Rational design of a solvatochromic fluorescent uracil analogue with a dual-band ratiometric response based on 3-hydroxychromone

Article abstract of DOI:10.1002/chem.201303399

Fluorescent nucleoside analogues with strong and informative responses to their local environment are in urgent need for DNA research. In this work, the design, synthesis and investigation of a new solvatochromic ratiometric fluorophore compiled from 3-hy

Full text of DOI:10.1002/chem.201303399

DOI: 10.1002/chem.201303399
Full Paper
&
Fluorescent Probes
Rational Design of a Solvatochromic Fluorescent Uracil
Analogue with a Dual-Band Ratiometric Response Based on
3-Hydroxychromone
Dmytro Dziuba,[a, b] Iuliia A. Karpenko,[a, c] Nicolas P. F. Barthes,[a] Benoꢀt Y. Michel,[a]
Andrey S. Klymchenko,[d] Rachid Benhida,[a] Alexander P. Demchenko,[e] Yves Mꢁly,[d] and
Alain Burger*[a]
Dedicated to Professor J.-F. Biellmann
Abstract: Fluorescent nucleoside analogues with strong and
informative responses to their local environment are in
urgent need for DNA research. In this work, the design, syn-
thesis and investigation of a new solvatochromic ratiometric
fluorophore compiled from 3-hydroxychromones (3HCs) and
uracil fragments are reported. 3HC dyes are a class of multi-
parametric, environment-sensitive fluorophores providing
a ratiometric response due to the presence of two well-re-
solved bands in their emission spectra. The synthesized con-
jugate demonstrates not only the preservation but also the
improvement of these properties. The absorption and fluo-
rescence spectra are shifted to longer wavelengths together
with an increase of brightness. Moreover, the two fluores-
cence bands are better resolved and provide ratiometric re-
sponses across a broader range of solvent polarities. To un-
derstand the photophysical properties of this new fluoro-
phore, a series of model compounds were synthesized and
comparatively investigated. The obtained data indicate that
uracil and 3HC fragments of this derivative are coupled into
an electronic conjugated system, which on excitation attains
strong charge-transfer character. The developed fluorophore
is a prospective label for nucleic acids.
erties and interactions of nucleic acids.[1] Artificial fluorescent
nucleosides can be site-selectively incorporated into nucleic
acid strands by an enzymatic polymerization reaction or by
chemical synthesis. As a result, a well-defined location and ge-
ometry of the fluorophore within the nucleic acid structure
can be achieved together with the ability to follow its func-
tional transformations.[2] This opens many possibilities that
cannot be realized with other types of covalent fluorescent
labels. Advanced fluorescent nucleosides allow for the sensing
of nucleic acid hybridization and conformational changes,[3]
typing of single-nucleotide polymorphisms (SNPs),[4] and sens-
ing the activity of DNA-binding proteins and enzymes.[5,6]
These results stimulate new efforts to synthesize advanced
emissive nucleosides based on new mechanisms of response.
