyanoethyl phos-
phoramidite 6 is shown in Scheme 1. Thus, 5-iodo-20-deoxyuridine
was coupling with N-trifluoroacetyl propynylamide in dry DMF
under Sonogashira coupling protocols7 to yield nucleoside 2, which
was cyclized to the furano derivative 3 by following the literature
procedure.8,9 50-Hydroxyl group was then protected with standard
tritylation method to get nucleoside 4. Next, the ammonia ex-
change method of Woo et al.10 was employed to give 20-deoxy-50-O-
(4,40-dimethoxytrityl)-6-(N-methylamino)-pyrrolocytidine, which
upon treatment with ethyl trifluoroacetate in methanol afforded
amino protected pyrrolo-dC analogue 5. All the synthesized nu-
cleosides were well characterized by NMR and HRMS spectrometer.
Phosphoramidite 6 was incorporated into DNA by automated DNA
synthesizer. Finally, fluorophores were post-synthetically in-
corporated into MB1 to afford MB2 and MB3, with PyI- and PyII-
labeled fluorophores, respectively (see Scheme S1, Supplementary
data) and were used to evaluate their ability in sensing target DNA.
As a target loop strand, we also synthesized ODN 9 (Table 1).
* Corresponding authors.
E-mail address: saitoy@chem.ce.nihon-u.ac.jp (Y. Saito).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.10.093

Y. Saito et al. / Tetrahedron 65 (2009) 934–939
935
(a)
Molecular beacon (MB):
Loop
+
Stem
Target DNA
Quencher
FAM
"on" state
(b) Ends free and quencher free MB:
(c)
Chemical structures of the fluorophore and pyrrolocytidine:
Figure 1. Illustration of (a) molecular beacon, (b) ends free and quencher-free MB, and (c) chemical structures of the fluorophores.
To prove our concept for the fluorescence quenching by the
opposite guanosine, we have first synthesized 13 mer linear ODNs 1
and 2 with free alkylamino substituent in which the fluorophores
were incorporated via post synthetic modification to generate
pyrene labeled ODNs 3 and 4 (Table 1). We examined the thermal
melting behavior, which showed an enhanced stability of the fully
matched duplex, ODN 3/5 (N¼G) and ODN 4/6 (N¼G) as compared
with the unmodified duplexes (ODN 7/5, ODN 8/6) (Table 2). We
checked the fluorescence response of the ODNs upon duplex for-
mation with the complementary sequences. Thus, from the fluo-
rescence spectra (Fig. 2), it is clear that the emission from the
pyrene-hooked pyrrolocytidine labeled probes is greatly decreased
in the presence of complementary strand ODN 5 (N¼G) or ODN 6
(N¼G) containing guanosine base (G) opposite to the labeled nu-
cleotide as compared with other complementary ODNs containing
A, T, C mismatched nucleotides. Thus, our probes are able to sense
the opposite matched base G from its target DNA by a drastic de-
crease of the fluorescence intensity. The close proximity of the
fluorophore and the opposite G in the duplexes is responsible for
the decrease in fluorescence intensity, which was again reflected
from the higher duplex stability compared with the control du-
plexes ODN 7/5 (N¼G) and ODN 8/6 (N¼G) (Table 2).
With the finding that the fluorescence of the pyrene-hooked
pyrrolocytidine is efficiently quenched by the opposite G, we
turned out our attention to evaluate the sensitivity of the designed
MBs in response to the duplex formation with the target DNA by
comparing the signal generated from the fluorophores. Thus, in the
resulting fluorescence spectra of MB2 and MB3, we observed
a large difference in the fluorescence of the hairpin MBs and their
duplexes with complementary loop strands (MB2/ODN 9; MB3/
ODN 9). For example, the hybridization of hairpin MB2 with its
complementary strand ODN 9 displayed a higher thermal stability
(Table 2) and a 3.4-fold enhancement in emission intensity relative
to that observed for hairpin state (Fig. 3a). MB3 is exceptionally
excellent, showing almost no background signal and thus allowing
us to detect the target DNA with high efficiency (Fig. 3c). This is due
to the pyrene carbonyl group, which is more sensitive to quenching
by electron transfer from opposite G, leading to a negligible back-
ground signal.4a Therefore, efficient quenching of hairpin state lead
to the fluorescence ‘off’ state and upon duplex formation strong
fluorescence appeared at ‘on’ state due to no contact with G, to
result in an efficient detection of the target DNA.
