Phenyl-imidazolo-cytidine analogues: Structure-photophysical activity relationship and ability to detect single dna mismatch

Article abstract of DOI:10.1021/jo5011944

To expand the arsenal of fluorescent cytidine analogues for the detection of genetic material, we synthesized para-substituted phenyl-imidazolo-cytidine (^PhImC) analogues 5a-g and established a relationship between their structure and fluoresce

Full text of DOI:10.1021/jo5011944

Article
pubs.acs.org/joc
Phenyl-imidazolo-cytidine Analogues: Structure−Photophysical
Activity Relationship and Ability To Detect Single DNA Mismatch
Marina Kovaliov, Michal Weitman, Dan Thomas Major, and Bilha Fischer*
Department of Chemistry, Gonda-Goldschmied Medical Research Center and the Lise-Meitner-Minerva Center of Computational
Quantum Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
S
* Supporting Information
ABSTRACT: To expand the arsenal of fluorescent cytidine
analogues for the detection of genetic material, we synthesized
para-substituted phenyl-imidazolo-cytidine (PhImC) analogues
5a−g and established a relationship between their structure and
fluorescence properties. These analogues were more emissive
than cytidine (λem 398−420 nm, Φ 0.009−0.687), and excellent
−
correlation was found between Φ of 5a−g and σp of the
substituent on the phenyl-imidazolo moiety (R2 = 0.94).
Calculations suggested that the dominant tautomer of PhImC in
methanol solution is identical to that of cytidine. DFT
calculations of the stable tautomer of selected PhImC analogues
suggested a relationship between the HOMO−LUMO gap and
Φ and explained the loss of fluorescence in the nitro analogue.
Incorporation of the CF3-PhImdC analogue into a DNA probe resulted in 6-fold fluorescence quenching of the former. A 17-fold
reduction of fluorescence was observed for the G-matched duplex vs ODN(CF3-PhImdC), while for A-mismatched duplex, only a
2-fold decrease was observed. Furthermore, since the quantum yield of ODN(CF3-PhImdC):ODN(G) was reduced 17-fold vs
that of a single strand, whereas that of ODN(CF3-PhImdC):ORN(G) was reduced only 3.8-fold, ODN(CF3-PhImdC) appears to
be a DNA-selective probe. We conclude that the ODN(CF3-PhImdC) probe, exhibiting emission sensitivity upon single
nucleotide replacement, may be potentially useful for DNA single nucleotide polymorphism (SNP) typing.
ties of the natural nucleosides were significantly improved by
extending the purine/pyrimidine π-system.1−4,12−18 Several
fluorescent pyrimidine analogues with extended fluorophores
have been reported (Figure 1). For example, imidazolo-
cytidine, 1, exhibited a longer emission wavelength and higher
quantum yield as compared to cytidine (λem 390 nm; Φ 0.051
vs λem 303 nm; Φ 0.00008).19 Emission properties of
compound 1 enabled studies on permeation and inhibition of
human nucleoside transporters.20−22
INTRODUCTION
■
Fluorescence-based methods have been instrumental in
advancing biophysical assays and high throughput screening
techniques, particularly for identification and quantification of
nucleic acids.1−4 For the latter application, nucleic acids are not
useful, as native nucleobases are practically nonemissive.5−7
Hence, conjugation of oligonucleotide probes with various dye
molecules has become a common procedure. However, most
dye molecules are large and hydrophobic and have limited
water solubility. Furthermore, a large dye molecule attached to
a nucleotide alters the efficiency of enzymatic incorporation
into DNA/RNA, resulting in different levels of labeling and
prohibits quantification of nucleic acids. End labeling with
common fluorescent tags (e.g., cyanine dyes) is synthetically
simple yet is far from being optimal since end labels are not
necessarily sensitive to remote binding events and such
fluorophores are large, charged, and frequently noninnocent.8
Currently, there is a great need for new, improved fluorescent
nucleoside analogues with minimal structural perturbation yet
with high quantum yields and relatively long emission
wavelength.
Figure 1. Fluorescent fused-cytidine (C) and 2′-deoxycytidine (dC)
nucleoside analogues: imidazolo-C analogue, 1; pyrrolo-dC, 2;
phenylpyrrolo-dC, 3; thiophene-yl-pyrrolo-dC, 4.
Over the past decade, a number of intrinsically fluorescent
nucleoside analogues that can replace natural DNA bases as
oligonucleotide probes have been reported as alternatives to
dye-substituted nucleosides.9−11 Thus, the fluorescent proper-
Received: May 29, 2014
Published: July 3, 2014
© 2014 American Chemical Society
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Pyrrolo-2′-deoxycytidine, 2 (λabs 353 nm, λem 448 nm, Φ
0.038 in Tris buffer) and phenyl-pyrrolo-2′-deoxycytidine, 3
(λabs 360 nm, λem 465 nm, Φ 0.31 in phosphate buffer), have
found use as fluorimetric reporting groups.23 The substitution
of the phenyl group on the pyrrole ring not only yielded a
much more emissive compound but also contributed to the
stabilization of the duplexes.11,24,25 Pyrrolo-2′-deoxycytidine
was also extended via conjugation to a thiophene ring, 4,
resulting in improved fluorescence properties (λabs 369 nm, λem
469 nm, Φ 0.41 in H2O).26
Recently, we have reported on new fluorescent pyrimidine
nucleosides, phenyl-imidazolo-cytidine analogues (PhImC) 5a−
d, based on fusion of the imidazolo moiety to cytidine and
further conjugation of the para-phenyl substituted ring (Figure
2).27 These compounds fluoresce in the blue region and exhibit
quantum yields and maximum emission that are 7000-fold
larger and ca. 110 nm red-shifted vs that of cytidine.
Specifically, we report on the synthesis and photophysical
properties of a series of para-substituted-PhImC analogues, 5a−
g. We analyzed the relationship between the fluorescence
properties of several PhImC analogues and the nature of the
substituents as reflected by several Hammet constants. In
addition, we analyzed this relationship by density functional
theory (DFT) calculations. In particular, we identified the most
stable tautomers of the PhImCs, compared their computed and
experimental absorption spectra, and correlated the exper-
imental Φ values with the computed HOMO−LUMO gaps. In
addition, we synthesized and studied the fluorescent properties
of CF3-PhIm-cytosine, 6. Based on the established structure−
activity relationship, we synthesized p-CF3-imidazolo-2′-deoxy-
cytidine (CF3-PhImdC), 7, and incorporated it into DNA.
Finally, we evaluated the influence of 7 on duplex stability and
its ability for single mismatch detection.
Substituted PhImdC analogues could be used in a wide range
of applications, including but not limited to single nucleotide
polymorphism (SNP) detection.31−35 The novel compounds
may be employed as useful reporters of sequence and structure
of nucleic acids, e.g., the resolution of hybridization events,36
folding,37 conformational change,38 as well as enzyme action.39
In addition, the novel analogues may be used for micro-
environmental probing studies for detection of nucleobase
damage,40 depurination/depyrimidation,41 and base flipping.37
RESULTS AND DISCUSSION
■
Synthesis of PhImC and Its Derivatives. We have
previously reported the synthesis of para-substituted PhImC
analogues 5a−d, which exhibited improved photophysical
properties as compared to imidazolo-cytidine, 1.27 Here, we
targeted the synthesis of additional analogues representing a
wider chemical space (5a−g).
Synthesis of analogues 5a−g was achieved by condensation
of per-benzoylated 5-aminocytidine, 8,42,43 with para-substi-
tuted benzaldehyde 9a−g, in the presence of p-TsOH (20 mol
%), in DMF at 80 °C, to give 10a−g in 36−47% yield.44
Addition of 1 M NaOH in MeOH, at room temperature for 1
h, yielded 5a−g in 80−85% yield (Scheme 1).