Fluorescence offers a limited number of parameters for quanti-
tative detection. Typically, the events occurring in the microen-
vironment of fluorophores may be monitored by changes in:
1) fluorescence intensity; 2) fluorescence anisotropy; 3) excit-
ed-state lifetime; and 4) position of the emission band maxi-
mum (emission color).[7] Analytical assays based on fluorescent
nucleosides commonly use one of these modalities. A classic
example is 2-aminopurine (2AP), a constitutional isomer of ad-
enine, changing emission intensity based on its local environ-
ment. 2AP fluoresces brightly in water (F=0.68), whereas
stacking interactions with neighboring nucleobases within the
nucleic acid strands strongly quenches 2AP fluorescence.[8] As
a consequence, quantitative determination of fluorescence in-
Introduction
Fluorescent analogues of non-emissive natural nucleosides
found broad applications in studying structure, dynamic prop-
[a] Dr. D. Dziuba, I. A. Karpenko, N. P. F. Barthes, Dr. B. Y. Michel, Dr. R. Benhida,
Prof. Dr. A. Burger
Institut de Chimie de Nice, UMR 7272
Universitꢀ de Nice Sophia Antipolis, CNRS
Parc Valrose, 06108 Nice cedex 2 (France)
E-mail: burger@unice.fr
[b] Dr. D. Dziuba
Present address: Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Flemingovo nam. 2, CZ-16610, Prague 6 (Czech Republic)
[c] I. A. Karpenko
Present address: Laboratoire d’Innovation Thꢀrapeutique
UMR 7200, Universitꢀ de Strasbourg, CNRS, Facultꢀ de Pharmacie
74 route du Rhin, 67401 Illkirch (France)
[d] Dr. A. S. Klymchenko, Prof. Dr. Y. Mꢀly
Laboratoire de Biophotonique et Pharmacologie, UMR 7213
Facultꢀ de Pharmacie, Universitꢀ de Strasbourg, CNRS
74 Route du Rhin, 67401 Illkirch (France)
[e] Prof. Dr. A. P. Demchenko
A.V. Palladin Institute of Biochemistry
9 Leontovicha Street, Kiev 01030 (Ukraine)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303399 (including H/13C NMR spectra of
all compounds, representative H–1H COSY, H–13C HMQC and H–13C HMBC
spectra, Figure S1, and Tables S1 and S2 from steady-state fluorescent
measurements).
1
1
1
1
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tensity may be problematic because it depends on the concen-
tration and is sensitive to unaccountable quenching effects.
Application of color-changing nucleosides can only partially re-
solve this problem; typically they are molecules that possess
a highly dipolar excited state and exhibit polarity-dependent
shifts in their emission together with the intensity changes,
which can be rather strong. Both parameters can monitor
changes occurring in their microenvironment.[9] Meanwhile,
sensing based on fluorescent intensity or spectral shifts suffers
from several limitations, related mainly to the problems of the
calibration of the fluorescence response, which is commonly
displayed in relative units. To overcome these limitations, an
approach based on the l-ratiometric sensing can be used.[10]
Based on recording of the fluorescence signal as the ratio of
intensities at two or more wavelengths, this sensing has under-
gone rapid development during recent years. Such simple
two-channel probing makes possible to record an intrinsically
calibrated analytical signal that does not depend on local
probe concentration or instrumentation settings. Analytical
assays for nucleic acids based on l-ratiometry are known. The
vast majority of them exploit double-dye systems, such as fluo-
rescence resonance energy transfer (FRET) pairs and formation
of excimers.[11] Nevertheless, this method requires double label-
ing to obtain informative signals. An attractive alternative
using a single dye is still poorly explored because of the very
limited number of dyes that can display this property and of
the difficulties in their synthesis.[10,12] In this context, the focus
of our research is the synthesis of fluorescent nucleosides con-
taining single ratiometric emitters. With this aim, we concen-
trated our efforts on fluorescent nucleosides incorporating 3-
hydroxychromone fluorophores as single-dye ratiometric re-
porters.
3-Hydroxychromones (3HCs) are a prospective class of fluo-
rescent dyes offering exceptional solvatochromism together
with dual-band fluorescence suitable for ratiometric detection.
The two emission bands originate from a reaction occurring in
the excited state: the excited-state intramolecular proton
transfer (ESIPT, Figure 1).[13] Upon excitation, 3HC molecules
Figure 1. Origin of the dual emission of 3-hydroxychromones (ESIPT=excit-
ed state intramolecular proton transfer; BPT=ground state back-proton
transfer; N and T denote the normal and tautomer forms, respectively).
attain the normal excited form (N*), which then undergoes an
ESIPT reaction to produce the tautomer (T*) excited form. Both
states are highly emissive, the T* band being typically 80–
100 nm redshifted compared with the N* band (Figure 1).
