Fluorescence enhancement of oligodeoxynucleotides modified with green fluorescent protein chromophore mimics upon triplex formation

Article abstract of DOI:10.1039/C6OB01278G

Green fluorescent protein (GFP)-based molecular-rotor chromophores were attached to the 5-positions of deoxyuridines, and subsequently, incorporated into the middle positions of oligodeoxynucleotides. These oligonucleotides were designed to form triplex D

Full text of DOI:10.1039/C6OB01278G

Organic &
Biomolecular Chemistry
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Fluorescence enhancement of
oligodeoxynucleotides modified with green
fluorescent protein chromophore mimics upon
triplex formation†
Cite this: DOI: 10.1039/c6ob01278g
Takashi Kanamori, Akihiro Takamura, Nobuhiro Tago, Yoshiaki Masaki,
Akihiro Ohkubo, Mitsuo Sekine* and Kohji Seio*
Green fluorescent protein (GFP)-based molecular-rotor chromophores were attached to the 5-positions
of deoxyuridines, and subsequently, incorporated into the middle positions of oligodeoxynucleotides.
These oligonucleotides were designed to form triplex DNA in order to encapsulate the GFP chromo-
phores, mimicking GFP structures. Upon triplex formation, the embedded GFP chromophores exhibited
fluorescence enhancement, suggesting the potential application of these fluorescent probes for the
detection of nucleic acids.
Received 14th June 2016,
Accepted 23rd December 2016
DOI: 10.1039/c6ob01278g
www.rsc.org/obc
when the internal rotation of the bond is restricted, the radia-
tive relaxation pathways become dominant, and the dyes emit
Introduction
Fluorescent oligonucleotides have been extensively used as strong fluorescence. Thus, oligonucleotides incorporating
fluorescent probes for the detection and structural determi- molecular rotor-type fluorescent residues can be used as fluo-
1
nation of biologically functional nucleic acids. One of the rescent oligonucleotide probes which emit stronger fluo-
essential properties of the fluorescent probes is that they emit rescence when they form complexes with target nucleic acids
fluorescence only when they bind to their target molecules or or proteins in which the conformation of the molecular rotor
5
form specific structures. To further explore the potential of is restricted.
these probes, fluorescent nucleotides having switchable emis-
sive properties must be developed.
Interestingly, the naturally occurring green fluorescent
6
protein (GFP) contains a molecular rotor-type fluorophore
7
The emissions of fluorescent nucleosides can be controlled having a (4-hydroxybenzylidene)imidazolin-3-one skeleton. In
by various mechanisms. For example, the popular fluorescent such proteins, this residue is forced to become planar due to
2
nucleoside 2-aminopurine riboside undergoes fluorescence its interaction with the surrounding amino acid residues, and
3
8
changes in response to microhydration and stacking thus, emits strong fluorescence. Therefore, if (4-hydroxybenzy-
4
with nearby nucleobases. Recently, a group of fluorescent lidene)imidazolin-3-one or its derivatives were introduced into
molecules known as “molecular rotors” has widely been the appropriate positions of oligonucleotides, such oligo-
5
investigated.
nucleotides would become useful molecular rotor-type fluo-
Molecular rotor-type fluorescent dyes contain a rotatable rescent probes. Indeed, Hocek and co-workers have reported
chemical bond which is crucial for their fluorescence pro- deoxycytidine incorporating a (3,5-difluoro-4-hydroxybenzy-
perties. Under the conditions in which the bond can rotate, lidene)-2-methylimidazolin-3-one (FBI) residue as a molecular
5
c
the dyes emit only very weak fluorescence because the dyes in rotor, which exhibits fluorescence enhancement by 2.0–3.5
their excited states lose energy through nonradiative pathways times upon its interaction with DNA binding proteins. In
which involve the internal rotation around the bond. However, addition, Stafforst and Diederichsen reported peptide nucleic
acids (PNAs) incorporating a (4-hydroxybenzylidene)-2-methyl-
imidazolin-3-one (HBI) residue and their fluorescence
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta-Cho,
Midori-ku, Yokohama 226-8501, Japan. E-mail: msekine@bio.titech.ac.jp,
kseio@bio.titech.ac.jp
enhancement by ca. 2.2 times upon the formation of PNA–
DNA duplexes. Thus, the incorporation of FBI and HBI resi-
9
dues affords oligonucleotides which emit fluorescence in
†
Electronic supplementary information (ESI) available: Experimental pro-
5c,d
various structural contexts such as DNA–protein interactions
1
13
cedures, H, and C NMR charts; ESI-TOF mass data for the compounds and
MALDI-TOF mass data and HPLC charts for the oligonucleotides. See DOI:
and duplex formation. However, the increase of the fluo-
rescence intensity upon binding to the targets is at best 2 to
10.1039/c6ob01278g
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HBI
FBI
Fig. 1 (A) Structures of dU
and dU residues, and (B) the illustration
of their triplex-induced fluorescence configurations.
Scheme 1 Synthesis of intermediates: (4-hydroxybenzylidene)-2-
methylimidazolinone derivative (3a), (3,5-difluoro-4-hydroxybenzyli-
dene)-2-methylimidazolinone derivative (3b), and 5-aminodeoxyuridine
4
times greater. A higher fluorescence increase is preferable
for further applications.
(
6).
To obtain higher fluorescence on/off rates, we tried to
combine the HBI-type chromophore with the previously
5
e
reported fluorescence system using a DNA triplex (see below).
Here, we designed deoxyuridine derivatives having an HBI or
FBI residue directly attached to the 5-position of the uracil ring
derivative 5 in 82% yield in the presence of 10% Pd/C.