PyI
To assess whether the design of
,
PyIIpC-G base pair could be
used irrespective of the stem sequence and the position in the stem,

936
Y. Saito et al. / Tetrahedron 65 (2009) 934–939
CF3
NH
CF3
O
O
O
NH
O
N
O
F3C
N
H
O
O
I
NH
NH
N
N
O
N
O
(a)
(b)
(c)
N
O
N
O
HO
HO
DMTrO
HO
O
O
O
O
OH
OH
OH
1
OH
2 (88%)
3 (84%)
4 (67%)
CF3
NH
CF3
NH
O
O
NH
NH
N
N
(d)
(e)
DNA
Synthesis
N
O
ODNs
N
O
DMTrO
DMTrO
O
N
O
O
P
OH
6
NC
5 (50%)
O
Scheme 1. Synthesis of alkylamino substituted pyrrolocytidine and the incorporation into short oligonucleotides. Reagents and conditions: (a) DMF/Pd(PPh3)4/CuI/Et3N/tri-
fluoroacetylpropargylamider/rt, overnight; (b) CuI/MeOH/Et3N/60 ꢀC, 3 h; (c) pyridine/DMTrCl/DMAP/rt, 12 h; (d) (i) aq NH3/MeOH/55 ꢀC, 24 h, (ii) ethyltrifluoroacetate/MeOH, rt,
12 h; (e) 2-cyanoethyldiisopropylchlorophosphoramidite/CH2Cl2/Et3N.
we have synthesized MB4 and MB7 in which the NH2pC-G pair was
placed three and two bases apart, respectively, from the 30-stem
terminus and having no terminal G-C base pair.4a Thus, MB5 and
MB6 were synthesized from MB4 and MB8 from MB7 via post
synthetic modification. The thermal stability (Table 2) and the
fluorescence response in the presence of target DNA (Fig. 3b, d, e)
was found to be quite similar to that of other stem sequences
(Fig. 3a and c). The emissive colors of MB6 with the target are
depicted in Figure 3f. Thus, it is clear that our MBs with fluorophore
labeled pC-G base pair are capable of detecting the target DNA with
an excellent efficiency, irrespective of the sequence as well as the
position in the stem.
sequences and the length of the stem with high efficiency. The
designed ends free MBs shown here produced excellent ‘on’/‘off’
signals that are thus widely usable as a sensitive probe. MB3,
MB6, and MB8 are of particular importance with almost no
background signals and thus capable of sensing specific target
DNA sequence with an excellent selectivity and sensitivity. Our
designed MBs may be used in heterogeneous assay and on
microarray via immobilization on a solid surface through the free
ends.
4. Experimental
4.1. General
3. Conclusion
1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz)
were measured with Bruker Avance 400F spectrometer. Coupling
constant (J value) are reported in hertz. The chemical shifts are
shown in parts per million downfield from tetramethylsilane, using
In conclusion, we have developed conceptually new quencher-
free MBs. We were able to detect the target DNA irrespective of
residual chloroform (
d
7.26 in 1H NMR,
d
77.2 in 13C NMR) and di-
Table 1
methyl sulfoxaide (
d
2.50 in 1H NMR,
d
39.5 in 13C NMR) as an in-
DNA sequences used in this study
ternal standard. ESI-TOF masses were recorded on a Mariner (5076)
spectrometer, Applied Biosystems.