The dual emission of 3HCs is highly sensitive to the polarity
of the surrounding medium.[14] In particular, the ratio of the
two emission bands changes with respect to the dielectric con-
stant and H-bond donor ability of the environment.[15] High
values of dielectric constant and strong H-bond acidity of the
solvent stabilize the N* state and inhibit the ESIPT reaction,
thus increasing the N*/T* intensity ratio. Therefore, the ratio of
the two emission bands provides information about the micro-
environment of the dye. In addition to the N*/T* ratio, informa-
tion may be extracted from the relative positions of the two
bands, so that 3HCs are multiparametric fluorescent probes.[15]
Over the past two decades, 3HCs have found applications in
sensing polarity of normal[16] and supercritical liquids,[17] prob-
ing lipid membranes[18] and proteins.[19] However, applications
of 3HC-based probes to nucleic acid sensing remain meager.
The work presented herein expands our earlier efforts in the
use of 3HC for nucleic acid probing. We showed that the H-
bond-sensitive fluorophore 2-furyl-3-hydroxychromone conju-
gated to a polyamine chain can distinguish single- from
double-stranded DNA through its two-band ratiometric re-
sponse.[20] Subsequently, we synthesized a fluorescent nucleo-
side, which included the environment-sensitive 2-thienyl-3-hy-
droxychromone as a nucleobase mimic.[21] Incorporation of the
nucleoside into oligonucleotides induced only a small destabi-
lization of the duplex structure. Most importantly, the 3HC nu-
cleoside showed significant changes in its ratiometric response
upon binding to a complementary strand or as the result of
the conformational changes caused by the chaperone viral nu-
Abstract in Ukrainian:
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cleocapsid protein.[22] According to these data, 3HCs are pro-
spective fluorophores for probing nucleic acids and their inter-
molecular interactions.
In this work, we developed a new 3HC-based ratiometric
fluorophore with improved properties for the purpose of nu-
cleic acid probing. We designed and synthesized a fluorophore
composed of 3HC and uracil aromatic fragments assembled
into a conjugated electronic system through an electron-con-
ducting acetylenic linker and investigated its solvatochromic
properties. The fluorophore showed improved fluorescence
properties, including redshifted absorption and emission,
higher brightness, and a new sensitivity profile to H-bond in-
teractions. The dye constitutes a promising building block for
incorporation into nucleic acids.
Figure 2. Proposed structure of the electronically connected 2-thienyl-3-hy-
droxychromone and uridine fragments.
uracil (Figure 2). ICT was shown to play an important role in
modulating the environment-sensing properties of 3HC
dyes.[14a,24] Furthermore, it has been shown for 2-thienyl-3-hy-
droxychromones that their 5’-position is highly sensitive to
electron-donating groups.[25]
Results and Discussion
To better understand the photophysics of this new com-
pound, we compared it with two analogues, namely the
parent 2-thienyl-3-hydroxychromone 2 (TC) and a TC bearing
an acetylene moiety 3 (TCe; Figure 3).
In addition, we have synthesized and characterized for fur-
ther comparison the methylated analogues of these three
compounds 4, 5 and 6 (named MTC, MTCe, and MTCeU, re-
spectively) that are unable to undergo ESIPT.
Design of the fluorophore
Design of new fluorophores with predefined photophysical
characteristics is a great challenge. To reach this aim, the ap-
proach based on modification of known fluorescent cores ap-
pears particularly fruitful.[23] For designing a new fluorophore
based on the 3-hydroxychromone core for nucleic acid label-
ing, several requirements need to be met: 1) the target mole-
cule should contain a 3HC moiety with an unprotected hydrox-
yl group to use ESIPT as the principal sensing mechanism;
2) the fluorophore should be connected with one of the natu-
ral nucleobases to allow its incorporation into DNA as a corre-
sponding nucleoside; 3) the 3HC and nucleobase parts should
be connected through a short rigid linker to ensure a defined
localization of the fluorophore with respect to the nucleic acid
main axis. Based on these requirements, the following chemi-
cal structure of the fluorophore 1 (named TCeU) was pro-
posed.