Subsequently, the acetyl groups were removed by treatment
with aqueous ammonia in pyridine to give 5-amino-2′-deoxy-
(Fig. 1). We prepared DNA duplexes incorporating these
12
HBI
uridine (6) in 89% yield. By the use of 3a, 3b, and 6, dU
uridine derivatives, and studied their fluorescence enhance-
ment in the presence of triplex-forming oligodeoxynucleotides
FBI
(
8a) and dU (8b) were synthesized as shown in Scheme 2.
10
Using previously reported conditions, compound 3a or 3b
was condensed with 6 in pyridine at 60 to 90 °C for 3a or 40 °C
for 3b to give POM-protected dU
(TFOs). We have previously reported the TFO-induced fluo-
rescence of DNA duplexes incorporating 5-(3-methyl-
HBI
FBI
(7a) in 42% and dU (7b)
MBF 5e
benzofuran-2-yl)deoxyuridine (dU
), and the application of
in 55%, respectively. The subsequent deprotection of the POM
groups of 7a and 7b gave the desired 8a and 8b, respectively.
In addition, for oligonucleotide synthesis, 7a and 7b were
converted to the phosphoramidites 10a and 10b via the
the TFO-induced fluorescence to a model experiment for the
detection of micro RNAs. In this system, the single stranded
target DNA strand reverse-transcribed from the microRNA is
MBF
captured by the single stranded ODN containing dU
5
′-DMTr derivatives 9a and 9b, respectively. The phosphorami-
forming Watson–Crick base pairs, and the fluorescence of
dites were directly used for oligonucleotide synthesis after
usual work-up.
MBF
dU
was enhanced by the formation of the DNA triplex,
using the TFO-incorporating C3 residue.
In this triplex, the methylbenzofuran moiety protrudes into
the major groove and is covered by the nucleobases around the
abasic site in the TFO. The formation of the triplex enhanced
fluorescence by 16 times in comparison with that observed in
the absence of the TFO. Therefore, if the oligonucleotides
modified with HBI and FBI show similar triplex-induced large
fluorescence enhancement as shown in Fig. 1, they might be
Fluorescence properties of nucleosides
The fluorescence properties of 8a and 8b were studied (Fig. 2).
Compound 8a shows a maximum absorbance in both metha-
nol and glycerol–methanol solutions at around 376 nm, which
utilized for the detection of biologically important nucleic
MBF
acids in combination with dU
.
Results and discussion
Synthesis of nucleoside phosphoramidites
First,
the
protected
(4-hydroxybenzylidene)-2-methyl-
imidazolinone (3a) was prepared (Scheme 1). The hydroxy
group of 4-hydroxybenzaldehyde (1a) was protected using a
pivaloyloxymethyl (POM) group to give 2a in 66% yield. Under
7
a,10
previously reported conditions,
compound 2a was con-
densed with N-acetylglycine to give the oxazoline derivative 3a
in 33% yield. Similarly, the protected 3,5-difluoro-4-hydroxy-
benzylidene derivative 3b was synthesized from 3,5-difluoro-4-
hydroxybenzaldehyde (1b). 3′,5′-Di-O-acetyl-5-nitro-deoxyuri-
Scheme 2 HBI- and FBI-modified deoxyuridines (8a and 8b, respect-
1
1
dine (4) was separately hydrogenolyzed to the 5-amino ively), and their phosphoramidites.
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than 8a does, which is attributed to the deprotonated form of
the difluorophenol ring (Fig. 2E).
Thermal stabilities of DNA triplexes incorporating HBI and
FBI residues
Next, to study the fluorescence properties of ODNs and DNA
HBI
FBI
triplexes incorporating dU
and dU , ODNs were syn-
thesized on an automated DNA/RNA synthesizer using phos-
phoramidites 10a and 10b. The sequences of the synthesized
and purchased oligonucleotides are shown in Fig. 3. ODN-1 is
an oligodeoxynucleotide containing only canonical deoxy-
HBI
nucleotides. ODN-2-H and ODN-2-F incorporate dU
and
FBI
dU at position Y, respectively, each of which forms an anti-
parallel duplex with ODN-1. We also synthesized TFO-C3 and
TFO-A, which form parallel triplexes with duplexes ODN-2-H/
ODN-1 or ODN-2-F/ODN-1. TFO-C3 has a propylene linker, C3,
at the position opposite to Y. In the TFO, 2-aminopyridine (Py)
residues, with pK
a
= 5.9, were introduced instead of cytosine so
Fig. 2 Photophysical properties of 8a and 8b. The absorption spectra
of (A) 8a and (C) 8b (30 μM) in glycerol–methanol (95 : 5, v/v) or metha-
nol solution, and the fluorescence spectra of (B) 8a (100 μM, λex 380 nm)
and (D) 8b (1 μM, λex 430 nm) in the glycerol–methanol or methanol
solution. (E) Structures of the protonated and de-protonated forms of
HBI and FBI residues.
that the TFOs would bind to the duplexes even under neutral
1
4
pH conditions.
Using the ODNs, we first studied triplex formation. The UV
melting experiments of a solution of ODN-1, ODN-2-H, and
TFO-C3 (2 μM each) showed two transitions near 35 and 62 °C
(
Fig. S6A†). The transition at a lower temperature corresponds
to the melting of the ODN-1/ODN-2-H/TFO-C3 triplex to the
ODN-1/ODN-2-H duplex and TFO-C3, and the other transition
corresponds to the further melting of the ODN-2-H/ODN-1
duplex into single strands. In the case of the triplex containing
TFO-A, the same experiment gave the Tm of 41 °C (Fig. S6C†).
By carrying out similar experiments, we determined the
melting temperatures of the triplex ODN-1/ODN-2-F/TFO-C3
and ODN-1/ODN-2-F/TFO-A as 34 °C and 39 °C, respectively
1
3
is close to that of HBI (Fig. 2A). It also shows a smaller absor-
bance at around 260 nm which is close to that of uracil. Upon
irradiation at 380 nm, the glycerol–methanol solution of 8a
emits fluorescence at around 453 nm, whereas the methanol
solution emits only very weak fluorescence (Fig. 2B). It had
been reported that HBI derivatives emitted strong fluorescence
9
signals in viscous glycerol. Thus, these results suggest that
(
m
Fig. S6B and S6D†). The higher T s of the triplexes containing
the molecular-rotor like properties of HBI are retained even
after its placement on the uracil ring.