Figure 2. Phenyl-imidazolo-cytosine analogues studied here.
These fluorescent nucleoside derivatives have been well
characterized and an effect of the phenyl ring substituent on
fluorescence properties of the parent compound was observed
(e.g., 5d; λabs 346 nm; λem 411 nm; Φ 0.617 vs 5a; λabs 342 nm;
λem 402 nm; Φ 0.198 in MeOH), but no clear dependence of
their fluorescence on the electronic character of the substituent
was established. These data led us to extend our research on
structure−activity relationship of the imidazolo-cytidine series.
Although there are numerous reports on the structural
dependence of fluorescence properties,28−30 i.e., λem and Φ
values, there are a limited number of reports on the effect of
substituents on the fluorescence of nucleoside analogues.
Recently, we have explored the relationship between the nature
of substituents on cinnamyl moiety in 8-(substituted
cinnamyl)adenosine and the fluorescence properties of the
latter. We found excellent correlation between λab and Φ of the
analogues and the computed HOMO and LUMO energy levels
of 8-vinyl-9-methyladenine (fluorescent molecule) and sub-
stituted toluene rings (fluorescence modulators).18
Scheme 1. Synthesis of PhImC Analogues 5a−g
Here, we first targeted the establishment of relationship
between the chemical nature of the substituent on the phenyl-
imidazolo moiety and the fluorescence characteristics of the
resulting imidazolo-cytidine analogues 5a−g. Next, we selected
one of the most promising fluorescent nucleosides, CF3-PhImC,
5d, and investigated the effect of its incorporation into an
oligonucleotide on its photophysical properties.
To examine the effect of ribose and 2′-deoxyribose on the
photophysical properties of PhImC analogues we synthesized
the cytosine analogue, 6, related to the highly emissive
compound 5d (Scheme 2).27 5-Nitrocytosine, 12, was readily
prepared from cytosine 11 in the presence of fuming nitric
acid45 and was then subjected to hydrogenation to provide 5-
aminocytosine, 13.46 Condensation of 13 with p-CF3-
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Scheme 2. Synthesis of p-CF3-PhIm-cytosine
centrated solution of 20 that was diluted with CH3CN to a
formal concentration of 0.1 M and immediately used for the
automated synthesis of the oligonucleotide. This procedure
allowed us to carry out the synthesis of the DNA probe without
any loss of the phosphoramidite 20 in the purification step.
Absorption and Fluorescence Spectra. The photo-
physical properties of para-substituted PhImC compounds 5a−g
were studied in methanolic solutions. The fluorescence
quantum yields were determined using quinine sulfate in 0.1
M H2SO4 (λex 350 nm, λem 446 nm, Φ 0.54) as a reference.49
The concentrations of the samples were in the linear range in
which the Beer−Lambert law applies, and the optical density
(OD) was less than 0.05 to avoid the inner filter effect.50 The
fluorescent properties of analogues 5a−g are summarized in
Table 1. The absorption spectra, fluorescence intensity, and
normalized fluorescence are presented in Figures 3 and 4. The
conjugation of the para-substituted phenyl ring to the parent
imidazolo-cytidine, 1 (λex 322 nm, λem 390 nm, Φ 0.051 in
methanol),20−22 indeed improved the photophysical properties,
e.g. 5d (λex 346 nm, λem 411 nm, Φ 0.617) and 5g (λex 350 nm,
λem 420 nm, Φ 0.687).
The various p-substituents in 5a−g had a minor effect on the
wavelength of the absorption maxima, which were in the range
of 342−350 nm, except for the −NO2, analogue, 5e (λab 368
nm). The electron-withdrawing substituents evoked more
efficiently bathochromic shift compared to electron-donating
substituents (e.g., −NO2 analogue, 5e, vs −OCH3 analogue, 5c,
and the parent compound, 5a: 368 and 347 vs 342 nm).
Although the absorption maxima were rather insensitive to
the alteration of the substituents on the phenyl ring, the
emission maxima were more affected by the substituents
(Figure 4). The compounds having the largest Stoke’s shift are
5d and 5g (65 and 70 nm), and the smallest shift was observed
for 5e (30 nm) (Table 1).
benzaldehyde in the presence of p-TsOH afforded compound 6
in 55% yield.
Next, we prepared CF3-PhImdC phosphoramidite, 20, to be
incorporated into an oligonucleotide (Scheme 3). First, 2′-
deoxyuridine, 14, was acetylated and then nitrated with
nitrosonium tetrafluoroborate in dimethylformamide to give
15.47 Next, compound 15 was transformed into the
corresponding cytidine analogue 16 using triisopropylbenzene-
sulfonyl chloride (TPSCl) followed by treatment with
NH4OH.48 After hydrogenation of 16, protected 5-amino-2′-
deoxycytidine, 17, was reacted with p-CF3-benzaldehyde in the
presence of p-TsOH to afford compound 18 in 50% yield.
Deacetylation of 18 with sodium methoxide afforded nucleo-
side 7 in 80% yield. The corresponding phosphoramidite 20
was obtained in two steps: 5′ protection using 4,4′-
dimethoxytrityl chloride in anhydrous pyridine followed by
phosphorylation with 2-cyanoethyl N,N,N′,N′-tetraisopropyl-
phosphorodiamidite in the presence of tetrazole. This gave,
after filtration of diisopropylammonium tetrazolide, a con-
The longest λem was observed for analogue 5g bearing a
−CN group (420 nm). Generally, electron-withdrawing
a
Scheme 3. Synthesis of CF3-PhImdC Phosphoramidite 20
a
Reaction conditions: (a) Ac2O in pyridine, overnight, rt; (b) NOBF4 in DMF, 30 min, rt; (c) TPSCl, DMAP, TEA in CH3CN, 3 h, rt; (d) 33%
NH4OH in CH3CN, 2 h, rt; (e) 10% Pd/C, 1.5 atm, THF, AcOH, 6 h, rt; (f) p-CF3-benzaldehyde, p-TsOH in DMF, 4 h, 80 °C; (g) NaOMe in
MeOH, 30 min, rt; (h) DMTCl in pyridine, 2.5 h, rt; (i) N,N,N′,N′-tetraisopropylphosphorodiamidite, tetrazole in CH3CN, 45 min, rt.
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Table 1. σ Values and Photophysical Properties of Analogues 5a−g in MeOH
51
52
+
53
−
a
R
σp
σp
σp
λab (nm)
λem (nm)
Stoke’s shift (nm)
Φ
εmax (cm−1 M−1
)
5a
5b
5c
5d
5e
5f
H
0
0
0
342
344
347
346
368
347
350
402
403
405
411
398
404
420
60
59
58
65
30
57
70
0.215
0.198
0.233
0.617
0.009
0.312
0.687
7400
8800
CH3
OCH3
CF3
NO2
F
−0.170
−0.268
0.541
−0.311
−0.778
0.612
−0.17
−0.26
0.65
9900
11250
13100
10540
14150
0.778
0.790
1.27
0.062
−0.073
0.659
−0.03
1.00
5g
CN
0.660
a
Quantum yields were determined using quinine sulfate as a standard.
various σ values of the substituents. Previous reports indicated a
linear correlation between sigma values and fluorescent
properties of several organic compounds.54−56 Thus, we
attempted to correlate quantum yields and emission data with
the electronic properties of the phenyl-imidazolo- moiety in
analogues 5a−g as reflected by the σ Hammett constant, σp, of
the phenyl substituents.
The σ constant σ− is designated in the case where negative
charge buildup in the transition state occurs, and the rate of the
reaction is consequently accelerated by electron-withdrawing
groups.57 On the other hand, σ+ constants were developed for
substituents conjugated with a reaction center which could
effectively delocalize a positive charge. Hence, an electron-
donating group will accelerate the rate of the reaction by
resonance stabilization.58 To elucidate the formation of any
charge in PhImC system in the excited state, we explored the
Figure 3. UV−vis spectra of para-substituted PhImC analogues in
MeOH (5.5 μM).