Synthesis
Synthesis of the fluorophore 1 TCeU and the model com-
pounds is depicted in Scheme 1 and Scheme 2. Several syn-
thetic strategies are reported to elaborate the 3HC scaffold. In
our study, the Algar–Flynn–Oyamada (AFO) reaction was em-
ployed as the most facile and abundant reaction for construct-
ing the 3-hydroxychromone core. Appreciable yields of the
target chromones were achieved by using a “soft” one-pot
protocol of the AFO reaction (Scheme 1).[26] An important fea-
ture of this protocol is the possibility to obtain pure product
with minimal manipulation. According to this procedure, 2-hy-
droxyacetophonone 7 was treated with thiophene-aldehydes 8
and 9 in ethanol in the presence of sodium hydroxide with
subsequent addition of hydrogen peroxide and acidic work-up
to give 3-hydroxychromones 2 (TC) and 10 in 50 and 48%
Fluorophore 1 is composed of two fragments, namely 2-
thienyl-3-hydroxychromone, which was previously shown to
be a good ratiometric reporter sensitive to H bonding[21] and
uracil, as the simplest member of the genetic alphabet. 1,3-Di-
methyl uracil was used to exclude any prototropic tautomerisa-
tion in the nucleobase part, which could trigger side reactions
such as 5-endo cyclization
during fragment assembly. An
acetylenic linker was introduced
between the 5’ position of the
thienyl ring and the 5 position
of uracil. Such a modification of
uracil is commonly used in the
synthesis of fluorescent nucleo-
sides with new photophysical
characteristics.[1b,9i]
Moreover,
the selected configuration of
the nucleobase and chromone
moieties is expected to allow an
intramolecular charge-transfer
(ICT) between 4-carbonyl of
chromone and N-1 atom of
Figure 3. Structure of the designed fluorophore TCeU and model compounds.
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the model compounds TCe (3)
and MTCe (5) required for the
photophysical studies.
Concerning the preparation
of the nucleobase fragment,
5-iodouracil 13 was first bis-me-
thylated on its N-1 and N-3 posi-
tions as reported.[29] A Sonoga-
shira coupling in presence of
TMS-acetylene provided the
alkyne derivative 15 in good
yields. Removal of TMS by treat-
ment with tetrabutylammonium
fluoride (TBAF) led to the re-
quired fragment for the final as-
sembly. Sonogashira coupling
between fluorescent moieties 11
and 12 and the nucleobase part
16 gave the desired dye MTCeU
(6) and 17 in appreciable yields.
The Cbz masking the 3-hydroxyl
Scheme 1. Synthesis of 2-thienyl-3-hydroxychromones. Reagents and conditions: a) NaOH, EtOH, RT, 48 h; b) H2O2
(30% aq.); c) Me2SO4, KOH, 18-crown-6, CH2Cl2/H2O, RT, 1 h; d) Cbz-Cl, KOH, 18-crown-6, CH2Cl2/H2O, RT, 1 h;
e) [PdCl2(PPh3)2], CuI, NEt3, THF, reflux.
group was cleaved by a simple treatment with aqueous am-
monia in methanol at room temperature to afford the target
fluorophore TCeU (1).
Spectroscopic properties of the designed fluorophore
UV/Vis absorption and fluorescence properties of the designed
fluorophore as well as of the model compounds were investi-
gated in a set of eleven solvents varying in polarity (Table 1).
Table 1. Spectroscopic properties of TCeU in different solvents.
H[b]
[c]
[d]
[e]
[f]
Solvent
ET(30)[a]
Sb2
labs
lN*
lT*
IN*/IT*
F[g]
Scheme 2. Modular synthesis of 3HC-uracil fluorophores. Reagents and con-
ditions: a) Me2SO4, NaOH, H2O, 1008C, 2 h; b) TMS-acetylene, [PdCl2(PPh3)2],
CuI, DIPEA, THF, 558C, 4 h; c) TBAF, THF, RT, 1 h; d) 11 or 12, [PdCl2(PPh3)2],
CuI, DIPEA, THF, 558C; e) NH4OH, MeOH, RT, 1 h.