The reagents for DNA synthesis were purchased from Glen
Research. Mass spectra of oligodeoxynucleotides were determined
with a MALDI-TOF MS (Shimadzu AXIMA-LNR, positive mode)
ODNs
Sequences
1
2
3
4
5
6
7
8
50-d(CGCAATNH2pCTAACGC)-30
50-d(CGCAACNH2pCCAACGC)-30
50-d(CGCAATPyIpCTAACGC)-30
50-d(CGCAACPyIpCCAACGC)-30
50-d(GCGTTA N ATTGCG)-30 [N¼A, T, G, C]
50-d(GCGTTG N GTTGCG)-30 [N¼A, T, G, C]
50-d(CGCAATCTAACGC)-30
Table 2
Thermal melting properties and the fluorescence quantum yields
50-d(CGCAACCCAACGC)-30
50-d(TTCAATCTTGGATAC)-30
Duplexes
Tm (ꢀC)
FF
Duplexes
Tm (ꢀC)
FF
9
10 (MB1)
11 (MB2)
12 (MB3)
13 (MB4)
14 (MB5)
15 (MB6)
16 (MB7)
17 (MB8)
50-d(GAGGGGTATCCAAGATTGAACNH2pCCTC)-30
50-d(GAGGGGTATCCAAGATTGAACPyIpCCTC)-30
50-d(GAGGGGTATCCAAGATTGAACPyIIpCCTC)-30
50-d(TGAGGAGTATCCAAGATTGAATCNH2pCTCA)-30
50-d(TGAGGAGTATCCAAGATTGAATCPyIpCTCA)-30
50-d(TGAGGAGTATCCAAGATTGAATCPyIIpCTCA)-30
50-d(TGAGGAGTATCCAAGATTGAATCTNH2pCCA)-30
50-d(TGAGGAGTATCCAAGATTGAATCTPyIIpCCA)-30
ODN 3
ODN 4
ODN 7/5 [N¼G]
MB2
MB3
MB5
d
0.030
0.023
d
ODN 3/5 [N¼G]
ODN 4/6 [N¼G]
ODN 8/6 [N¼G]
MB2/ODN 9
MB3/ODN 9
MB5/ODN 9
56.0
63.0
61.1
52.8
47.4
53.9
50.4
52.2
0.009
0.004
d
d
53.7
50.4
45.7
46.7
47.5
49.5
0.018
0.001
0.020
0.008
0.005
0.048
0.037
0.061
0.130
0.120
MB6
MB8
MB6/ODN 9
MB8/ODN 9

Y. Saito et al. / Tetrahedron 65 (2009) 934–939
937
6
(a)
(b) 2.5
A
A
T
G
C
ss
T
G
C
ss
3
1.25
0
0
350
350
475
Wavelength (nm)
600
475
Wavelength (nm)
600
Figure 2. Fluorescence spectra of ODN 3 (a) and ODN 4 (b) (2.5
phosphate, 0.1 M sodium chloride, pH 7.0, rt). Excitation wavelength was 346 nm. ‘ss’ denotes single stranded ODN.
m
M) and the duplexes formed by hybridization with ODN 5 and 6 (N¼A, T, G, C), respectively (2.5
mM, 50 mM sodium
with 20,30,40-trihydroxyacetophenone as a matrix. Calf intestinal
alkaline phosphatase (Promega), Crotalus adamanteus venom
phosphodiesterase I (USB), and Penicillium citrinum nuclease P1
(Roche) were used for the enzymatic digestion of ODNs. All
aqueous solutions utilized purified water (Millipore, Milli-Q sp
UF). Reversed-phase HPLC was performed on CHEMCOBOND 5-
ODS-H columns (10ꢁ150 mm, 4.6ꢁ150 mm) with a JASCO Chro-
matograph, Model PU-2080, using a UV detector, Model UV-2075
plus at 260 nm.
J¼5.6 Hz, 1H), 8.80 (s, 1H), 6.66 (s, 1H), 6.15 (dd, J¼6.1, 6.1 Hz, 1H),
5.12 (t, J¼5.3 Hz, 1H), 4.46 (d, J¼5.6 Hz, 2H), 4.23 (m, 1H), 3.93 (m,
1H), 3.70–3.59 (complex, 2H), 2.41 (ddd, J¼4.2, 6.1, 13.5 Hz, 1H),
2.04 (m,1H); 13C NMR (100 MHz, DMSO-d6): dC 36.8, 41.8, 61.4, 70.4,
88.3, 88.9, 103.3, 106.2, 116.4 (q, J¼286.2 Hz), 139.2, 152.5, 154.3,
157.1 (q, J¼36.5 Hz), 171.8; ESI-MS m/z 378.1 [MþH]þ; HR-ESIMS
m/z 400.0743 [MþNa]þ calcd for C14H14F3N3O6Na, 400.0732.
