Synthesis and properties of novel base-discriminating fluorescent (BDF) nucleosides: A highly polarity-sensitive fluorophore for SNP typing

Article abstract of DOI:10.1016/j.tetlet.2004.09.003

We have developed novel alkanoylpyrene-labeled BDF nucleosides, ^AMPyU and ^MPyU. These nucleosides exhibit strong fluorescence emission at long wavelength that is highly sensitive to solvent polarity. BDF probes containing ^AMPy

Full text of DOI:10.1016/j.tetlet.2004.09.003

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7827–7831
Synthesis and properties of novel base-discriminating
fluorescent (BDF) nucleosides: a highly polarity-sensitive
fluorophore for SNP typing
Yoshio Saito,a Yohei Miyauchi,b Akimitsu Okamotob and Isao Saitoa,*
aNEWCAT Institute, School of Engineering, Nihon University and SORST, Japan Science and Technology Agency,
Tamura, Koriyama 963-8642, Japan
bDepartment of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Kyoto 615-8510, Japan
Received 9 July 2004;revised 1 September 2004;accepted 1 September 2004
Abstract—We have developed novel alkanoylpyrene-labeled BDF nucleosides, AMPyU and MPyU. These nucleosides exhibit strong
fluorescence emission at long wavelength that is highly sensitive to solvent polarity. BDF probes containing AMPyU selectively emit
fluorescence only when the base opposite BDF nucleoside is adenine and act as effective reporter probes for homogeneous SNP
typing.
Ó 2004 Elsevier Ltd. All rights reserved.
Fluorescent molecules, whose emission spectra and
quantum yields are markedly sensitive to solvent polar-
ity, are widely used as reporter probes for investigating
chemical, biochemical, and biological phenomena.1
1-Pyrenoyl derivatives are one of such attractive fluo-
rophores since they possess favorable photochemical pro-
perties, such as high stability, high quantum yield, and
high sensitivity to solvent polarity.2 Oligonucleotides
(ODN) possessing such fluorescent molecules can be
used for detecting the change in the DNA microenviron-
ment, such as that which occurs in hybridization and
conformational change, and can be used for single
nucleotide polymorphisms (SNPs) typing as well.
cence emission, there is still great demand for further
improvement of a fluorescent probe possessing long
wavelength emission. Therefore, we report herein novel
BDF probes containing highly polarity-sensitive fluores-
cent nucleosides, 1-pyrenoyl-labeled 20-deoxyuridine
AMPyU 1, for discrimination of adenine base on a target
DNA at longer wavelength. We also synthesized other
polarity-sensitive fluorescent nucleoside MPyU 2 to eval-
uate the effect of rigid acetylene linker on the discrimina-
tion of the targeted bases opposite BDF nucleosides.
The synthesis of a novel BDF nucleoside AMPyU 1 is out-
lined in Scheme 1. Pyrenecarbonyl derivative prepared
from pyrene and 4-pentinoic acid was coupled with 5-
iodo-20-deoxyuridine 5 under Sonogashira conditions
using Pd(PPh3)4 to afford 1.5 Protection of the deoxy-
ribose 50-hydroxy group with the 4,40-dimethoxytrityl
group afforded 6. After conversion to phosphoramidite,
7 was incorporated into oligonucleotides by an auto-
mated DNA synthesis (Fig. 1).
In our continuous efforts to develop base-discriminating
fluorescent (BDF) nucleosides, we devised a new homo-
geneous assay method that provides a clear distinction
of the base on the complementary strand by fluorescence
change.3 Particularly, we have recently reported novel
pyrenecarboxamide-labeled BDF nucleosides which are
sensitive to solvent polarity, and indicated that the
BDF probes selectively emit fluorescence only when
the complementary bases are perfectly matched bases.4
However, for easy and clearer detection of the fluores-
The synthesis of MPyU 2 has been accomplished accord-
ing to Scheme 2. Pyrenylpropyn 9 which was prepared
according to the protocol of Shi Shun et al.6 was cou-
pled with 30,50-diprotected 5-iodo-20-deoxyuridine 8 by
Pd(PPh3)4 to afford 10. Compound 10 was then hydro-
genated over 10% Pd/C in methanol to give 11, which
was subsequently oxidized by MnO2 to provide 1-pyr-
enoyl derivative 12. Treatment of 12 with TBAF
Keywords: BDF;Pyrene;Oligodeoxynucleotide;Fluorescence.