H2O
MeOH
EtOH
BuOH
CH3CN
DMSO
acetone
CH2Cl2
EtOAc
THF
63.1
55.4
51.9
49.7
45.6
45.1
42.2
40.7
38.1
37.4
33.9
0.35
0.47
0.48
0.48
0.32
0.88
0.49
0.05
0.45
0.48
0.14
401
395
397
400
392
402
393
397
394
396
399
500
501
488
481
472
477
465
458
451
448
441
–
–
0.08
0.22
0.18
0.19
0.15
0.25
0.15
0.17
0.15
0.18
0.15
558
574
571
576
582
581
577
579
585
587
1.20
0.63
0.50
0.54
1.43
0.53
0.31
0.36
0.73
0.13
yields, respectively.[27] Derivative 4 (MTC) was obtained in 97%
yield after methylation of TC by dimethyl sulfate under the
phase-transfer catalysis (PTC) conditions reported previously.[28]
Compounds 2 (TC) and 4 (MTC) were used for spectroscopic
characterizations. Bromo-chromone 10 was converted under
the same conditions into the methylated derivative 11 needed
for the synthesis of the model compound MTCe.
The synthesis of the designed fluorophore TCeU requires an
appropriate protection for the 3-OH group of the chromone.
We selected a carbonate-type protecting group, the benzyloxy-
carbonyl group (known as Cbz or Z), which was successfully
employed in our recent work for the synthesis of 3HC-contain-
ing nucleosides.[22] The Cbz group was introduced onto
bromo-chromone 10 by treatment with Cbz-Cl under PTC con-
ditions. Sonogashira coupling with TMS-acetylene and com-
pounds 10 and 11 resulted in moderate to excellent yields of
PhCH3
[a] Reichardt’s empirical solvent polarity index.[30] [b] H-bond basicity.[15,31]
[c] Position in nm of the absorption maximum. [d] Position in nm of the
maximum of the normal emission band. [e] Position in nm of the maxi-
mum of the tautomer emission band. [f] Ratio of the intensities at the
two emission maxima. [g] Fluorescence quantum yield measured using 3-
hydroxyflavone in toluene (F=0.29) as a reference.[32]
Four protic solvents (water, methanol, ethanol, n-butanol) and
seven aprotic solvents (acetonitrile, dimethyl sulfoxide, ace-
tone, ethyl acetate, THF, dichloromethane, toluene) were se-
H
lected. According to their H-bond basicity (Sb2 ) values, three
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of the aprotic solvents may be classified as neutral (toluene, di-
chloromethane, acetonitrile), whereas the other aprotic sol-
vents are rather basic.[15,31]
Comparison of TCeU with model compounds
The absorption and fluorescence properties of TC and TCe
were investigated and compared with those obtained for
TCeU in the same set of solvents to get further insight regard-
ing the effect of the electronic conjugation in the extended
chromophore (Table 2). The absorption spectrum shifts to red
The compound TCeU exhibits a single absorption band cen-
tered at 392–401 nm. The positions of the maxima correlate
poorly with the solvent polarity showing a little positive solva-
tochromism. In all solvents except for water, TCeU showed
dual emission, in which the short-wavelength and long-wave-
length bands can be attributed to the N* and T* states, respec-
tively. The N*/T* ratio as well the position of the N* emission
band were strongly solvent-dependent (Table 1, Figure 4).
Table 2. Spectroscopic characteristics[a] of TC, TCe, and TCeU in different
solvents.
Compound
Solvent
labs
lN*
lT*
IN*/IT*
F
MeOH
EtOH
359
360
354
354
357
358
378
379
373
374
379
379
395
397
392
394
397
399
424
424
414
412
412
412
442
439
431
430
432
431
501
488
472
451
458
441
540
542
540
543
537
546
561
563
563
566
561
572
558
574
576
579
577
587
0.72
0.34
0.07
0.05
0.02
0.01
0.43
0.22
0.10
0.12
0.05
0.03
1.20
0.63
0.54
0.36
0.31
0.13
0.07
0.08
0.08
0.12
0.30
0.24
0.13
0.14
0.14
0.15
0.19
0.20
0.23
0.18
0.15
0.15
0.17
0.15
CH3CN
EtOAc
CH2Cl2
PhCH3
MeOH
EtOH
CH3CN
EtOAc
CH2Cl2
PhCH3
MeOH
EtOH
TC
TCe
CH3CN
EtOAc
CH2Cl2
PhCH3
TCeU
[a] See the footnotes [c]–[g] in Table 1 for the definitions of these param-
eters.
in the following sequence: TC!TCe!TCeU (Figure 5). Extinc-
tion coefficients increase upon extending the p-electronic
system of the fluorophore. Values are 23500, 30500, and
35000mꢀ1 cmꢀ1 in methanol for TC, TCe, and TCeU, respective-
ly.