4.2.3. 20-Deoxy-50-O-(4,40-dimethoxytrityl)-6-(N-trifluoroacetyl
methylamido)-furanouridine (4)
4.2. Procedure for the synthesis of compounds 2–6
To a solution of 3 (635.6 mg, 1.686 mmol) and N,N-dimethyla-
minopyridine (catalytic amount) in anhydrous pyridine (10 ml) was
added 4,40-dimethoxytrityl chloride (685.5 mg, 2.02 mmol) and
stirred at room temperature for overnight. After completion of the
reaction, MeOH (1.0 ml) was added to the reaction mixture. The
solvent was then evaporated and the crude mixture was purified by
silica gel column chromatography (CHCl3/MeOH/Et3N¼40:1:1) to
yield 4 (960.9 mg, 1.41 mmol, 84%) as a colorless solid. 1H NMR
(400 MHz, DMSO-d6): dH 10.11 (s, 1H), 8.62 (s, 1H), 7.40–7.38
(complex, 2H), 7.32–7.21 (complex, 8H), 6.94–6.89 (complex, 4H),
6.15 (dd, J¼5.0, 6.3 Hz, 1H), 6.02 (s, 1H), 5.42 (d, J¼4.8 Hz, 1H), 4.42
(s, 2H), 4.33 (m, 1H), 4.03 (m, 1H), 3.74 (s, 6H), 3.35–3.27 (complex,
2H), 2.46 (m, 1H), 2.19 (m, 1H); 13C NMR (100 MHz, DMSO-d6): dC
36.7, 46.3, 55.7 (2C), 60.4, 63.4, 69.8, 86.6, 88.2, 102.6, 106.1, 113.9
(4C), 116.4 (q, J¼286.2 Hz), 127.4, 128.2 (2C), 128.6 (2C),130.4 (2C),
130.4 (2C), 135.9, 136.1, 138.7, 145.5, 152.7, 154.2, 157.3 (q,
J¼36.5 Hz), 158.8 (2C), 171.7; ESI-MS m/z 680.3 [MþH]þ; HR-ESIMS
m/z 702.2066 [MþNa]þ calcd for C35H32F3N3O8Na, 702.2039.
4.2.1. 5-[3-(Trifluoroacetamido)propynyl]-20-deoxyuridine (2)
5-Iodo-20-deoxyuridine (1, 1.0 g, 2.8 mmol) was dissolved in dry
DMF (20 ml). N-Trifluoroacetyl propynylamide (676.8 mg,1.5 mmol),
Pd(PPh3)4 (970.6 mg, 0.84 mmol), CuI (160.0 mg, 0.84 mmol), and
Et3N (1.2 ml, 8.46 mmol) were added to the reaction mixture and
was stirred at room temperature under argon for overnight.11 After
the reaction is over, the reaction mixture was evaporated and pu-
rified by silica gel column chromatography (CHCl3/MeOH¼8:1) to
yield 2 (938.1 mg, 2.49 mmol, 88%) as a colorless solid. 1H NMR
(CD3OD, 400 MHz) dH 8.32 (s, 1H), 6.22 (dd, J¼6.5, 6.6 Hz, 1H), 4.38
(ddd, J¼3.7, 6.6, 9.6 Hz,1H), 4.26 (s, 2H), 3.93 (ddd, J¼3.0, 5.1, 6.6 Hz,
1H), 3.80 (dd, J¼3.0, 12.0 Hz, 1H), 3.72 (dd, J¼5.1, 12.0 Hz, 1H), 2.30
(ddd, J¼3.7, 6.5, 13.6 Hz, 1H), 2.19 (m, 1H); 13C NMR (CD3OD,
100 MHz) dC 29.5, 40.4, 61.4, 70.9, 76.7, 85.9, 87.1, 87.7, 98.5, 116.2 (q,
J¼284.9 Hz), 144.5, 149.9, 157.3 (q, J¼37.2 Hz), 163.4; ESI-MS m/z
378.1 [MþH]þ; HR-ESIMS m/z 400.0745 [MþNa]þ calcd for
C14H14F3N3O6Na, 400.0732.
4.2.4. 20-Deoxy-50-O-(4,40-dimethoxytrityl)-6-(N-trifluoroacetyl-
methylamido)-pyrrolocytidine (5)
4.2.2. 20-Deoxy-6-(N-trifluoroacetyl methylamido)-
furanouridine (3)
A two necked round-bottomed flask equipped with a reflux
condenser was charged with 20-deoxy-50-O-(4,40-dimethoxytrityl)-
6-(N-trifluoroacetyl methylamido)-furanouridine (4, 96.2 mg,
0.142 mmol), concentrated NH4OH (3 ml), and MeOH (4 ml), and
then stirred at 55 ꢀC. Progress of the reaction was followed by TLC.