+
−
possible correlation of σp , σp constants of the substituents in
5a−g with the quantum yields and emission maxima of these
analogues (Table 1).
substituents, e.g., −CF3 and −F, led to a bathochromic shift of
the emission maxima (see 5d and 5f, Table 1). Analogues 5d
and 5g bearing a −CF3 or −CN group exhibited the highest
quantum yields (0.671 and 0.687, respectively), exceeding also
those of the related phenyl-pyrolo-cytidine analogues.11,25
Although analogue 5e bears the most electron-withdrawing
group, −NO2, in this series of compounds, it was practically
nonfluorescent with quantum yield of 0.009. Analogues 5b and
5c bearing an electron-donating group, −CH3 or −OCH3,
exhibited quantum yields of 0.198 and 0.233, respectively,
similar to the parent compound, 5a (Φ 0.215).
The dependencies of quantum yields or emission wave-
+
−
lengths on Hammet substituent constants (σp, σp , σp ) are
given in Figure 5. An excellent correlation was found between
the quantum yield values and σp− constants (R2 = 0.94) (Figure
5C), similar to that of σp (Figure 5A) and much better than
+
correlation with σp constants (Figure 5B). The data point for
the −NO2 substituted analogue 5e, significantly deviates from
−
all correlation graphs. The correlation of Φ values with σp
constants implies that the electronic density redistribution in
the excited states of the molecules generates partial negative
charge.
Next, we analyzed the substituent-dependent photophysical
properties of analogues 5a−g by seeking first correlation with
Figure 4. Fluorescence spectra (A) and normalized fluorescence spectra (B) for the series of para-substituted PhImC analogues in methanol (3 μM),
λex 350 nm.
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+
−
Figure 5. Correlation of quantum yields (A−C) or emission maxima (D−F) with σ constants (σp, σp , σp ).
A plot of emission wavelengths vs σp− (Figure 5F), indicated
a linear dependence (R2 = 0.87). Again the −NO2-substituted
analogue is an exception to this correlation. Previously, it was
found that fluorescence decreases or even disappears upon
introduction of a nitro group to fluorescent compounds.18,59−61
The mechanism of quenching was related to the greatly
lowered LUMO energy levels of the aromatic compounds due
to the strong electron-withdrawing effect of nitro groups.62 This
finding is echoed in our results.
Effect of the Glycosyl Moiety on Photophysical
Properties of 5d. In order to investigate the effect of the
ribose ring on the fluorescence properties of phenyl-imidazolo-
cytidine compounds, we synthesized imidazolo-cytosine and 2′-
deoxy-cytidine analogues. Specifically, we synthesized analogues
6 and 7 because of the promising photophysical properties of
Figure 6. Fluorescence spectra for p-CF3-substituted PhImC analogues
(3 μM) in methanol (except for 6, which was measured also in water
at the same concentration).
CF3-PhImC, 5d. Their quantum yields, absorption, and emission
maxima are summarized in Table 2, and emission spectra are
shown in Figure 6.
Table 2. Fluorescent Properties of PhImC Analogues 5d, 6,
and 7
that in methanolic solution. λem of 6 in water shifted to 416 nm
vs 406 nm in methanol, and quantum yields were 0.712 in
water vs 0.736 in methanol. We suggested that hydrogen
bonding to 6 might play an important role in lowering the
quantum yield. In methanol the less polar solvent, that less
suited for hydrogen bonding with water, may explain the
decrease of the quantum yield and the shift of λem. When we
compared the fluorescence properties of 5d to 2′-deoxycytosine
analogue, 7, as expected we observed very small change, due to
the additional OH group.
a
analogue
solvent
λab (nm) λem (nm)
Φ
ε (cm−1M−1
)
5d
6
methanol
water
346
332
344
347
411
416
406
405
0.617
0.712
0.736
0.642
11250
9100
6
methanol
methanol
9350
7
10850
a
Quantum yields were determined using quinine sulfate as a standard.
The ribose and 2′-deoxyribose rings reduced the quantum
yield of the base analogue 6 in methanol by up to 17% (0.617
and 0.642 vs 0.736). However, only a minor change was
observed in the emission wavelength due to the ribose/2′-
deoxyribose ring (411 and 405 nm for 5d and 7 vs 406 nm for
6).
The improved photophysical properties of 6 may suggest its
potential use for the synthesis of fluorescent PNA (peptide
nucleic acids) probes. In addition, the promising fluorescent
properties of the 2′-deoxy-cytidine analogue 7 encouraged us to
incorporate it into an oligonucleotide and to evaluate the
potential of the latter as a probe for SNP detection (see below).
Theoretical Calculations. To better understand the effect
of the substituents on the fluorescent properties of PhImC
Due to the solubility of 6 in water fluorescence spectrum was
also measured in an aqueous solution, showing small changes vs
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compounds, we explored the correlation between Φ of the
latter compounds and their HOMO−LUMO energy gaps.
Specifically, we studied model compounds N-Me-R-PhImC
21a−e and their four possible tautomers A−D (Figure 7). We
more stable even in solution, and are expected to be the
dominant forms.
Next, we explored the correlation between Φ and HOMO−
LUMO energy gap of model compounds 21a−e.63 HOMO and
LUMO orbital energies were calculated for the ground state in
methanol. All calculations were performed on the most stable
tautomer in methanol (tautomer A for −H, −CF3, −CH3 and
tautomer C for −NO2).
Table 4 presents experimental and computed absorption
wavelengths and energies of HOMO and LUMO orbitals for
compounds 21a−e. In general, absorbance is related to the gap
between HOMO and LUMO levels, and indeed the electronic
transitions obtained from time-dependent (TD) DFT calcu-
lations are mainly composed of HOMO → LUMO transition
(Table S41, Supporting Information). Yet, additional excita-
tions also contribute to the first excited state. The oscillator
strengths suggest that these are allowed transitions.
Figure 7. Four possible tautomers (A−D) of N-Me-R-PhImC, 21a−e.
Derivatives 5d and 5e (−CF3 and −NO2 substituents,
respectively) exhibited extremely high and low Φ values,
respectively (Table 1). In an attempt to understand the effect of
the substituents on the fluorescence of the nucleobases, one
may analyze the frontier orbital energy levels and their spatial
distribution. In a previous study on a series of 8-(substituted
cinnamyl)-adenosine derivatives, we have shown that the
electron density of LUMO orbitals of nitro-compounds is
largely localized on the benzene ring, due to the strong electron
withdrawing effect of the substituent.18 Here, we observed the
same pattern of large localization of electron density on the
benzene ring in LUMO of derivative 21e due to the nitro group
(Figure 8).62
The small energy gap of the HOMO−LUMO levels (Table
4) of the nitro derivative leads to a possible quenching channel
of the fluorescence. Thus, we suggest that the HOMO−LUMO
gap in model compound 21e, which is smaller than for all other
model compounds, explains its Φ value which was found to be
an order of magnitude lower than that of all other compounds.
Incorporation of PhImC Analogue 7 into an Oligonu-
cleotide. To explore potential applications of PhImC
compounds, we studied the photophysical properties of
derivative 5d within a DNA environment. We selected p-CF3-
substituted analogue, 5d, which showed one of the highest
quantum yields and relatively long absorption and emission
wavelengths (λex 346 nm, λem 411 nm, Φ 0.617). Previously, we
showed by NMR studies, that analogue 5d preserves cytidine
natural hydrogen bond pattern with guanosine.27 Here we
extended our studies and explored the tautomeric equilibrium
of 5d in methanol solution by calculations at M062X/6-311+
+G(d,p) level (Table 3). Tautomer A, which resembles cytidine
in Watson−Crick base-pairing with guanosine, was found to be
the most stable among the possible tautomers (Figure 7).