Figure 4. Fluorescence spectra of TCeU normalized at the maximal intensity
This sequential redshift corresponds well to the gradual in-
crease in the conjugation length of the fluorophores. The data
also suggest that the chromone and uridine parts in TCeU are
fused into a single conjugated electronic system as evidenced
by the disappearance of the characteristic absorption band of
in: A) protic, and B) aprotic solvents.
The N* band demonstrates positive solvatochromism, which
is typical for this class of compounds.[33] The position of the T*
band maximum is blueshifted in protic solvents, as compared
with aprotic solvents, probably due to the formation of
a H bond between the phenoxide oxygen of TCeU in the excit-
ed tautomer state and the solvent, as it was proposed for 2-
furyl-3HC.[34] The Stokes shifts for the first and second emission
bands are comprised between 2387–5356 and 7395–
8027 cmꢀ1, respectively. The large Stokes shift obtained for the
second emission band is characteristic for ESIPT probes. As
a general trend, the N*/T* ratio increases with an increase in
solvent polarity. Thus, the highest values of N*/T* ratio are ob-
served for highly polar protic methanol and aprotic DMSO,
whereas the lowest values are observed for the most apolar
toluene. The fluorescence quantum yields are rather good in
all solvents (0.15–0.25), except water (0.08).
Figure 5. Absorption spectra of compounds TC, TCe and TCeU in ethyl ace-
tate. Absorptions are proportional to their absorption coefficient.
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uracil at about 260 nm (Figure 5). Interestingly, our designed
fluorophore exhibits suitable absorption properties with an ab-
sorption band around 400 nm making possible its selective ex-
citation in the presence of proteins and nucleic acids with con-
ventional commercial lasers.
To further investigate the contribution of ICT to the photo-
physical behavior of the synthesized ESIPT dyes, their methy-
lated analogues MTC, MTCe, and MTCeU were characterized in
the same set of solvents (Supporting Information, Table S2).
MTC, MTCe, and MTCeU showed comparable absorption coef-
ficients to the parent non-methylated dyes and absorption
bands centered at 337–341, 362–368, and 375–388 nm, respec-
tively. When compared with the corresponding non-methylat-
ed chromones, the methylated analogues exhibit a significant
blueshift of their absorption maximum. This significant blue-
shift may be explained by a possible distortion of the planar
structure of the fluorophores due to an increased steric hin-
drance between the methoxyl group and the thienyl substitu-
ent in positions 3 and 2 of the chromone ring.[37]
Similar to TCeU, the model compounds TC and TCe display
dual emission, indicating that all three of them undergo ESIPT.
The positions of the emission bands shift to the red with the
increase in the fluorophore conjugation: TC!TCe!TCeU. For
each solvent studied, the value of the N*/T* ratio of TCeU is
much higher than those of TC and TCe (Table 2). Furthermore,
the dual emission of these dyes depends differently on the sol-
vent polarity.
In contrast to TCeU, compounds TC and TCe show only lim-
ited positive solvatochromism of the N* band. Indeed, going
from toluene to MeOH, the N* band shifts to the red by only
12 nm for TC and TCe, whereas for TCeU the redshift reaches
60 nm. The T* band of TC and TCe also does not reveal clear
variations with solvent polarity. Remarkably, all three com-
pounds showed an increase in the N*/T* ratio with increasing
As expected, no tautomer band in the emission spectra was
observed due to blocked ESIPT. Notably, no significant blue-
shift in the emission maxima was observed, when compared
with the N* band of non-methylated chromones. This indicates
that the geometry of the relaxed excited state is close for both
methylated and non-methylated compounds. The emission
bands of the methylated 3HCs were centered at 426–450 nm
for MTC and at 432–442 nm for MTCe, showing a poor de-
pendence on solvent polarity. At the same time, compound
MTCeU exhibited a strong solvent dependence of the position
of its emission band, which ranges from 451 nm in ethyl ace-
tate to 511 nm in water (Figure 7). Thus, the methylated com-
Figure 6. Normalized fluorescence spectra of TC (c), TCe (a), and TCeU
(g) in: A) methanol, B) ethanol, C) ethyl acetate, and D) dichloromethane.