The photophysical properties of 8b were similarly studied.
As shown in Fig. 2C, compound 8b exhibits a maximum
absorption at around 377 nm, which is very close to that of 8a,
dA in comparison with those containing the C3 linker are
probably because the backbone conformation of TFO-A is less
flexible than that of TFO-C3 owing to the presence of a cyclic
structure in the deoxyribose residue.
and a second absorption at around 430 nm in both solvents. Fluorescence properties of DNA triplexes incorporating
The absorption at a longer wavelength is expected to be that of HBI and FBI residues
the deprotonated form of 8b having a difluorophenolate
anion, because the wavelength is very close to the maximum
Next, we studied the fluorescence properties of ODN-2-H and
ODN-2-F in their single-strand states, as duplexes with ODN-1,
wavelength of 8a or 8b in the presence of sodium methoxide
and as triplexes. As shown in Fig. 4A, ODN-2-H emits only
(see Fig. S5†). Thus, in glycerol–methanol solution, compound
weak fluorescence in the single-strand and duplexed states,
8
b is expected to be in an equilibrium between the deproto-
nated and protonated forms of the difluorophenol moiety.
Upon excitation at 430 nm, the glycerol–methanol solution of
8
b shows a maximum fluorescence at 500 nm, a longer wave-
length in comparison with that of 8a (Fig. 2D). It should be
noted that the above fluorescence spectra were measured
using a 100 μM solution of 8a and 1 μM solution of 8b. Thus, a
comparison of the observed fluorescence intensity of the
1
1
00 μM solution of 8a, which was about 200, and that of the
μM solution of 8b, which was about 550, suggests that 8b
emits much stronger fluorescence. In addition, because of the
lower pK of the phenolic proton, 8b emits a longer wavelength Fig. 3 Sequences of ODNs and TFO.
a
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single strand, whereas the fluorescence of the ODN-1/ODN-2-
H/TFO-C3 triplex is ca. 5.8 times higher than that of single-
strand ODN-2-H. The emission observed at 502 nm suggested
that the FBI residue was in the deprotonated form in the
single stranded, duplex, and triplex states. This observation is
reasonable because the pK
a
of the phenolic proton of FBI is ca.
7
a
5.5, which is much lower than the pH 7 solution conditions
used in these experiments.
Conclusions
In this paper, we synthesized the new switchable fluorescent
HBI
FBI
nucleosides, dU
and dU , and oligonucleotides incorpor-
ating them as fluorophores, to mimic those of GFPs. We
demonstrated their switchable fluorescence in response to
DNA triplex formation. The results reported in this paper are
the first examples of the fluorescence enhancement of HBI or
FBI residues in combination with DNA triplex formation. The
particular usefulness of the triplex system is demonstrated by
the enhancement of the fluorescence of ODN-2-H by 5.8 times,
which is greater than the previously reported oligonucleotide
Fig. 4 Fluorescence spectra of (A) ODN-2-H and (B) ODN-2-F in their
single-strand states (ss), as duplexes with ODN-1 (ds), as triplexes
ODN-1/ODN-2-Y/TFO-C3 (C3), and as triplexes ODN-1/ODN-2-Y/
TFO-A (A). Conditions: 2 μM oligonucleotides, 10 mM cacodylate buffer
(
2
pH 7.0), 10 mM MgCl , 500 mM NaCl.
5
e,9
analogs modified with HBI.
Because DNA triplexes have been widely used in the devel-
probably because the HBI residue can freely rotate. However,
upon the addition of TFO-C3 to form the ODN-1/ODN-2-H/ opment of the DNA nanostructures/nanomachines and DNA
1
6
5
e,17
HBI
TFO-C3 triplex, the fluorescence intensity at 465 nm increases biosensors,
the triplex-induced fluorescence of dU
and
FBI
by ca. 5.8 times. This result suggests that the molecular rotor- dU could also be applied to such nucleic acid technologies.
like properties of the HBI residue can be controlled by DNA For example, by using two ODN probes incorporating dU or
triplex formation, as expected. The emission observed at the previously reported dU
FBI
MBF 5e
,
each of which is complemen-
4
65 nm suggested that the HBI residue retained its protonated tary to a different target nucleic acid, and appropriately
form in the single stranded, duplex, and triplex states. It is designed TFOs, we can develop a detection system which can
difficult to deprotonate the phenolic proton of HBI while quantify two different target nucleic acids in a single test tube
FBI
retaining the triplex structure because it requires raising the by monitoring the fluorescence at different wavelengths (dU
MBF
pH of the solution to 8. Under such conditions, the Py residues at 500 nm and dU
are also deprotonated and the triplex is destabilized.