However, tautomer C, which resembles uridine in Watson−
calculated properties of models 21a−e containing N-methyl
group instead of a ribose group since the ribose group does not
significantly affect the fluorescent properties of the modified
base (Table 2).
The calculations focused on five substituents of PhImC that
represent different electronic effects: strong electron with-
drawing groups (-NO2 and −CF3), electron-donating groups
(−OMe and −Me), and the parent compound.
Since PhImC has four possible tautomers, we first calculated
their relative stabilities to determine their respective pop-
ulations. We optimized each tautomer in the gas phase. For all
substituents, tautomer C was the most stable. Tautomer A has
higher energy than tautomer C (up to 2.6 kcal/mol for the
−NO2 substituent, 21e). Tautomers B and D are destabilized
by about 10 kcal/mol as compared to tautomer C (Table 3).
This could possibly be due to repulsion between 1,3-nitrogen
lone-pair electrons. Optimization of the tautomers in a
continuum methanol solvent revealed that tautomer A is
expected to be the dominant tautomer in methanol, while
tautomer C is less stable than A by up to 1.8 kcal/mol (except
for 21e, where a mixture of tautomers A and C is expected).
Tautomers B and D are the least stable forms also in
methanolic solution (Table 3).
In addition, we computed the dipole moments for each
tautomer both in gas and solution phases in an attempt to
understand the above-mentioned effect of the solvent on
tautomeric equilibrium. In all cases, tautomer B had the largest
dipole moment, ca. 9−10 D, resulting in considerable
stabilization of this tautomer in a polar solvent (methanol).
This in turn induces a solvent effect on the tautomeric
equilibrium (Table S41, Supporting Information). Indeed, the
solvent induced change in the relative tautomerism energy is ca.
9 kcal/mol for all B analogues, resulting in similar energies for
tautomers A, B, and C. Nonetheless, tautomers A and C remain
Table 3. Relative Energies (kcal/mol) in the Gas Phase and in Methanol Solution for 21a−e Obtained from Calculations
Performed at M062X/6-311++G(d,p) level
gas phase
methanolic solution
R
A
B
C
D
A
B
C
D
21a
21b
21c
21d
21e
H
0.47
0.72
1.38
2.09
2.59
9.54
9.22
0.00
0.00
0.00
0.00
0.00
10.46
10.55
10.58
10.06
9.85
0.00
0.00
0.00
0.00
0.00
2.71
2.65
1.28
3.53
3.11
1.45
1.81
1.28
0.95
0.18
9.55
9.95
9.29
8.95
7.75
CH3
OCH3
CF3
9.70
11.18
12.11
NO2
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Table 4. Absorption Wavelengths (λabs), Oscillator Strengths (f), Molecular Orbital Energies, and Molecular Orbital Energy
Gaps, Obtained from Calculations Performed at the M062X/6-311++G(d,p) Level in Methanol Solution
analogue
R
λabs (nm) expt
λabs (nm) calcd
f
HOMO (eV)
LUMO (eV)
Δ (eV)
Φ expt
21a
21b
21c
21d
21e
H
342
344
347
346
368
314
314
317
317
345
0.526
0.571
0.620
0.567
0.764
−0.2715
−0.2695
−0.2648
−0.2763
−0.2777
−0.0498
−0.0472
−0.0450
−0.0579
−0.0855
0.220
0.222
0.220
0.218
0.190
0.215
0.198
0.233
0.617
0.019
CH3
OCH3
CF3
NO2
ing. These results imply that 5d may be a good candidate for
mismatch detection.
Next, we incorporated CF3-PhImdC, 7, into a 15-mer
oligonucleotide (Figure 9). Solid-phase oligonucleotide syn-
thesis was performed by employing phosphoramidite 20 and
standard building blocks. We incorporated this monomer at the
center of oligodeoxynucleotide (ODN) in order to study its
photophysical properties both in a single stranded DNA and in
the corresponding duplexes with either DNA or RNA
(oligoribonucleotide (ORN)). Furthermore, we investigated
the influence of 7 on the stability of both complementary and
noncomplementary (mismatched) duplexes. For this purpose,
we used the oligonucleotides shown in Figure 9.
The absorption and fluorescence spectra of ODN-
(CF3‑PhImdC) probe and the corresponding duplexes were
measured at 25 °C at 2 μM concentration in PBS buffer at pH
7.4. ODN(CF3‑PhImdC) exhibited an absorption band at 343
nm similar to 7, and upon duplex formation the absorption
maxima shifted slightly to a longer wavelength (350 nm)
(Figure 10).
The emission spectrum of ODN(CF3‑PhImdC) shows a
signal at 406 nm, however, its quantum yield decreased 6-fold
as compared to that of 2′-deoxy nucleoside 7 (0.102 vs 0.642)
(Table 5). In the duplexes of ODN(CF3‑PhImdC), with
matched and mismatched strands, the fluorescence of 7 was
strongly dependent on the purine base paired to it. Thus, the
quantum yield of mismatched ODN(CF3‑PhImdC):ODN(A)
was reduced 2-fold vs that of ODN(CF3‑PhImdC), while
mismatched ODN(CF3‑PhImdC):ODN(C)/(T) resulted in
6.5-fold reduction of fluorescence. The most impressive effect
was observed for matched ODN(CF3‑PhImdC):ODN(G),
where the fluorescence of the duplex was reduced 16.5-fold
vs that of the single strand, ODN(CF3‑PhImdC). Interestingly,
the corresponding duplex with RNA, ODN-
(CF3‑PhImdC):ORN(G), showed a 11.5-fold higher quantum
yield vs its DNA counterpart (0.027 vs 0.006). These
observations suggest that ODN(CF3‑PhImdC) may be used as
a sensitive, DNA-selective, SNP probe.
Figure 8. LUMO orbitals of compounds 21a, 21d, and 21e: (A) 21a
(−H) (B) 21d (−CF3), (C) 21e (−NO2).
Crick base-pairing with adenosine, was also quite stable with
relative energy of 0.95 kcal/mol. To obtain experimental
support for the compatibility of 5d to cytidine in DNA, we
prepared phosphoramidite monomer 20 for incorporation into
DNA probe.
Prior to the spectroscopic studies of DNA modified with
CF3-PhImdC, 7, we wanted to investigate the potential
quenching of 5d in the presence of various nucleobases. For
this purpose we performed Stern−Volmer titration of 5d with
all 5′-monophosphate nucleotides (NMPs) (Figure S3,
Supporting Information). We observed minor fluorescence
quenching of 5d with CMP and no quenching with UMP. AMP
and particularly GMP induced significant fluorescence quench-
Figure 9. Sequences of matched and mismatched ODNs used for study of ODN(CF3‑PhImdC).
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indicate that ODN(CF3‑PhImdC) is appropriate for discrim-
ination between natural base-pairing and mismatched pairing.
Furthermore, ODN (CF3−PhImdC) was found to be highly
selective for DNA vs RNA.
CONCLUSIONS
■
We reported here on the synthesis of a series of fluorescent
PhImC analogues. Next, we established a correlation between
the nature of para-substituents of the phenyl ring and the
fluorescent properties of these analogues, 5a−g. A linear
dependence was observed between Φ or emission wavelength
values and σp values (R2 = 0.94 and 0.87, respectively).
−
DFT calculations of model compounds 21a−e helped
identify the most stable tautomers in methanol solution, and
provided rational for the low fluorescence of the nitro
derivative. The calculations suggest two dominant tautomers
(A and C) in methanol solution. Moreover, the computed
absorption lines are in fair agreement with experiment, and the
low fluorescence of the nitro compound is possibly due to the
narrow HOMO−LUMO energy gap, which could open a
nonradiative decay channel.