Figure 7. Normalized fluorescence spectra of MTCeU in representative sol-
vents of different polarities.
solvent polarity (Figure 6). However, the N* band emission of
TCeU is significant in aprotic solvents of different polarity,
which is not the case of TC and TCe. Both compounds show
only a marginal contribution of their N* form to the emission
spectra in apolar solvents, such as ethyl acetate and dichloro-
methane (Figure 6C and D).
pound MTCeU, similarly to its parent 3-hydroxychromone
TCeU, shows a strong solvatochromism of its emission band,
confirming our hypothesis on the charge-transfer character of
the N* state. Our data confirm the presence of an ICT state
due to the interaction of the chromone and uracil moieties.
A significant increase in the fluorescence quantum yield was
observed for MTCeU (10–50%) as compared with MTC (0.5–
2%) and MTCe (2–20%). The high quantum yields of MTCeU
together with its solvatochromic properties compare favorably
with other color-changing nucleoside analogues[38] and make
this fluorophore a prospective candidate for incorporation in
DNA and further investigations.
Both the strong solvatochromism of the N* band of TCeU
and its high intensity in aprotic solvents are in line with
a charge-transfer character of its N* state. A very similar
charge-transfer character of the N* state was previously ob-
served for 4’-(dialkylamino)-3-hydroxyflavone[14a] and its ana-
logues having a push–pull structure.[35] It was hypothesized
that in aprotic solvents, the strong interaction of the highly
polar N* state with the environment may stabilize it dielectri-
cally and thus shifts the ESIPT equilibrium N*QT* towards to
the N* state.[36]
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Table 3. Time-resolved decay parameters[a] for TC, TCe, and TCeU in polar solvents.
N*
N*[b]
N*
N*[b]
T*
T*[b]
T*
T*[b]
Compound
Solvent
t1
a1
t2
a2
t1
a1
t2
a2
[ns][a]
[ns][a]
[ns][a]
[ns][a]
EtOH
MeOH
0.10ꢁ0.01
0.11ꢁ0.01
1.00
1.00
–
–
–
–
0.08ꢁ0.01
0.10ꢁ0.01
ꢀ0.45
ꢀ0.47
1.2ꢁ0.1
0.55
0.53
0.76ꢁ0.05
EtOH
MeOH
0.11ꢁ0.01
0.13ꢁ0.01
1.00
1.00
–
–
–
–
0.09ꢁ0.03
0.13ꢁ0.02
ꢀ0.53
ꢀ0.15
2.6ꢁ0.9
0.47
0.85
2.00ꢁ0.01
EtOAc
EtOH
MeOH
0.12ꢁ0.01
0.22ꢁ0.01
0.43ꢁ0.01
1.00
0.54
0.79
–
–
0.46
0.21
0.10ꢁ0.01
0.30ꢁ0.13
0.35ꢁ0.08
ꢀ0.47
ꢀ0.12
ꢀ0.25
2.4ꢁ0.2
1.9ꢁ0.1
1.55ꢁ0.1
0.53
0.88
0.75
0.50ꢁ0.01
0.91ꢁ0.02
[a] The short- (t1) and long- (t2) lived decay times for the N* and T* emission bands, respectively. [b] The corresponding pre-exponential coefficients for the
N* and T* emission bands, respectively.