After completion of the reaction (24 h), the solvent was removed
under vacuum and dried. The crude reaction mixture was treated
with 5 ml of MeOH containing ethyl trifluoroacetate (72.2 mg,
A round-bottomed flask was charged with 5-(N-trifluoroacetyl-
propynylamido)-20-deoxyuridine (2, 151.2 mg, 0.4 mmol) and
MeOH (3 ml), and CuI (15.3 mg, 0.08 mmol) was then added along
with Et3N (2 ml). This mixture was heated at 60 ꢀC for 3 h until the
cyclization reaction was completed as monitored by TLC. The
solvent was removed and the residue was dried under vacuum for
1 h. The resulting residue was purified by silica gel column
chromatography using gradient elution with CHCl3/MeOH (8:1 to
5:1) to yield desired compound 3 (116.7 mg, 0.309 mmol, 77%) as
a colorless foam. 1H NMR (400 MHz, DMSO-d6): dH 10.13 (t,
23 ml, 0.191 mmol) and was stirred for 12 h at room temperature.
After completion of the reaction, the solvent was removed and the
residue was further dried under vacuum. The resulting residue was

938
Y. Saito et al. / Tetrahedron 65 (2009) 934–939
(a)
4
2
4
(b)
MB5/
MB2/
ODN 9
MB5
ODN 9
MB2
2
0
0
350
475
Wavelength (nm)
600
350
475
Wavelength (nm)
600
(d)
7
(c)
MB3/
ODN 9
MB3
MB6/
ODN 9
MB6
2
3.5
1
0
0
380
480
Wavelength (nm)
580
380
480
Wavelength (nm)
580
(e)
7
(f)
MB8/
ODN 9
MB8
3.5
0
380
480
Wavelength (nm)
580
MB6
MB6/
ODN 9
Figure 3. Fluorescence spectra of hairpin MB2 (a), MB5 (b), MB3 (c), MB6 (d), and MB8 (e) and the duplexes formed by hybridization with ODN 9 (2.5
phosphate, 0.1 M sodium chloride, pH 7.0, rt). Excitation wavelength was 346 nm for MB2 and MB5 and was 369 nm for MB3, MB6, and MB8. ‘MB’ denotes hairpin state. (f)
Photograph displaying the emission behavior at 25 ꢀC of hairpin MB6 alone and of the duplex with target ODN 9 (2.5
M) upon illumination at 302 nm.
mM, 50 mM sodium
m
purified by silica gel column chromatography using CHCl3/MeOH/
Et3N¼100:10:1 to yield desired compound 5 (48.1 mg, 0.071 mmol,
50%) as a colorless foam. 1H NMR (400 MHz, DMSO-d6): dH 11.30 (br,
1H), 9.90 (br, 1H), 8.46 (s, 1H), 7.42–7.40 (complex, 2H), 7.32–7.22
(complex, 8H), 6.93–6.88 (complex, 4H), 6.26 (dd, J¼5.9, 6.1 Hz,1H),
5.38 (d, J¼4.2 Hz, 1H), 4.34 (complex, 3H), 4.00 (dd, J¼4.4, 7.7 Hz,
1H), 3.73 (s, 6H), 3.29 (complex, 2H), 2.40 (m, 1H), 2.11 (m, 1H); 13C
NMR (100 MHz, DMSO-d6): dC 37.0, 46.3, 55.7 (2C), 63.7, 70.1, 86.3,
86.6, 87.3, 98.0, 108.8, 113.9 (4C), 116.5 (q, J¼286.4 Hz), 124.5, 128.3
(2C), 128.4 (2C), 130.4 (4C), 135.8, 135.9, 137.8, 145.5, 154.3, 157.1 (q,
J¼36.1 Hz), 158.8 (2C), 159.8, 172.7; ESI-MS m/z 679.2 [MþH]þ; HR-
ESIMS m/z 701.2206 [MþNa]þ calcd for C35H33F3N4O7Na, 701.2199.