*
Corresponding author. Tel.: +81 24 956 8911;fax: +81 24 956 8924;
e-mail: saito@mech.ce.nihon-u.ac.jp
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.09.003

7828
Y. Saito et al. / Tetrahedron Letters 45 (2004) 7827–7831
O
O
O
O
N
X
HN
HN
O
N
O
HO
O
HO
O
X = NH: previously
reported BDF
X = CH2: AMPyU 1
OH
OH
MPyU 2
Figure 1. Structure of highly polarity sensitive fluorescent nucleosides, AMPyU and MPyU.
O
O
a, b
OH
3
4
O
O
N
O
N
I
HN
HN
O
O
c
HO
O
HO
O
OH
O
OH
5
1
O
Py
O
Py
O
HN
HN
O
N
O
N
d
e
DMTrO
NC
O
DMTrO
O
O
N
P
OH
7
6
O
Scheme 1. Reagents and conditions: (a) SOCl2, 100°C, 2h;(b) AlCl 3, pyrene, CH2Cl2, rt, overnight, 39% (in two steps);(c) 4, Pd(PPh3)4, CuI, Et3N,
DMF, rt, 6h, 88%;(d) DMTrCl, pyridine, rt, 8h, 75%;(e) ( i-Pr2N)2 PO(CH2)2CN, 1H-tetrazole, CH3CN, rt, 1h;DNA synthesis (ODN( AMPyU),
16 %).
provided target nucleoside 2.5 By employing a reaction
similar to that described for compound 7, we obtained
phosphoramidite 14 from 2. The newly synthesized
ODNs are summarized in Table 1.
noylpyrene, in water binary mixture with methanol.2c
In pure methanol, the fluorescence emission was around
436nm, but was shifted to 447nm when the water con-
centration was increased to 35% of methanol. They also
indicated that the fluorescence intensity increases with
increasing molar fraction of water. These results suggest
that BDF nucleosides containing an alkanoylpyrene
fluorophore should be highly polarity-sensitive. Impor-
tantly, the wavelength of 1-heptanoylpyrene is about
40–50nm longer than that reported for previous pyrene-
carboxamide fluorophore.4,7
There are numerous reports for fluorescence properties
of pyrenecarbonyl fluorophore. It is well known that
the monomer fluorescence of pyrene-1-carboxaldehyde
shows a strong dependency on solvent polarity. The flu-
orescence in polar solvent is quite high (UF in etha-
nol = 0.15), although the fluorescence in nonpolar
solvents such as n-hexane is very weak (UF < 0.001).2a,b
For pyrene-1-carboxaldehyde, fluorescence is emitted
from the n–p* state in nonpolar solvents. When the sol-
vent polarity increases, the p–p* state that lies slightly
above the n–p* state is brought below the n–p* state
by solvent relaxation during the lifetime of the excited
state, and thus becomes the emitting state. Further,
Armbruster and co-workers reported the fluorescence
properties of a pyrene carbonyl fluorophore, 1-hepta-
Since these reported alkanoylpyrene fluorophores show
highly polarity-sensitive fluorescence emission at longer
wavelength, we expected that the synthesized alka-
noylpyrene-labeled nucleosides AMPyU and MPyU would
also exhibit polarity-sensitive fluorescence emission.