Figure 10. Fluorescence spectra for CF3‑PhImdC-labeled ODN and the
corresponding duplexes. All measurements were carried out at 2 μM in
PBS buffer (pH 7.4) at room temperature, λex 350.
Table 5. Photophysical Properties of ODN(CF3‑PhImdC)
p-CF3−PhImdC, 7, which proved to be one of the most
promising fluorescent probes in ImC series, was incorporated
into an oligonucleotide. This fluorescent cytidine analogue
exhibited 8025-fold higher quantum yield (Φ 0.642) as
compared to cytidine. Analogue 7 did not interfere with the
hybridization process (ΔTm = 1 °C between ODN-
(CF3‑PhImdC):ODN(G), and the native duplex), thus
supporting the predominance of tautomer A of analogue 7.
Moreover, A 17-fold reduction of fluorescence was observed for
the G-matched duplex vs ODN(CF3‑PhImdC), while for A-
mismatched duplex, only a 2-fold decrease was observed.
Usually, the most common mutation is the transition that
exchanges a purine for a purine, A to G or vice versa. Hence,
our data suggest that ODN(CF3‑PhImdC) probe can be used to
detect DNA single mismatch. Furthermore, since quantum
yield of ODN(CF3‑PhImdC):ODN(G) was reduced 17-fold vs
that of single strand, whereas quantum yield of the
corresponding ODN(CF3‑PhImdC):ORN(G) was reduced
only 3.8-fold, ODN(CF3‑PhImdC) appears to be a DNA-
selective probe.
a
and the Corresponding Duplexes
λabs
(nm)
λem
(nm)
Tm
(°C)
Φ
single strand ODN(CF3‑PhImdC)
duplex with ODN(G)
duplex with ODN(A)
duplex with ODN(C)
duplex with ODN(T)
duplex with ORN(G)
unmodified DNA duplex
343
350
348
347
348
348
406
405
403
402
403
401
0.102
0.006
0.049
0.014
0.016
0.027
43
39
39
41
46
44
a
All measurements were carried out at 2 μM in PBS buffer (pH 7.4) at
room temperature.
The Tm values of the matched and mismatched duplexes
compared with the unmodified duplex, are presented in Table
5. Apparently, CF3-PhImdC can form stable base pairing
interactions in both DNA and RNA duplexes. The Tm values
of matched ODN(CF3‑PhImdC):ODN(G)/ORN(G) duplexes
were similar to unmodified duplex (43 and 46 vs 44 °C), while
Tm of mismatched duplexes was lower than that of unmodified
duplex (39 and 41 °C vs 44 °C).
The above calculations indicated that more stable tautomer
of CF3‑PhImdC fluorescent analogue is A, similar to cytidine
structure. We assumed that tautomer A is also stable in duplex
environment. This assumption was confirmed by the fact that
the matched duplex ODN(CF3‑PhImdC):ODN(G) had Tm,
almost identical to unmodified duplex. The mismatched duplex
with ODN(A) had lower Tm than the matched duplex possibly
indicating that the base-paired tautomer C, that is more similar
to uridine structure, is less stable in duplex environment. In
addition, we observed a larger decrease of quantum yield in
ODN(CF3‑PhImdC):ODN(G) matched duplex vs mismatched
duplexes indicating stronger hydrogen bonding. Since hydrogen
bonding has significant effect on quantum yield, we concluded
that the more stable tautomer of CF3−PhImdC in duplex
environment is A.
To conclude, CF3-PhImdC-labeled probes have a potential for
SNP typing; however, more studies are required to establish
their potential. In particular, the required studies include
incorporation of additional phenyl-imidazolo-cytidine ana-
logues into different positions in a DNA sequence and analysis
of their photophysical properties. These studies will be reported
in due course.
EXPERIMENTAL SECTION
■
General Methods. Reagents and solvents were purchased from
commercial sources and were used without further purification. All
moisture-sensitive reactions were carried out in flame-dried reaction
flasks with rubber septa, and the reagents were introduced with a
syringe. All reactants in moisture-sensitive reactions were dried
overnight in a vacuum oven. Progress of the reactions was monitored
by TLC on precoated silica gel plates. Visualization was accomplished
by UV light. Flash chromatography was carried out on silica gel.
Medium pressure chromatography was carried out using automated
1
flash purification system. H NMR spectra were obtained using a 200,
300, 400, or 600 MHz spectrometer. 13C NMR spectra were obtained
at 75, 100, or 150 MHz spectrometer. Chemical shifts are expressed in
ppm downfield from Me4Si (TMS) used as internal standard. The
values are given in δ scale. New compounds were analyzed under ESI
Only a few methods of mismatched detection based on
fluorescence of the opposing nucleotide have been reported in
the literature, and even fewer exhibited a significant change in
quantum yield and emission wavelengths.64−67 Our data
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(electron spray ionization) conditions on a Q-TOF microinstrument.
High resolution MS-MALDI-TOF spectra were recorded with TOF/
TOF instrument. Absorption spectra were measured on a UV
instrument using a 1 cm path length quartz cell. Emission spectra
were measured using luminescence spectrometer, and the emission
was corrected for the monochromator sensitivity according to the
standard procedure. Oligonucleotides were synthesized by standard
automated solid-phase method.
General Procedure for Synthesis of Protected Para-
Substituted Phenyl-imidazolo-cytidine Derivatives 10e−g.
Protected 5-NH2-cytidine, 8 (100 mg, 0.17 mmol), and the
appropriate para-substituted benzaldehyde (0.20 mmol) were
thoroughly mixed in DMF (2 mL), then p-TsOH (7 mg, 0.034
mmol) was added, and the solution was heated and stirred at 80 °C for
2−4 h and monitored by TLC (CHCl3/MeOH 9.5:0.5). When the
reaction was completed, the solution was cooled to rt. The reaction
mixture was added dropwise with vigorous stirring into a mixture of
Na2CO3 (0.034 mmol) and H2O (10 mL). The crude product was
extracted into EtOAc. The organic phase was washed with H2O and
brine and dried (Na2SO4). Evaporation of solvent gave the crude
product, which was purified by column chromatography over silica gel
(9:1 DCM/EtOH).
p-Nitrophenyl-imidazolo-cytidine-2′,3′,5′-O-tribenzoate
(10e). Product 10e was obtained by condensation of 8 (100 mg, 0.17
mmol) with p-nitrobenzaldehyde 9e (31 mg, 0.20 mmol) with p-
TsOH (7 mg, 0.034 mmol) as a catalyst, according to the general
procedure. Product 10e was obtained as a white solid in 40% yield (48
mg). Mp: 172−174 °C. 1H NMR (600 MHz, CDCl3): δ 8.74 (s, 1H),
8.63 (d, 2H), 8.30 (d, J=8.4 Hz, 2H), 8.12 (d, J=7.7 Hz, 2H), 8.07 (d,
J=7.7 Hz, 2H), 7.94 (d, J=7.7 Hz, 2H), 7.55 (m, 3H), 7.42 (m, 4H),
7.36 (m, 2H), 6.47 (bs, 1H), 6.15 (m, 1H), 5.90 (m, 1H), 4.88 (m,
3H) ppm. 13C NMR (150 MHz, CDCl3): δ 166.1, 165.2, 165.0, 162.7,
154.4, 149.4, 135.5, 133.8, 133.4, 129.9, 129.8, 129.5, 129.2, 128.9,
128.7, 128.6, 128.5, 128.4, 128.2, 126.2, 124.2, 91.9, 80.5, 75.0, 70.1,
63.1 ppm. HRMS (MALDI-TOF) m/z: calcd for C37H27N5O10Na
724.1655 [M + Na+], found 724.1648 [M + Na+].