When the TFO is changed to TFO-A, the fluorescence inten- multiplex nucleic acid detection systems might be developed.
at 428 nm). Moreover, by developing
other fluorescent nucleosides that emit at various wavelengths,
HBI
FBI
sity of the triplex decreased to levels comparable to those of We are now investigating derivatives of dU
and dU , incor-
the duplex. This result suggested that the large fluorescence porating various substituents on the phenol rings or changing
enhancement of the DNA triplex incorporating HBI required the positions of OH groups, to realize such multiplex detection
TFO incorporating the abasic C3 site. Probably, the HBI systems. Our results will be reported in due course.
residue is inserted into the space formed by the C3 linker and
the upper and lower base as shown in Fig. 1. It should be
noted that some molecular-rotor fluorophores such as SYBR
green interact with the minor groove of the DNA duplex. The
observation that the HBI and FBI residues in the duplexes did
Experimental
Materials
1
5
not emit fluorescence suggested that the mechanisms of the Reagents and dry solvents used for the synthesis of modified
HBI
FBI
fluorescence enhancement of dU
from that of these fluorophores.
and dU
are different nucleosides and their phosphoramidite units were purchased
from commercial sources. The dry solvents were stored over
In the case of ODN-2-F shown in Fig. 4B, the fluorescence molecular sieves 4A. Reagents for DNA synthesis and unmodi-
unexpectedly decreases upon duplex formation. However, the fied DNA phosphoramidite units (dA, T, dG, dC) and CPG-
fluorescence intensity at 502 nm of the triplex composed of columns were purchased from Glen Research. Sep-Pak C18
ODN-1/ODN-2-F/TFO-C3 is ca. 1.8 times higher than that of cartridges for DNA purification were purchased from Waters.
the single-strand state of ODN-2-F. The fluorescence intensity Unmodified oligodeoxynucleotides were purchased from
1
13
is much higher than that of the ODN-1/ODN-2-H/TFO-C3 Sigma-Aldrich. H and C NMR spectra were obtained at 500
triplex. As a result, the fluorescence intensity of ODN-2-F in and 126 MHz, respectively. The chemical shifts were measured
1
the triplex state is only ca. 1.8 times higher than that of the from DMSO in DMSO-d (2.49 ppm) for H NMR, 39.52 ppm
6
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1
3
1
for C NMR and CHCl
7
(
(
2
3
in CDCl
3
(7.26 ppm) for H NMR, ing the target compound were concentrated. The residue was
precipitated by the addition of methanol and filtered to give
1
3
7.16 ppm for C NMR.
1
2
1
4-Formylphenoxy)methyl pivalate (2a). Sodium iodide compound 6 (3.85 g, 89%); H NMR (500 MHz, DMSO-d)
4.05 g, 27.0 mmol) and chloromethyl pivalate (4.07 g, δ 11.29 (s, 1H), 6.92 (s, 1H), 6.19 (t, J = 7.0 Hz, 1H), 5.21 (d, J =
7.0 mmol) were dissolved in dry acetone (50 mL) and stirred 4.2 Hz, 1H), 4.89 (t, J = 5.5 Hz, 1H), 4.19 (s, 1H), 4.09 (m, 2H),
at room temperature under an argon atmosphere. After 7 h, 3.71 (m, 1H), 3.52 (m, 2H), 2.00 (m, 2H).
sodium carbonate (2.86 g, 27.0 mmol) and 4-hydroxybenzalde-
5-[(Z)-4-(4-((Pivaloyloxy)methoxy)benzylidene)-2-methyl-5-oxo-
hyde (1a) (3.00 g, 24.6 mmol) were added. The mixture was imidazolin-1-yl]-2′-deoxyuridine (7a). 5-Amino-2′-deoxyuridine
1
2
stirred for 19 h. After the completion of the reaction, diethyl (6) (727 mg, 3.00 mmol) and compound 3a (949 mg,
ether was added and the precipitates were removed by fil- 3.00 mmol) were dissolved in dry pyridine (12 mL) and stirred
tration. The filtrate was evaporated and purified by C-200 silica at 60 °C for 22 h, then the temperature was raised to 90 °C and
gel chromatography with n-hexane–ethyl acetate (96 : 4, v/v) to stirred for further 24 h. The reaction mixture was evaporated
1
give compound 2a (3.84 g, 66%); H NMR (500 MHz, chloro- and purified by C-200 silica gel chromatography with dichloro-
form-d) δ 9.93 (s, 1H), 7.87 (d, J = 9.0 Hz, 2H), 7.16 (d, J = methane–methanol (95 : 5, v/v). The eluted fraction containing
1
3
9
.0 Hz, 2H), 5.84 (s, 2H), 1.20 (s, 9H); C NMR (126 MHz, the target compound was evaporated and purified again by
chloroform-d) δ 191.0, 177.3, 161.8, 132.1, 131.6, 116.3, 84.8, C-200 silica gel chromatography with n-hexane–ethyl acetate to
+
+
1
3
9.2, 27.1. ESI-MS calcd for C H NNaO [M + Na] 259.0941; give compound 7a (686 mg, 42%); H NMR (500 MHz, DMSO-
13 16 4
found 259.0934.
6
d ) δ 11.89 (m, 1H), 8.29 (d, J = 5.1 Hz, 1H), 8.24 (d, J = 8.7 Hz,
(Z)-[4-((2-Methyl-5-oxo-oxazolin-4-ylidene)methyl)phenyl]oxy- 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.04 (s, 1H), 6.14 (m, 1H), 5.84 (s,
methyl pivalate (3a). Compound 2a (3.77 g, 16.0 mmol), 2H), 5.28 (m, 1H), 5.03 (m, 1H), 4.22 (m, 1H), 3.79 (s, 1H),
1
3
N-acetylglycine (1.87 g, 16.0 mmol) and sodium acetate (1.31 g, 3.62–3.48 (m, 2H), 2.24–2.06 (m, 5H), 1.12 (s, 9H); C NMR
6.0 mmol) were dissolved in acetic anhydride (16 mL) and (126 MHz, DMSO-d ) δ 176.6, 160.4, 158.0, 150.0, 141.4, 141.3,
1
6
stirred at 100 °C for 13 h. The reaction mixture was diluted 136.9, 134.1, 128.6, 125.8, 116.3, 108.1, 87.9, 85.3, 84.9, 70.1,
with ethyl acetate (400 mL) and washed with water. The 70.0, 61.1, 61.1, 38.6, 26.8, 15.6, 15.5. ESI-MS calcd for
+
+
organic layer was collected, evaporated and dried over Na
2
SO
4
.
C
26
H
30
N
4
NaO
9
[M + Na] 565.1905; found 565.1900.