Thus, we initially measured the fluorescence spectra of
monomer AMPyU and MPyU in solvents of different
polarities. With excitation of both AMPyU and MPyU at

Y. Saito et al. / Tetrahedron Letters 45 (2004) 7827–7831
7829
OH
OH
OH
Py
O
N
O
O
N
I
9
HN
HN
HN
b
O
a
O
N
O
TBSO
TBSO
O
TBSO
O
O
11
8
OTBS
OTBS
Py
10
OTBS
O
O
N
O
HN
O
HN
Py
O
f
c
O
N
R1O
O
DMTrO
NC
O
OR2
12 R1 = R2 = TBS
2 R1 = R2 = H
d
e
14
Py: pyrene
O
N
P
O
13 R1 = DMTr, R2 = H
Scheme 2. Reagents and conditions: (a) Pd(PPh3)4, CuI, Et3N, DMF, rt, 6h, 90%;(b) Pd/C, H 2, dioxane, rt, 17h, 76%;(c) MnO 2, CH2Cl2, frorisil, rt,
7h, 80%;(d) TBAF, THF, 0 °C, 1h, 92%;(e) DMTrCl, pyridine, rt, 8h, 75%;(f) ( i-Pr2N)2PO(CH2)2CN, 1H-tetrazole, CH3CN, rt, 1h;DNA synthesis
(ODN(MPyU), 13%).
Table 1. Oligonucleotides (ODNs) used in this study
water
MeOH
DMF
MeCN
EtOAc
7
6
5
4
3
2
1
water
MeOH
DMF
MeCN
EtOAc
6
5
4
3
2
1
Sequences
ODN(AMPyU)
ODN(MPyU)
ODN(natT)
ODN(N)
50-d(CGCAATAMPyUTAACGC)-30
50-d(CGCAATMPyUTAACGC)-30
50-d(CGCAATTTAACGC)-30
50-d(GCGTTANATTGCG)-30,
N = T, C, G, A
ODNALDH2
(
AMPyU) 50-d(TTTTCACTTAMPyUAGTG-
0
TATGCC)-30
0
375
475
575
375
475
575
ODNALDH2(A)
ODNALDH2(G)
ODNbcr/abl(
AMPyU)
50-d(GGCATACACTAAAGTGAAAA)-30
50-d(GGCATACACTGAAGTGAAAA)-30
50-d(TGAAGGGCTTAMPyUTGA-
ACTCTG)-30
(a)
(b)
wavelength (nm)
wavelength (nm)
Figure 2. (a) Fluorescence spectra of AMPyU (10lM) in water,
methanol, DMF, acetonitrile, and ethyl acetate. (b) Fluorescence
spectra of MPyU (10lM) in water, methanol, DMF, acetonitrile, and
ethyl acetate. Excitation wavelength was at 372nm.
ODNbcr/abl(A)
ODNabl(G)
50-d(CAGAGTTCAAAAGCCCTTCA)-30
50-d(CAGAGTTCAGAAGCCCTTCA)-30
372nm in water, strong fluorescence emission at 462nm
was observed as shown in Figure 2a and b. Upon exci-
tation of AMPyU and MPyU in methanol, we observed
medium emission at 443nm. In contrast, the fluores-
cence emission of both AMPyU and MPyU in less polar
solvents like DMF, acetonitrile, and ethyl acetate was
very weak. As expected, these alkanoylpyrene-labeled
nucleosides showed high sensitivity to the solvent polar-
ity. It is noteworthy that the wavelength of the emitted
light is about 70nm longer than that reported for previ-
ous pyrenecarboxamide-labeled BDF nucleosides
(397nm in water).4
mide fluorophore is attached to uracil at the C-6 posi-
tion, via a rigid acetylene linker, the fluorophore is
extruded to the outside of the groove due to base pairing
with adenine into a highly polar aqueous phase and,
therefore, strong fluorescence should be observed. In
contrast, when the pyrene fluorophore is inside of the
duplex due to the lack of base-pairing (mismatched),
the BDF base exhibits no emission due to the location
of the pyrene fluorophore at a highly hydrophobic site
in the groove.4a We assumed that these solvent polarity-
dependent fluorescence changes of AMPyU and MPyU
would be useful for the detection of the change in the