p-Fluorophenyl-imidazolo-cytidine-2′,3′,5′-O-tribenzoate
(10f). Product 10f was obtained by condensation of 8 (100 mg, 0.17
mmol) with p-fluorobenzaldehyde 9f (25 mg, 0.20 mmol) with p-
TsOH (7 mg, 0.034 mmol) as a catalyst, according to general
procedure. Product 10f was obtained as a white solid in 45% yield (51
mg). Mp: 183−185 °C. 1H NMR (300 MHz, CDCl3): δ 8.75 (s, 1H),
8.52 (m, 2H), 8.24 (d, J=7.8 Hz, 2H), 8.13 (m, 2H), 8.07 (m, 4H),
7.68 (m, 2H), 7.56 (m, 7H), 7.26 (m, 2H), 6.55 (m, 1H), 6.21 (m,
1H), 6.04 (m, 1H), 5.03 (m, 3H) ppm. 13C NMR (75 MHz, CDCl3):
δ 166.1, 165.2, 165.0, 163.3, 162.6, 134.1, 133.7, 133.6, 133.3, 130.4,
129.8, 129.1, 128.6, 128.5, 128.4, 123.9, 116.4, 116.1, 115.8, 92.3, 80.4,
75.1, 70.4, 63.4 ppm. HRMS (MALDI-TOF) m/z: calcd for
C37H28FN4O8 675.1889 [M + H+], found 675.1886 [M + H+].
p-Cyanophenyl-imidazolo-cytidine-2′,3′,5′-O-tribenzoate
(10g). Product 10g was obtained by condensation of 8 (100 mg, 0.17
mmol) with p-cyanobenzaldehyde 9g (27 mg, 0.20 mmol) with p-
TsOH (7 mg, 0.034 mmol) as a catalyst, according to the general
procedure. Product 10g was obtained as a white solid in 41% yield (56
mg). Mp: 183−185 °C. 1H NMR (400 MHz, CDCl3): δ 8.83 (s, 1H),
8.60 (m, 2H), 8.25 (d, J=7.4 Hz, 2H), 8.10 (m, 4H), 7.82 (d, J=7.7 Hz,
2H), 7.80 (m, 3H), 7.71 (m, 6H), 6.54 (m, 1H), 6.24 (m, 1H), 6.04
(m, 1H), 5.06 (m, 3H) ppm. 13C NMR (100 MHz, CDCl3): δ 166.1,
165.2, 165.0, 162.4, 155.6, 154.5, 135.5, 133.9, 133.7, 133.4, 132.7,
129.9, 129.8, 129.7, 129.1, 128.9, 128.8, 128.6, 128.5, 128.4, 128.3,
126.2, 118.3, 114.7, 92.1, 80.6, 75.1, 70.3, 63.2 ppm. HRMS (MALDI-
TOF) m/z: calcd for C38H28N5O8 682.1938 [M + H+], found
682.1932 [M + H+].
purified by an automated medium pressure column chromatography
(8:2 CHCl3/MeOH).
p-Nitrophenyl-imidazolo-cytidine (5e). Product 5e was ob-
tained by treating protected nucleoside 10e (105 mg, 0.15 mmol) with
NaOH in MeOH according to general procedure, yielding 52 mg
(85%) of a white solid. Mp: >222 °C dec. 1H NMR (600 MHz,
DMSO-d6): δ 8.99 (s, 1H), 8.36 (d, J = 7.2 Hz, 2H), 8.24 (d, J = 7.2,
2H), 5.89 (d, J = 2.2 Hz, 1H), 5.55 (s, 1H), 5.41 (s, 1H), 5.03 (s, 1H),
4.05 (m, 2H), 3.97 (m, 1H), 3.84 (m, 1H), 3.67 (m, 1H) ppm. 13C
NMR (150 MHz, DMSO-d6): δ 155.4, 154.8, 148.8, 138.5, 135.6,
131.8, 126.2, 123.8, 92.2, 85.1, 75.1, 68.8, 59.9 ppm. HRMS (MALDI-
TOF) m/z: calcd for C16H15N5O7Na 412.0869 [M + Na+], found
412.0871 [M + Na+].
p-Fluorophenyl-imidazolo-cytidine (5f). Product 5f was
obtained by treating protected nucleoside 10f (100 mg, 0.15 mmol)
with NaOH in MeOH according to general procedure, yielding 47 mg
1
(87%) of a yellowish solid. Mp: >212 °C dec. H NMR (400 MHz,
DMSO-d6): δ 9.08 (s, 1H), 8.19 (m, 2H), 7.38 (m, 2H), 5.91 (d, J =
2.7 Hz, 1H), 5.54 (s, 1H), 5.38 (s, 1H), 5.06 (s, 1H), 4.06 (m, 2H),
3.97 (m, 1H), 3.84 (m, 1H), 3.68 (m, 1H) ppm. 13C NMR (100 MHz,
DMSO-d6): δ 164.8, 162.4, 162.2, 153.7, 134.3, 131.7, 131.6, 129.3,
129.2, 128.1, 126.0, 124.9, 116.2, 115.9, 114.8, 114.6, 91.2, 83.9, 75.1,
70.3, 69.1, 59.4 ppm. HRMS (MALDI-TOF) m/z: calcd for
C16H15FN4O5Na 385.0924 [M + Na+], found 385.0923 [M + Na+].
p-Cyanophenyl-imidazolo-cytidine (5g). Product 5g was
obtained by treating protected nucleoside 10g (102 mg, 0.15 mmol)
with NaOH in MeOH according to general procedure, yielding 46 mg
(80%) of a white solid. Mp: >205 °C dec. 1H NMR (400 MHz,
DMSO-d6): δ 8.89 (s, 1H), 8.41 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 8.1,
2H), 5.90 (d, J = 2.3 Hz, 1H), 5.61 (s, 1H), 5.42 (s, 1H), 5.04 (s, 1H),
4.04 (m, 2H), 3.99 (m, 1H), 3.90 (m, 1H), 3.73 (m, 1H) ppm. 13C
NMR (100 MHz, DMSO-d6): δ 163.4, 137.5, 131.6, 128.7, 128.6,
124.3, 123.4, 91.2, 83.7, 74.1, 67.5, 58.9 ppm. HRMS (MALDI-TOF)
m/z: calcd for C17H15N5O5Na 392.0971 [M + Na+], found 392.0990
[M + Na+].
5-Amino-cytosine (13). 5-Nitro-cytosine 12 (2 g, 12.8 mmol) and
10% Pd−C (140 mg) in H2O/EtOH (2:1, 20 mL) was shaken in Parr
apparatus under H2 atmosphere (55 psi) for 3 h. The suspension was
filtered over Celite and washed with hot water. The filtrate was
concentrated in vacuo. The crude product was recrystallized from
water. The green crystals was filtered and washed with ice-cold water
1
to give 1.2 g (75%) of product 13. Mp: 247−249 °C. H NMR (300
MHz, DMSO-d6): δ 10.01 (br. s, 2H), 6.74 (s, 1H), 6.62 (br. s, 1H),
3.80 (s, 2H) ppm. 13C NMR (150 MHz, DMSO-d6): δ 162.2, 156.1,
124.3, 115.3 ppm. HRMS (MALDI-TOF) m/z: calcd for C4H7N4O1
127.0620 [M + H+], found 127.0629 [M + H+].
p-(Trifluoromethyl)phenyl-imidazolo-cytosine (6). 5-Amino-
cytosine, 13 (300 mg, 2.36 mmol), and p-trifluoromethylbenzaldehyde
(330 mg, 3.00 mmol) were thoroughly mixed in DMF (15 mL), p-
TsOH (81 mg, 0.47 mmol) was added, and the solution was heated
and stirred at 80 °C for 6 h and monitored by TLC (CHCl3/MeOH,
8:2). When the reaction was completed, the solution was cooled to rt.