The crude product was purified by C-200 silica gel chromato-
5-[(Z)-4-(4-Hydroxybenzylidene)-2-methyl-5-oxo-imidazolin-1-
graphy with n-hexane–ethyl acetate (85 : 15, v/v). Furthermore, yl]-2′-deoxyuridine (8a). To a solution of compound 7a
the crude compound was dissolved in n-hexane–ethyl acetate (200 mg, 0.369 mmol) in pyridine (2 mL) was added 28% aq.
(90 : 10, v/v, 30 mL) at 50 °C and cooled to room temperature. NH3 (3 mL) and the reaction mixture was stirred for 7 h at
The precipitates were filtered to give compound 3a (1.70 g, room temperature. The reaction mixture was evaporated under
1
3
3%); H NMR (500 MHz, chloroform-d) δ 8.07 (d, J = 8.7 Hz, reduced pressure. Methanol (5 mL) was added to the remain-
H), 7.12–7.07 (m, 3H), 5.82 (s, 2H), 2.39 (s, 3H), 1.21 (s, 9H). ing residues and the precipitate was filtered. The residue was
C NMR (126 MHz, chloroform-d) δ 177.4, 168.1, 165.7, 159.2, dissolved in water and washed with dichloromethane. The
34.3, 131.4, 131.0, 128.0, 116.4, 85.0, 39.1, 27.1, 15.8. ESI-MS aqueous layer was collected and lyophilized to give compound
2
1
3
1
+
+
1
calcd for C H NaO [M + Na] 340.1155; found 340.1152.
8a (91 mg, 58%); H NMR (500 MHz, DMSO-d ) δ 11.83 (brs,
6
1
7
19
5
3
′,5′-Di-O-acetyl-5-amino-2′-deoxyuridine (5). To a solution of 1H), 10.19 (brs, 1H), 8.26 (s, 1H), 8.10 (d, J = 8.6 Hz, 2H), 6.96
1
1
compound 4 (1.60 g, 4.48 mmol) in methanol (800 mL), was (s, 1H), 6.84 (d, J = 8.5 Hz, 2H), 6.19–6.08 (m, 1H), 5.30–5.21
added 10% Pd–C (224 mg) under an argon atmosphere. Then (m, 1H), 5.02 (m, 1H), 4.23 (m, 1H), 3.78 (s, 1H), 3.61–3.47 (m,
the argon was replaced with H and the reaction mixture was 2H), 2.25–2.05 (m, 5H); C NMR (126 MHz, DMSO-d ) δ 169.3,
2 6
1
3
stirred vigorously for 1.5 h at room temperature. Then, Pd–C 169.2, 161.4, 161.2, 160.2, 160.0, 149.8, 149.6, 141.1, 141.0,
was filtered using Celite and the filtrate was evaporated and 135.2, 134.3, 126.8, 125.1, 115.9, 108.1, 87.7, 85.1, 84.9,
+
purified by C-200 silica gel chromatography with dichloro- 70.0, 69.8, 60.9, 15.3, 15.3. ESI-MS calcd for C20
methane–methanol (97 : 3, v/v) to give compound 5 (1.2 g, [M + Na] 451.1224; found 451.1220.
H
20
N
4
NaO
7
+
1
8
1
2
2%); H NMR (500 MHz, chloroform-d) δ 9.20 (s, 1H), 6.90 (s,
(2,6-Difluoro-4-formylphenoxy)methyl pivalate (2b). Sodium
H), 6.37 (dd, J = 8.6, 5.6 Hz, 1H), 5.20 (m, 1H), 4.46–4.29 (m, iodide (2.09 g, 13.9 mmol) and chloromethyl pivalate (2.10 g,
H), 4.29–4.13 (m, 1H), 3.48 (m, 2H), 2.44 (m, 1H), 2.19–2.12 13.9 mmol) were dissolved in dry acetone (25 mL) and stirred
1
3
(
m, 7H); C NMR (126 MHz, chloroform-d) δ 170.6, 160.5, at room temperature under an argon atmosphere. After 18 h,
1
49.0, 122.9, 116.2, 84.9, 84.9, 82.1, 74.2, 74.2, 64.0, 37.1, 21.1. potassium carbonate (1.93 g, 13.9 mmol) was added to the
+
+
ESI-MS calcd for C13
H
17
N
3
NaO
7
[M + Na] 350.0959; found reaction mixture. Then, the reaction mixture was placed on an
3
50.0961.
ice bath and 3,5-difluoro-4-hydroxybenzaldehyde (1b) (2.00 g,
5
-Amino-2′-deoxyuridine (6). To a solution of compound 5 12.7 mmol) was added. The mixture was stirred at room temp-
(5.84 g, 17.8 mmol) in pyridine (60 mL) was added 28% erature for 27 h. After the completion of the reaction, diethyl
NH OH (80 mL). The reaction mixture was stirred for 13 h at ether was added and the precipitates were filtered. The filtrate
4
room temperature. Then, it was concentrated and the crude was evaporated and purified by C-200 silica gel chromato-
product was purified by C-200 silica gel chromatography with graphy with n-hexane–ethyl acetate (96 : 4, v/v) to give com-
1
dichloromethane–methanol (85 : 5, v/v). The fractions contain- pound 2b (3.03 g, 88%); H NMR (500 MHz, chloroform-d)
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δ 9.85 (s, 1H), 7.46 (s, 1H), 7.45 (s, 1H), 5.76 (s, 2H), 1.16 (s, (m, 1H), 4.23 (m, 1H), 3.78 (s, 1H), 3.62–3.46 (m, 2H),
1
3
13
9
1
8
2
H); C NMR (126 MHz, chloroform-d) δ 188.9, 188.7, 177.4, 2.25–2.05 (m, 5H); C NMR (126 MHz, DMSO-d ) δ 169.0,
6
57.2, 155.2, 138.8, 132.5, 113.4, 113.4, 113.3, 113.2, 88.4, 88.3, 163.3, 160.1, 153.0, 152.9, 151.1, 151.0, 149.7, 141.2, 141.1,
8.0, 39.0, 26.9. ESI-MS calcd for C13
95.0752; found 295.0744.