DNA microenvironment, such as SNP typing. There-
fore, the measurement of the fluorescence spectra of
2.5lM of AMPyU-, MPyU-containing duplex in sodium
phosphate buffer (pH7.0) was performed. The
fluorescence spectra of AMPyU-, MPyU-containing ODNs
and the duplexes containing different bases opposite
BDF nucleosides were measured with excitation at
Such fluorescent nucleosides would be valuable for mon-
itoring the microenvironment of DNA duplex, such as
for sensing the difference in polarities between the inside
and outside of DNA duplexes. Previously, we reported
pyrenecarboxamide-labeled BDF nucleosides which
contain a acetylene linker.4a When the pyrenecarboxa-

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Y. Saito et al. / Tetrahedron Letters 45 (2004) 7827–7831
16
12
8
20
15
10
5
A
A
G
C
T
G
C
T
4
ss
ss
0
0
375 425 475 525 575
375 425 475 525 575
wavelength (nm)
wavelength (nm)
(a)
(b)
Figure 3. (a) Fluorescence spectra of ODN(AMPyU) hybridized with
2.5lM ODN(A), ODN(G), ODN(C), or ODN(T) and single-stranded
ODN(AMPyU). (b) Fluorescence spectra of ODN(MPyU) hybridized
with 2.5lM ODN(A), ODN(G), ODN(C), or ODN(T) and single-
stranded ODN(MPyU) (50mM sodium phosphate, 0.1M sodium
chloride, pH7.0, room temperature). Excitation wavelength was at
374nm.
374nm. In the case of AMPyU-containing ODN, the flu-
orescence spectrum of the perfectly matched duplex
[ODN(AMPyU)/ODN(A)] showed strong fluorescence
emission at 462nm (quantum yield UF = 0.351).7 In con-
trast, the fluorescence of the mismatched duplexes
(ODN(AMPyU)/ODN(N), N = C, G, T) and single-
stranded ODN[ODN(AMPyU)] showed weak emission
(UF = 0.177, 0.003, 0.235, and 0.063, respectively,
Fig. 3a). Although AMPyU-containing ODN indicated
A-selective fluorescence emission, no selectivity was ob-
served in MPyU-containing ODN which lack rigid acety-
lene linker. The fluorescence quantum yields of duplexes
(ODN(MPyU)/ODN(N), N = C, G, T, A) and single-
stranded ODN[ODN(MPyU)] were 0.130, 0.057, 0.144,
0.145, and 0.038, respectively (Fig. 3b). These results
indicate that a long and rigid acetylene linker is indis-
pensable to induce clear change of the microenviron-
ment around pyrenoyl fluorophore.
Figure 4. (a) Fluorescence spectra of 2.5lM ODNALDH2(
AMPyU)
hybridized with 2.5lM ODNALDH2(A) or ODNALDH2(G) (50mM
sodium phosphate, 0.1M sodium chloride, pH7.0, room temperature).
(b) Detection of fluorescence image.
ODNALDH2(A) or ODNALDH2(G) was hybridized with 2.5lM
ODNALDH2
AMPyU). Fluorescence was observed with a fluorescence
A
volume of 2.5lM
(
imager, Versa Doc Imaging System (BioRad), equipped with a 290–
365nm transilluminator. The image was taken through a 380nm long
pass emission filter. (c) Fluorescence spectra of 2.5lM ODNbcr/abl
(
AMPyU) hybridized with 2.5lM ODNbcr/abl(A) or ODNabl(G). (d)
Detection of fluorescence image. A volume of 2.5lM ODNbcr/abl(A) or
ODNabl(G) was hybridized with 2.5lM ODNbcr/abl
AMPyU).