Evaporation of the solvent gave the crude product, which was purified
by column chromatography over silica gel, yielding 364 mg (55%) of a
1
white solid. Mp: >218 °C dec. H NMR (600 MHz, DMSO-d6): δ
8.46 (s, 1H), 8.33 (d, J = 6.4 Hz, 2H), 7.89 (d, J = 6.4, 2H) ppm. 13C
NMR (150 MHz, DMSO-d6): δ 132.3, 131.2, 128.9, 128.3, 127.1,
127.0, 126.1 ppm. HRMS (MALDI-TOF) m/z: calcd for C12H8F3N4O
281.0650 [M + H+], found 281.0639 [M + H+].
5-Nitro-2′-deoxyuridine-3′,5′-O-diacetate (15). 2′-Deoxyuri-
dine, 14 (5 g, 0.02 mol), was treated with acetic anhydride (11 μL,
0.12 mol) in pyridine (200 mL) at room temperature overnight.
Volatiles were evaporated to dryness, and the residue was
coevaporated from toluene to remove traces of pyridine, acetic acid,
and acetic anhydride. Acetylated uridine was obtained as gammy solid
without purification, yielding 5.2 g, 95%. The acetylated nucleoside
was dissolved in dimethylformamide (150 mL) and treated with
nitrosonium tetrafluoroborate (14 g, 0.11 mol) for 30 min. The
reaction was terminated by addition of water (20 mL). The reaction
mixture was diluted with ethyl acetate (600 mL) and washed with
General Procedure for Synthesis of Imidazolo-cytidine
Derivatives 5e−g. Protected nucleoside (0.15 mmol, 1 equiv) was
added to a solution of NaOH (54 mg, 1.35 mmol, 9 equiv) in MeOH
(2 mL), and the resulting mixture was stirred at room temperature for
1 h. The residue was diluted with dichloromethane and extracted with
water, and the aqueous layer was freeze-dried. The crude product was
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aqueous sodium bicarbonate. The organic layer was separated and
dried over sodium sulfate. The crude product was purified on a silica
gel column using ethyl acetate/hexanes (1:1 v/v). Fractions containing
the titled product were combined and evaporated to give 7.2 g (92%).
Mp: 175−177 °C. 1H NMR (600 MHz, CDCl3): δ 9.11 (s, 1H), 8.78
(s, 1H), 6.28 (m, 1H), 5.26 (m, 1H), 4.40 (m, 3H), 2.71 (m, 1H) 2.30
(m, 1H), 2.16 (s, 3H), 2.13 (s, 3H) ppm. 13C NMR (150 MHz,
CDCl3): δ 170.3, 170.2, 153.4, 147.8, 143.7, 126.1, 86.8, 83.5, 73.7,
63.5, 39.1, 20.8, 20.7 ppm. HRMS (MALDI-TOF) m/z: calcd for
C13H15N3O9Na 380.0703 [M + Na+], found 380.0706 [M + Na+].
5-Nitro-2′-deoxycytidine-3′,5′-O-diacetate (16). 5-Nitro-uri-
dine-3′,5′-O-diacetate, 15 (1 g, 2.82 mmol), was dissolved in dry
acetonitryle (50 mL). Triethylamine (1 mL, 7.17 mmol) and 2-
mesitylenesulfonyl chloride (1.1 g, 5.41 mmol) were added, and the
mixture was left at room temperature for 3 h. Next, a mixture of 1:1
NH4OH/CH3CN (17 mL) was added and the reaction was stirred for
additional 3 h at room temperature. The reaction mixture was diluted
with dichloromethane and washed with 1 M KHSO4 solution. The
organic layer was separated, washed twice with water, dried over
sodium sulfate, and evaporated. The crude product was purified on a
silica gel column using ethyl acetate/methanol (9:1 v/v). Fractions
containing the titled product were combined and evaporated to give a
pure nucleoside as yellowish foam (678 mg, 68%). Mp: 178−179 °C.
1H NMR (CDCl3): δ 9.22 (s, 1H), 7.92 (s, 1H), 8.03 (s, 1H), 6.88 (s,
1H), 6.23 (m, 1H), 5.22 (m, 1H), 4.41 (m, 3H), 2.85 (m, 1H), 2.22
(m, 1H), 2.14 (s, 3H), 2.11 (s, 3H) ppm. 13C NMR (150 MHz,
CDCl3): δ 170.3, 170.2, 157.7, 152.1, 145.5, 119.6, 87.8, 83.3, 73.93,
63.3, 39.6, 20.8, 20.6 ppm. HRMS (MALDI-TOF) m/z: calcd for
C13H16N4O8Na 379.0866 [M + Na+], found 379.0865 [M + Na+].
5-Amino-2′-deoxycytidine-3′,5′-O-diacetate (17). To a sol-
ution of 16 (1 g, 2.8 mmol) in THF (104 mL), and glacial AcOH (8
mL) was added 10% Pd/C (625.5 mg). The solution was charged with
H2 (1.5 atm), and stirred for 7 h at room temperature. The reaction
was filtered through a pad of Celite, washed with an excess of MeOH,
and concentrated in vacuo. The crude material was purified by
automated column chromatography (20% MeOH in EtOAc) yielding
17 (450 mg, 50%). Mp: 188−190 °C. 1H NMR (600 MHz, CDCl3): δ
7.26 (s, 1H), 7.12 (s, 1H), 6.26 (m, 1H), 5.14 (m, 1H), 4.28 (m, 2H),
4.17 (m, 1H), 2.47 (m, 1H), 2.12 (m, 1H), 2.05 (s, 6H) ppm. 13C
NMR (150 MHz, CDCl3): δ 170.5, 170.4, 162.0, 154.6, 125.0, 115.6,
85.7, 81.7, 74.2, 63.8, 37.4, 20.8, 20.7 ppm. HRMS (MALDI-TOF) m/
z: calcd for C13H18N4O6Na 349.1124 [M + Na+], found 349.1119 [M
+ Na+].
8.38 (d, J = 7.27 Hz, 2H), 7.92 (d, J = 7.27 Hz, 2H), 6.25 (m, 1H),
5.27 (m, 1H), 4.28 (m, 1H), 3.74 (m, 1H), 3.67 (m, 1H), 3.64 (m,
2H), 2.42 (m, 1H), 2.15 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ
153.6, 135.1, 127.4, 125.8, 125.2, 87.9, 87.2, 69.4, 60.5, 41.4 ppm.
HRMS (MALDI-TOF) m/z: C17H16F3N4O4 397.1123 [M + H+],
found 397.1128 [M + H+].
p-(Trifluoromethyl)phenyl-imidazolo-(2′-deoxy-5′-O-dime-
thoxytrityl)-cytidine (19). Compound 7 (100 mg, 0.25 mmol) was
coevaporated twice with dry pyridine and then dissolved in the same
solvent (1 mL). 4,4′-Dimethoxytrityl chloride (102 mg, 1.51 mmol)
was dissolved in dry pyridine (1 mL) and added dropwise to the
solution which was stirred at room temperature for 2 h. Methanol (0.5
mL) was added, and the solution was evaporated. The residue was
coevaporated twice with toluene and then purified by column
chromatography over silica gel (DCM/MeOH, 95:5 containing 1%
1
pyridine) yielding 19 (110 mg, 65%). Mp: 215−217 °C. H NMR
(600 MHz, Acetone-d6): δ 8.78 (s, 1H). 8.48 (d, J = 8.7 Hz, 2H), 7.87
(d, J = 8.7 Hz, 2H), 7.54 (m, 2H), 7.46 (m, 2H), 7.42 (m, 2H), 7.32 (t,
J = 6.5, 2H), 7.21 (t, J = 6.1, 1H), 6.91 (m, 4H), 6.34 (t, J = 5.9 1H),
4.62 (m, 1H), 4.25 (m, 1H), 3.76 (s, 3H), 3.73 (s, 3H), 3.53 (m, 1H),
3.44 (m, 1H), 2.73(m, 1H), 2.39 (m, 1H) ppm. 13C NMR (150 MHz,
Acetone-d6): δ 162.6, 159.7, 159.6, 154.1, 145.9, 136.7, 136.6, 135.9,
134.4, 131.0, 130.9, 128.9, 128.7, 128.6, 127.5, 126.6, 126.5, 126.0,
114.1, 114.0, 89.16, 88.0, 87.5, 71.6, 64.3, 55,47, 55.44, 42.9 ppm.