+
+
H
14
F
2
NaO
4
[M + Na]
137.1, 124.3, 123.9, 115.5, 115.3, 107.7, 87.7, 85.1, 85.0,
69.9, 69.8, 60.9, 15.4, 15.4. ESI-MS calcd for C H F N NaO
7
+
2
0
18
2
4
+
(
Z)-[2,6-Difluoro-4-((2-methyl-5-oxo-oxazolin-4-ylidene)methyl) [M + Na] 487.1036; found 487.1028.
phenyl]oxymethyl pivalate (3b). Compound 2b (2.92 g, 5′-O-(4,4′-Dimethoxytrityl)-5-[(Z)-4-(4-((pivaloyloxy)methoxy)
2.4 mmol), N-acetylglycine (1.45 g, 12.4 mmol) and sodium benzylidene)-2-methyl-5-oxo-imidazolin-1-yl]-2′-deoxyuridine (9a).
acetate (1.01 g, 12.4 mmol) were dissolved in acetic anhydride Compound 7a (398 mg, 0.734 mmol) was co-evaporated with
12 mL) and stirred at 100 °C for 13 h. The reaction mixture dry pyridine. Then, compound 7a was dissolved in dry pyridine
was diluted with ethyl acetate (500 mL) and washed with aq. (7 mL) and 4,4′-dimethoxytrityl chloride (273 mg, 0.807 mmol)
NaHCO and brine. The organic layer was collected, evapor- was added. The reaction mixture was stirred at room tempera-
ated and dried over Na SO . The crude product was purified by ture for 6 h under an argon atmosphere. Then, the reaction
C-200 silica gel chromatography with n-hexane–ethyl acetate was quenched by adding methanol (1 mL) and, the solvents
1
(
3
2
4
1
(
95 : 5, v/v) to give compound 3b (2.47 g, 57%); H NMR were evaporated. The crude product was extracted with ethyl
(
1
500 MHz, chloroform-d) δ 7.72 (s, 1H), 7.70 (s, 1H), 6.93 (s, acetate and washed with water and then with brine. The
H), 5.75 (s, 2H), 2.42 (s, 3H), 1.19 (s, 9H); C NMR (126 MHz, organic layer was dried over Na SO and evaporated. The crude
2 4
1
3
chloroform-d) δ 177.5, 167.6, 167.3, 156.9, 156.8, 154.9, 154.8, product was purified by N-60 silica gel chromatography with
1
1
1
35.7, 135.6, 135.4, 134.1, 130.0, 129.9, 129.9, 128.1, 128.1, n-hexane–ethyl acetate (30 : 70, v/v). The fractions containing
28.0, 115.9, 115.9, 115.8, 115.7, 88.5, 88.5, 88.5, 39.1, 27.1, the target compound were evaporated and again purified by
+
+
6.0. ESI-MS calcd for C17
H
17NNaO
5
[M + Na] 376.0967; N-60 silica gel chromatography with dichloromethane–
found 376.0977.
-[(Z)-4-(3,5-Difluoro-2-methyl-4-((pivaloyloxy)methoxy)benzyl-
methanol (96 : 4, v/v) to give compound 9a (261 mg, 42%);
H NMR (500 MHz, DMSO-d ) δ 11.98 (s, 1H), 8.23 (d, J =
6
1
5
idene)-5-oxo-imidazolin-1-yl]-2′-deoxyuridine (7b). 5-Amino-2′- 8.2 Hz, 2H), 7.93 (s, 1H), 7.36–7.12 (m, 11H), 7.00 (m, 1H),
1
2
deoxyuridine (6) (688 mg, 2.83 mmol) and compound 3b 6.81 (m, 4H), 6.19 (t, J = 6.5 Hz, 1H), 5.85 (s, 2H), 5.37 (s, 1H),
1.0 g, 2.83 mmol) were dissolved in dry pyridine (13 mL) and 4.34–4.22 (m, 1H), 3.91 (m, 1H), 3.68 (s, 6H), 3.32–3.03 (m,
(
1
3
stirred at 40 °C for 3 days under an argon atmosphere. The 2H), 2.27 (m, 2H), 2.07–1.98 (m, 3H), 1.12 (s, 9H); C NMR
reaction mixture was evaporated and purified by C-200 silica (126 MHz, DMSO-d ) δ 176.4, 169.2, 169.0, 162.4, 160.2, 158.0,
gel chromatography with dichloromethane–methanol 157.7, 149.7, 144.7, 144.6, 136.6, 135.6, 135.1, 133.9, 129.7,
97.5 : 2.5, v/v). The eluted fraction containing the target com- 129.6, 129.5, 128.4, 127.9, 127.8, 127.6, 127.6, 126.7, 125.7,
6
(
pound was evaporated and purified again by C-200 silica gel 116.1, 113.2, 108.1, 85.9, 85.8, 85.6, 85.1, 84.9, 84.7, 70.5, 64.1,
+
chromatography with n-hexane–ethyl acetate and finally eluted 63.6, 55.0, 38.4, 26.6, 15.3. ESI-MS calcd for C H N NaO
11
4
7
48
4
+
with ethyl acetate to give 7b (901 mg, 55%). The analytical [M + Na] 867.3212; found 867.3192.