(
of the A/G (A-allele/G-allele) SNP sequence of alcohol
dehydrogenase 2 (ALDH2)8 by using AMPyU-containing
BDF probe. We added BDF probe ODNALDH2(
AMPyU)
to a solution of the target sequence, ODNALDH2(A) (A-
allele) and ODNALDH2(G) (G-allele), and incubated
these solutions at room temperature for 1min. The sam-
ple solutions were then illuminated at 360nm, and the
fluorescence images were taken through a 380nm cutoff
filter. A strong fluorescence emission was observed
In melting temperature measurements of the duplex,
a high duplex stability was observed for AMPyU- and
MPyU-containing duplexes. As shown in Table 2, the
melting temperatures of ODN(AMPyU)/ODN(A)
and ODN(MPyU)/ODN(A) were considerably higher
than that observed for other mismatched duplexes in
sodium phosphate buffer (pH7.0), suggesting that both
AMPyU and MPyU form a stable base pair with A.
for the ODNALDH2
whereas the mismatched
AMPyU)/ODNALDH2(G)] showed considerable fluores-
(
AMPyU)/ODNALDH2(A) duplex,
duplex [ODNALDH2
(
cence quenching (Fig. 4a and b). The fluorescence quan-
tum yield of the matched duplex (AMPyU/A UF = 0.110)
was approximately 10 times stronger than that observed
for the mismatched one (AMPyU/G, UF = 0.012).
The clear change in the fluorescence of AMPyU-contain-
ing ODN by the type of the base on the complementary
strand would be very useful for SNP typing and gene
sequence detection. First, we tested the SNP detection
Next, we examined the detection of bcr/abl cancer gene
sequence.9 As a result of the hybridization of BDF
probe with ODNbcr/abl(A) (cancer gene sequence), a
strong emission was obtained for the addition of
a,b
Table 2. Tm measurement of duplex containing AMPyU or MPyU
5'-CGC AAT X TAA CGC-3'
3'-GCG TTA N ATT GCG-5'
X = AMPyU or MPyU
ODNbcr/abl
ODNbcr/abl
(
AMPyU), whereas the emission from the
(
AMPyU)/ODNabl(G) (normal sequence) duplex
was negligible. This AMPyU-containing BDF probe facil-
itates the distinction of adenosine on a target DNA by a
drastic fluorescence change at longer wavelength than
that for previous pyrenecarboxamide-labeled BDF.4
The fluorescence quantum yield of the AMPyU-contain-
ing BDF probe is also enhanced as compared with that
of reported BDF probes, benzopyridopyrimidine
Entry
N
AMPyU, Tm (°C)
MPyU, Tm (°C)
1
2
3
4
A
G
C
T
54.4
48.5
47.9
52.3
59.9
53.8
50.7
48.1
a 2.5lm DNA in 50mM Na phosphate, 100mM NaCl, pH7.0.
b Tm for ODN(natT)/ODN(A) = 56.1°C.

Y. Saito et al. / Tetrahedron Letters 45 (2004) 7827–7831
7831
(BPP, U = 0.035)3a and naphthopyridopyrimidine (NPP,
U = 0.096).3d These fluorescence properties of
AMPyU are favorable for the application as an
SNP typing probe. However, when BDF probes con-
taining the -CAMPyUC- sequence were used, the fluores-
cence emission was strongly quenched by the flanking C/
G base pairs. This indicates that there are some limita-
tions for the sequence to use in this method using
AMPyU-containing BDF probe.
9297;(c) Okamoto, A.;Tainaka, K.;Saito, I. Chem. Lett.
2003, 32, 684–685;(d) Okamoto, A.;Tainaka, K.;Saito, I.
Tetrahedron Lett. 2003, 44, 6871–6874;(e) Okamoto, A.;
Ichiba, T.;Saito, I. J. Am. Chem. Soc. 2004, 126, 8364–
8365;(f) Okamoto, A.;Tanaka, K.;Fukuta, T.;Saito, I.
Chem. Bio. Chem. 2004, 5, 958–963.