HRMS (MALDI-TOF) m/z: calcd for C38H34F3N4O6 699.2430
(MH+), found 699.2439 (MH+).
p-(Trifluoromethyl)phenyl-imidazolo-(2′-deoxy-5′-O-dime-
thoxytrityl)-cytidine 3′-(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite (20). A 0.45 M solution of tetrazole (0.086 mmol,
0.200 mL) in anhydrous acetonitrile was added, with stirring under a
flow of argon, to a solution of 19 (60 mg, 0.086 mmol) in anhydrous
acetonitrile (0.430 mL), followed by 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphoramidite (0.086 mmol, 28 μL). TLC analysis
showed that the reaction was completed within 1 h, with the formation
of two diastereoisomers. The precipitated diisopropylammonium
tetrazolide was removed by filtration and the solution was diluted
with anhydrous acetonitrile (0.350 mL) to give 0.1 M solution of 20.
The solution was immediately used in the phosphorylation step on the
automated synthesizer, without further purification. Presence of the
product was confirmed by phosphorus NMR analysis. 31P NMR (80
MHz, acetone-d6): δ 149.61, 149.34 ppm. MS (ESI) m/z: calcd for
C47H51F3N6O7P 899 (MH+), found 899 (MH+).
Absorption Measurements. Absorption spectra of analogues 1
and 2 were determined in MeOH. The concentration of the samples
was in the range of 4−5 μM. The concentrations of all single-stranded
and double-stranded oligonucleotides were 3 μM in PBS buffer
containing NaC1 (8.0 g), KC1 (0.2 g), Na2HPO4 (1.15 g), KH2PO4
(0.2 g), in water (100 mL, pH 7.4) at 25 °C. Samples were measured
at room temperature in a 10 mm quartz cell with a 1 cm path length.
Fluorescence Measurements. Emission spectra of analogues
5a−g were determined in MeOH. The concentration of the samples
was in the range of 3−3.5 μM. The emission wavelengths of all the
single-stranded and double-stranded oligonucleotides were measured
in PBS buffer (pH 7.4) at the concentration of 3 μM. All the
measurement included 750 V sensitivity and a 5 nm slit and were
performed at room temperature, λex 350, λem 355−650 nm range.
Samples were measured in a 10 mm quartz cell with a 1 cm path
length. The fluorescence quantum yield (Φ) was determined relative
to quinine sulfate in 0.1 M H2SO4 (λex 350 nm, λem 446 nm, Φ 0.54).49
Oligonucleotide Synthesis, Workup, and Purification. The
oligonucleotides were assembled using a DNA/RNA synthesizer and
by the phosphoramidite method. CPGs (3 μmol, pore size 500 Å),
DNA reagents, and commercially available phosphoramidite mono-
mers were used. The synthesized, p-CF3-PhImdC-modified nucleoside
phosphoramidite, 20, was used at 0.1 M in dry acetonitrile. The
oligonucleotides were cleaved from the solid support with 1:1 (v/v)
33% NH4OH and 33% methylamine in ethanol at 65 °C, for 10 min.
The crude product was purified by RP-HPLC on C-18 column and
eluted with a solvent mixture of 0.1 M TEAA (pH 7), and a linear
gradient over 30 min from 5% to 40% acetonitrile at a flow rate of 3
p-(Trifluoromethyl)phenyl-imidazolo-2′-deoxycytidine-
3′,5′-O-diacetate (18). Protected 5-amino-2′-deoxycytidine 17 (540
mg, 1.65 mmol) and p-TsOH (70 mg, 0.33 mmol) were thoroughly
mixed in DMF (5.5 mL), p-(trifluoromethyl)benzaldehyde (260 μL,
1.90 mmol) then was added, and the solution was heated and stirred at
80 °C for 2 h. The reaction mixture was cooled to rt and added
dropwise with vigorous stirring into a mixture of K2CO3 (320 mg, 2.31
mmol) and H2O (10 mL). The crude product was extracted into
EtOAc, and the organic phase was washed with H2O and brine and
dried (Na2SO4). Evaporation of solvent gave the crude product, which
was purified by column chromatography over silica gel (1% MeOH in
EtOAc) yielding 18 (435 mg, 50%). Mp: 184−185 °C. 1H NMR (600
MHz, CDCl3): δ 8.71 (s, 1H), 8.55 (d, J = 6.4 Hz 2H), 7.78 (d, J = 6.4
Hz, 2H), 6.27 (m, 1H), 5.27 (m, 1H), 4.43 (m, 2H), 4.29 (m, 1H),
2.78 (m, 1H), 2.16 (m, 1H), 2.11 (s, 6H) ppm. 13C NMR (150 MHz,
CDCl3): δ 170.4, 170.2, 162.6, 161.5, 156.1, 154.6, 135.0, 134.6, 133.4,
133.1, 132.9, 132.7, 131.1, 129.9, 128.2, 128.1, 126.0, 125.4, 124.6,
122.8, 121.0, 88.6, 83.3, 73.7, 63.5, 39.2, 20.8, 20.7 ppm. HRMS
(MALDI-TOF) m/z: calcd for C21H20F3N4O6 481.1335 [M + H+],
found 481.1344 [M + H+].
p-(Trifluoromethyl)phenyl-imidazolo-2′-deoxycytidine (7).
Compound 18 (300 mg, 0.63 mmol) was dissolved in NaOH (75
mg, 1.87 mmol) solution in methanol (8 mL) and stirred at room
temperature for 3 h. The reaction was neutralized with acetic acid and
evaporated. The crude product was purified by column chromatog-
raphy over silica gel (DCM/EtOH, 4:3) yielding 7 (185 mg, 80%).
1
Mp: >205 °C dec. H NMR (400 MHz, DMSO-d6): δ 9.02 (s, 1H),
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dx.doi.org/10.1021/jo5011944 | J. Org. Chem. 2014, 79, 7051−7062

The Journal of Organic Chemistry
Article
mL/min. The oligomers were converted to the sodium salt using CM
Sephadex C-25 equilibrated in NaCl and washed well with water. The
identity of the oligomers was determined by MALDI-TOF mass
spectroscopy: ODN(CF3‑PhImdC) calcd for C156H171F3N66O84P14Na
[M + Na+] 4825.73, found 4829.56. The yield of the synthesized
(CF3−PhImdC)ODN after HPLC purification was 16% and the
unmodified oligonucleotide, 20%.
Hybridization of Oligonucleotides. Solutions of labeled single-
strands were mixed at room temperature with an equimolar amount of
the complementary single strand oligonucleotides in PBS buffer (pH
7.4). Samples were hybridized by heating to 90 °C for 5 min and
subsequently allowed to cool to room temperature over 2 h prior to
measurements.
AUTHOR INFORMATION
■
Corresponding Author
*Fax: 972-3-6354907. Tel: 972-3-5318303. E-mail: bilha.
fischer@biu.ac.il.
Notes
The authors declare no competing financial interest.
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