sample (86 mg) was obtained from the 100 mg of 7b after re- 5′-O-(4,4′-Dimethoxytrityl)-5-[(Z)-4-(3,5-difluoro-2-methyl-4-
cycling preparative GPC-HPLC; H NMR (500 MHz, DMSO-d ) ((pivaloyloxy)methoxy)benzylidene)-5-oxo-imidazolin-1-yl]-2′-
1
6
δ 11.83 (s, 1H), 8.29 (s, 1H), 8.09–8.05 (m, 2H), 7.03 (s, 1H), deoxyuridine (9b). Compound 7b (807 mg, 1.39 mmol) was
6
4
2
.13 (m, 1H), 5.75 (m, 2H), 5.21 (s, 1H), 5.00–4.92 (m, 1H), co-evaporated with dry pyridine. Then, compound 7b was dis-
.26–4.20 (m, 1H), 3.81 (d, J = 4.6 Hz, 1H), 3.64–3.49 (m, 2H), solved in dry pyridine (14 mL) and 4,4′-dimethoxytrityl chlor-
1
3
.23–2.10 (m, 5H), 1.12 (s, 9H); C NMR (126 MHz, DMSO-d
6
)
ide (520 mg, 1.53 mmol) was added. The reaction mixture was
δ 176.5, 169.0, 168.9, 165.4, 165.2, 160.1, 156.0, 156.0, 154.1, stirred at room temperature for 7 h under an argon atmo-
1
1
2
6
54.0, 149.8, 141.4, 141.3, 139.2, 133.6, 131.2, 122.6, 115.6, sphere. The reaction mixture was quenched by the addition of
15.5, 115.4, 107.5, 88.2, 87.7, 85.2, 85.0, 69.9, 69.8, 60.9, 38.3, methanol and evaporated. The crude product was extracted
+
+
6.6, 15.5, 15.5. ESI-MS calcd for C26
01.1717; found 601.1722.
H
28
F
2
N
4
NaO
9
[M + Na]
with ethyl acetate and washed with water and then with brine.
The organic layer was dried over Na SO and evaporated. The
2
4
5
-[(Z)-4-(3,5-Difluoro-4-hydroxybenzylidene)-2-methyl-5-oxo- crude product was purified by N-60 silica gel chromatography
imidazolin-1-yl]-2′-deoxyuridine (8b). To a solution of com- with dichloromethane–methanol (95 : 5, v/v) to give compound
1
pound 7b (67 mg, 0.116 mmol) in pyridine (0.58 mL) was 9b (770 mg, 63%); H NMR (500 MHz, DMSO-d ) δ 12.02 (s,
6
3
added 28% aq. NH (0.58 mL) and the reaction mixture was 1H), 8.09 (d, J = 9.8 Hz, 2H), 7.97 (s, 1H), 7.37–7.14 (m, 9H),
stirred for 6 h at room temperature. The reaction mixture was 7.01 (d, J = 7.5 Hz, 1H), 6.82 (m, 4H), 6.21 (t, J = 6.5 Hz, 1H),
evaporated under reduced pressure. The crude residues were 5.75 (s, 2H), 5.39 (d, J = 4.4 Hz, 1H), 4.31 (m, 1H), 3.97–3.89
dissolved in water and a small amount of acetonitrile, and (m, 1H), 3.69 (s, 6H), 3.37–3.05 (m, 2H), 2.30 (m, 2H), 2.09 (m,
1
3
then purified by C-18 reversed phase column chromatography 3H), 1.12 (s, 9H); C NMR (126 MHz, DMSO-d
6
) δ 176.5, 168.9,
with water–acetonitrile (4 : 1, v/v) to give compound 8b (22 mg, 168.8, 164.9, 164.9, 160.1, 160.0, 158.1, 158.0, 156.0, 154.1,
1
4
1
4
6
1%); H NMR (500 MHz, DMSO-d ) δ 11.92–11.83 (m, 1H), 149.7, 144.7, 144.4, 141.1, 140.8, 139.0, 135.7, 135.4, 135.1,
1.03 (brs, 1H) 8.27 (s, 1H), 7.97 (d, J = 9.9 Hz, 2H), 6.98 (d, J = 133.7, 133.6, 133.4, 131.3, 131.2, 129.7, 129.4, 127.9, 127.6,
.7 Hz, 1H), 6.14–6.10 (m, 1H), 5.32–5.23 (m, 1H), 5.08–4.97 126.7, 122.7, 115.6, 115.4, 113.2, 107.8, 88.2, 86.0, 85.8, 85.7,
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8
5.2, 84.8, 70.3, 69.9, 64.1, 63.5, 54.9, 38.3, 26.5, 15.5, 15.4. purified using a Sep-pak C18 cartridge. The DMTr group on
+
+
ESI-MS calcd for C H F N NaO [M + Na] 903.3023; found the 5′-end was removed in the cartridge using 2% TFA in
4
7
46
2
4
11
9
03.3009.
′-O-(4,4′-Dimethoxytrityl)-5-[(Z)-4-(4-((pivaloyloxy)methoxy) in water and then further purified by anion exchange HPLC
benzylidene)-2-methyl-5-oxo-imidazolin-1-yl]-2′-deoxyuridine (DNAPac PA100 9 × 250 mm) or reverse phase HPLC (XBridge
′-(2-cyanoethyl N,N-diisopropylphosphoramidite) (10a). C18 5 µm 4.6 × 150 mm).
water. The DNA mixture was eluted by using 25% acetonitrile
5
3
Compound 9a (50 mg, 0.059 mmol) was co-evaporated with dry
pyridine, dry toluene and dry dichloromethane respectively. Oligonucleotide synthesis containing 10a or 10b (ODN-2-Y)
Then, compound 9a was dissolved in dry dichloromethane
DNA was firstly synthesized until 10a or 10b was incorporated
(
(
2 mL) and diisopropylamine (5.6 μL, 0.040 mmol), 1H-tetrazole
2.7 mg, 0.039 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraiso-
by using the DNA/RNA synthesizer. After the synthesis, the
resin was dried under reduced pressure. 10a or 10b (15 µmol)
was co-evaporated 8 times with anhydrous acetonitrile and dis-
solved in anhydrous acetonitrile (150 µL) as solution A. 1H-
Tetrazole (75 µmol, 5.25 mg) was dried under reduced pressure
and then dissolved in anhydrous acetonitrile (150 µL) as solu-
tion B. Solutions A and B were added into the resin and then
reacted for 45 min. After the reaction, the resin was washed
with anhydrous acetonitrile and capped using 5% phenoxyace-
tic anhydride in THF–pyridine and 10% 1-methylimidazole in
THF for 2 min. After the reaction, the resin was washed with
propylphosphordiamidite (21.4 μL, 0.067 mmol) were added
respectively. The reaction mixture was stirred at room tempera-
ture for 16 h. Then, it was diluted with dichloromethane
(20 mL) and washed with water, saturated aq. NaHCO
3
, and
brine. The organic layer was dried over Na SO and evaporated.