4. (a) Okamoto, A.;Kanatani, K.;Saito, I. J. Am. Chem. Soc.
2004, 126, 4820–4827;(b) Saito, Y.;Miyauchi, Y.;Oka-
moto, A.;Saito, I. Chem. Commun. 2004, 1704–1705.
5. Spectroscopic data for selected compounds are provided.
Compound 1: 1H NMR (pyridine-d5, 400MHz) d 2.61–2.75
(m, 2H), 2.97 (t, 2H, J = 7.6Hz), 3.49 (t, 2H, J = 7.6Hz),
4.11 (dd, 1H, J = 2.4, 11.6Hz), 4.21 (dd, 1H, J = 2.8,
11.6Hz), 4.48 (ddd, 1H, J = 2.4, 2.8, 3.4Hz), 5.02 (m, 1H),
6.93 (dd, 1H, J = 6.0, 6.4Hz), 8.00–8.40 (m, 9H), 9.23 (d,
1H, J = 9.6Hz); 13C NMR (pyridine-d5, 100MHz) d 14.7,
40.9, 41.2, 61.3, 70.6, 73.8, 85.4, 88.5, 92.1, 99.9, 124.0,
124.1, 124.6, 124.8, 125.8, 126.1, 126.3, 126.4, 126.9, 128.9,
129.2, 129.2, 130.3, 130.8, 131.8, 133.4, 143.0, 150.3, 162.6,
201.7;FABMS (Glycerol/CH 3OH), m/z 509 ([M+H]+),
HRMS calcd for C30H25O6N2 ([M+H]+) 509.1712, found
In conclusion, we have developed novel alkanoyl-
pyrene-labeled BDF nucleosides, AMPyU and MPyU.
These nucleosides show a strong fluorescence depend-
ency on solvent polarity at long wavelength. BDF
probes containing AMPyU selectively emit fluorescence
only when the base opposite BDF base is adenine. The
homogeneous SNP typing method using AMPyU-con-
taining BDF probes is a powerful alternative to conven-
tional SNP typing as well as gene detection.
1
509.1709. Compound 2: H NMR (pyridine-d5, 400MHz)
d 2.67–2.71 (m, 2H), 3.11 (t, 2H, J = 7.6Hz), 3.60 (t, 2H,
J = 7.6Hz), 4.16 (dd, 1H, J = 2.4, 11.6Hz), 4.25 (dd, 1H,
J = 2.4, 11.6Hz), 4.49 (ddd, 1H, J = 2.4, 3.2, 3.4Hz), 5.06
(m, 1H), 7.06 (dd, 1H, J = 6.4, 6.8Hz), 8.01–8.50 (m, 9H),
9.22 (d, 1H, J = 9.2Hz); 13C NMR (pyridine-d5, 100MHz)
d 23.3, 41.3, 41.6, 62.3, 71.4, 85.5, 89.0, 113.6, 124.7, 124.7,
125.3, 125.6, 126.4, 126.7, 126.9, 127.1, 127.6, 129.6, 129.7,
129.8, 130.9, 131.5, 132.9, 134.0, 137.6, 151.8, 164.8, 203.7;
FABMS (DTT/CH3OH), m/z 485 ([M+H]+), HRMS calcd
for C28H25O6N2 ([M+H]+) 485.1717, found 485.1713.
6. Shi Shun, A. L. K.;Chernick, E. T.;Eisler, S.;Tykwinski,
R. R. J. Org. Chem. 2003, 68, 1339–1347.
7. The quantum yields (U) were calculated according to the
following reference: Morris, J. V.;Mahaney, M. A.;Huber,
J. R. J. Phys. Chem. 1976, 80, 969–974.
8. Hsu, L.;Tani, K.;Fujiyoshi, T.;Kurachi, K.;Yoshida, A.
Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3771–3775.
9. Shtivelman, E.;Lifshitz, B.;Gale, R. P.;Roe, B. A.;
Canaani, E. Cell 1986, 47, 277–284.
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