4.2.5. 20-Deoxy-30-(2-cyanoethyldiisopropylphosphoramidite)-50-
O-(4,40-dimethoxytrityl)-6-(N-trifluoroacetylmethylamido)-
pyrrolocytidine (6)
To a solution of 5 (77.2 mg, 0.114 mmol) and Et3N (1 ml) in an-
hydrous CH2Cl2 (3 ml) was added 2-cyanoethyldiisopropylchloro-
phosphorodiamidate (80
ml) under argon atmosphere. The
resulting mixture was stirred at room temperature for 1 h and was

Y. Saito et al. / Tetrahedron 65 (2009) 934–939
939
then evaporated to dryness. The residue was dissolved in EtOAc and
was washed with aq NaHCO3 and brine. The organic layer was
separated, dried over anhydrous Na2SO4 and evaporated to dryness
and was subsequently used for oligodeoxynucleotide synthesis
without further purification.
UV-2550 spectrophotometer at room temperature using 1 cm path
length cell.
4.3.4. Fluorescence measurements
ODN solutions were prepared as described in Tm measurement
experiment. Fluorescence spectra were obtained using a Shimadzu
RF-5300PC spectrophotometer at 25 ꢀC using 1 cm path length cell.
The excitation and the emission bandwidth was 1.5 nm.
4.3. Oligonucleotide synthesis and characterization
All the reagents for DNA synthesis were purchased from Glen
Research. ODNs were synthesized by a conventional phosphor-
amidite method by using an Applied Biosystems 3400 DNA/RNA
synthesizer. ODNs were purified by reverse phase HPLC on a 5-
ODS-H column (10ꢁ150 mm, elution with 50 mM ammonium for-
mate buffer (AF), pH 7.0, linear gradient over 45 min from 3% to 40%
acetonitrile at a flow rate 2.0 ml/min). ODNs containing modified
nucleotides were fully digested with calf intestine alkaline phos-
phatase (50 U/ml), and P1 nuclease (50 U/ml) at 37 ꢀC for 12 h.
Digested solutions were analyzed by HPLC on a CHEMCOBOND 5-
ODS-H column (4.6ꢁ150 mm, elution with a solvent mixture of
50 mM AF buffer, pH 7.0, flow rate 1.0 ml/min). The concentration
of each ODNs was determined by comparing peak areas with
a standard solution containing dA, dC, dG, and dT at a concentration
of 0.1 mM. Mass spectra of ODNs purified by HPLC were determined
with a MALDI-TOF mass spectrometer.
4.3.5. MALDI-TOF mass spectral data for the ODNs
See Table 3.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific re-
search of MEXT, Japanese Government. Author S.S.B. is thankful to
JSPS for an individual fellowship.
Supplementary data
Some photophysical spectra, copies of 1H, 13C NMR spectra of
selected compounds. This material is available free of charge via the
Internet at http://elsevier.com. Supplementary data associated
with this article can be found in the online version, at doi:10.1016/
j.tet.2008.10.093.
4.3.1. Synthesis of modified G-quenched molecular beacons (MB)
The fluorophores were post-synthetically incorporated into
alkylamino substituted MB to get desired molecular beacons, MB2,
3, 5, 6, and 8. Thus, active esters (1.0 mg) were dissolved in a small
References and notes
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ml) and added to amino modified MBs
(20 l) in a total volume of 150
m
m
l of 1.0 M NaHCO3 and incubated
for 8 h at 37 ꢀC. Purification and the characterization of the prod-
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All Tms of the ODNs (2.5 mM, final duplex concentration) were
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Tm values.
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ODN solutions were prepared as described in Tm measurement
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Table 3
MALDI-TOF mass spectral data for the ODNs
ODNs
MALDI-TOF mass
MALDI-TOF mass
calcd [MþH]þ
found [MþH]þ
ODN 1
ODN 2
ODN 3
ODN 4
ODN 10 MB1
ODN 11 MB2
ODN 12 MB3
ODN 13 MB4
ODN 14 MB5
ODN 15 MB6
ODN 16 MB7
ODN 17 MB8
3957.68
3927.66
4185.93
4155.90
7745.15
7973.40
8041.48
8361.57
8589.83
8657.90
8361.57
8657.90
3956.97
3928.47
4186.72
4156.27
7745.14
7972.97
8041.12
8361.04
8590.02
8658.83
8361.62
8657.09
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