2
4
The crude product was purified by N-60 silica gel chromato-
graphy with n-hexane–ethyl acetate (50 : 50, v/v). The fractions
containing the target compound were concentrated (22.7 mg)
and directly applied for solid phase DNA synthesis.
5
′-O-(4,4′-Dimethoxytrityl)-5-[(Z)-4-(3,5-difluoro-2-methyl-4-
(pivaloyloxy)methoxy)benzylidene)-5-oxo-imidazolin-1-yl]-2′-
deoxyuridine 3′-(2-cyanoethyl N,N-diisopropylphosphoramidite)
10b). Compound 9b (31 mg, 0.035 mmol) was co-evaporated
acetonitrile and then oxidation was performed using 0.02 M I
2
(
in THF–pyridine–water for 1 min. The resulting resin was
washed with acetonitrile, dried, and re-packed into a synthesis
column for automated DNA synthesis. The rest of the synthesis
was performed according to the general oligonucleotide syn-
thesis protocol shown above except for the cleavage and
deprotection conditions. Cleavage and deprotection were
performed by using 28% aqueous ammonia solution for 2 h at
ambient temperature for ODN-2-Y.
(
with dry pyridine, dry toluene and dry dichloromethane
respectively. Then, compound 9b was dissolved in dry di-
chloromethane (0.41 mL) and diisopropylamine (3.24 μL,
0
.023 mmol), 1H-tetrazole (1.6 mg, 0.023 mmol) and 2-cyano-
ethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (14.5 μL,
.045 mmol) were added respectively. The reaction mixture
0
was stirred at room temperature for 2.5 h, then the same
amount of diisopropylamine, 1H-tetrazole, and 2-cyanoethyl-
N,N,N′,N′-tetraisopropylphosphordiamidite was added to the
reaction mixture and stirred for an additional 1.5 h to com-
plete the reaction. Then the reaction mixture was diluted with
dichloromethane (20 mL) and washed with water. The organic
Measurements of UV and fluorescence spectra of nucleoside
The UV spectra were measured at ambient temperature using
3
(
1
0 μM solution of 8a and 8b in methanol or glycerol–methanol
95 : 5, v/v). The fluorescence spectra were measured using
00 μM solution of 8a and 1 μM solution of 8b prepared using
the same solvents. The excited wavelengths were 380 nm for 8a
and 430 nm for 8b.
2 4
layer was dried over Na SO and evaporated. The crude
product was purified by N-60 silica gel chromatography with
n-hexane–ethyl acetate (50 : 50, v/v). The fractions containing
the target compound was concentrated (29.5 mg) and directly
applied for solid phase DNA synthesis.
Measurements of UV and fluorescence spectra of
oligonucleotides
The oligonucleotide concentrations were determined by the
1
8
General oligodeoxynucleotide synthesis
standard method by assuming that the molar absorption
coefficients of 8a and 8b were identical to that of thymidine.
The solutions of 2 μM of each oligodeoxynucleotide in 10 mM
sodium cacodylate containing 500 mM NaCl and 10 mM
The synthesis of oligodeoxynucleotides was carried out using
an automated DNA/RNA synthesizer (ABI 392 DNA/RNA
synthesizer or nS8-II) using a general protocol of ultra-mild
DNA synthesis (deblocking reagent: 3% trichloroacetic acid in
MgCl were prepared. Before the measurements, the solutions
2
were heated at 85 °C for 5 min, and then cooled to ambient
temperature. The solutions were cooled at 5 °C for 10 min and
used for the measurements.
2 2
CH Cl , activator: 0.25 M 5-benzylthio-1H-tetrazole in anhy-
drous MeCN, capping reagents: 5% phenoxyacetic anhydride
in THF–pyridine and 10% 1-methylimidazole in THF, oxidizing
reagent: 0.02 M I
performed on the 1 µmol scale except for ODN-2-Y, which was
2
in THF–pyridine–water). The synthesis was
Measurements of UV-melting curves
performed on the 0.2 µmol scale. The synthesized DNA was The solutions of 2 μM of each oligodeoxynucleotide in 10 mM
reacted with 28% aqueous ammonia solution for 12 h at sodium cacodylate containing 500 mM NaCl and 10 mM
2
ambient temperature for deprotection and cleavage from resin. MgCl were prepared. The solutions were heated at 85 °C
The elution was evaporated under reduced pressure and then for 5 min, and then cooled to 5 °C. The solutions were heated
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at a rate of 1 °C min− . During the melting reactions, the UV
1
7 (a) J. S. Paige, K. Y. Wu and S. R. Jaffrey, Science, 2011, 333,
642; (b) C. L. Walker, K. A. Lukyanov, I. V. Yampolsky,
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absorbances at 260 nm were monitored.
2
015, 27, 64; (c) U. Wenge and H.-A. Wagenknecht,
Acknowledgements
Synthesis, 2011, 502.
This work was supported by JSPS KAKENHI Grant Number
8 S. R. Meech, Chem. Soc. Rev., 2009, 38, 2922.
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