Time-resolved studies of ESIPT
the ESIPT process is slower in TCeU in comparison with TC
and TCe in the same solvents. Indeed, an increase in the
charge-transfer character of the N* state in TCeU due to the
electron-donating uracil group may further strengthen the in-
termolecular solvent–solute H bond, thus additionally slowing
down the ESIPT reaction.[39] The longer component in the N*
emission, however, is not similar to the long-lived decay com-
ponent in the T* emission, which one would expect in the
case of reversible ESIPT.[36]
Time-resolved investigation of the ESIPT reaction in com-
pounds TC, TCe, and TCeU was performed to understand the
dramatic differences in the sensitivity of these compounds to
solvent polarity. Time-resolved measurements were conducted
in polar solvents in which both the normal and tautomer fluo-
rescence bands were well separated (Table 3). Except for TCeU
in the two protic solvents, the decay of the N* band of the
three compounds was characterized by a single short-lived
component (t1N*), whereas the T* band decay was character-
ized by a fast rise component (t1T*) with a lifetime value close
to that of t1N*, followed by a slower decay component (t2T*). A
similar behavior was already reported for (2-furyl)-3-hydroxy-
chromone[39] and for 3-hydroxyquinolones,[40] the aza-ana-
logues of 3HCs, strongly suggesting an irreversible ESIPT
mechanism that is kinetically controlled. In this mechanism,
Thus, the proton transfer in TCeU in polar protic solvents
likely remains irreversible, whereas the longer component of
the N* emission decay may be assigned to the fluorescence of
a different form, probably the intermolecular H-bonded species
that is generally non-emissive in 3HCs, but was shown to emit
in 3-hydroxyquinolones[40] and 4’-dialkylamino-3-hydroxyfla-
vones.[41] The presence of this new emissive species is consis-
tent with the higher quantum yield measured for TCeU in
methanol and ethanol as compared with TC and TCe.
N*
T*
the t1 and t1 values describe the kinetics of the proton
transfer, whereas the long-lived decay component of the T*
state (t2T*) describes the lifetime of the T* form. For TCe, the
ESIPT is slightly slower than in TC. For both compounds, the
short-lived lifetimes are slightly higher in methanol, confirming
that the ESIPT process is slower in this more H-bond-donating
solvent.[39]
Correlation of the spectroscopic properties of TCeU with sol-
vent polarity
In the prospect of using TCeU as an environment-sensitive
fluorophore for DNA probing, it is important to establish the
relationships between the photophysical parameters of the
fluorophore (position of absorption/emission bands, and N*/T*
ratio) and quantitative parameters of the environment polarity.
In this work, we used the Reichardt’s polarity scale ET(30),[30] an
empirical scale commonly used in the analysis of dye solvato-
chromism. It has also been established as a reliable scale for
analyzing emission of 3HCs.[16,35]
In the case of TCeU, the data indicate an apparent change
in the ESIPT mechanism in polar protic solvents. Whereas the
N* emission in ethyl acetate is characterized by monoexponen-
tial kinetics, the N* emission in EtOH and MeOH shows biexpo-
nential decay with commensurable amplitudes. The shorter
component in these solvents still matches well with the corre-
sponding rise component of the T* emission band, so it can
be assigned to the ESIPT process. Interestingly, the value of
this component drastically rises with the increase of the H-
bond-donating ability of the solvent, indicating a slowing
down of the ESIPT. The slowing down of ESIPT can be assigned
to the formation of an intermolecular H bond between the 3-
hydroxyl group of the dye with the solvent, which disrupts the
intramolecular H bond with the carbonyl oxygen. Remarkably,
We observe that the positions of the absorption band and
the T* emission band of TCeU show a poor correlation with
ET(30). In contrast, a strong correlation is observed for the posi-
tion of the N* band, which undergoes a bathochromic shift
with an increase in solvent polarity (Figure 8). Obviously, this
solvatochromism is caused by the strong dipole moment of
the N* state, which possesses an ICT character. The strong sol-
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yields observed in H-bond-acceptor solvents (Table 1). Finally,
the good correlation observed with all other solvents
(Figure 8), indicates that the N*/T* ratio depends both on the
polarity, H-bonding acidity and basicity of the solvent.
It should be noted that the strong variation of the dual
emission of TCeU is observed in a broad range of solvent po-
larities, ranging from apolar toluene up to polar methanol. This
is an important difference with its analogues TC, TCe, and the
previously reported 2-furyl-3-hydroxychromone, which show
strong variation of their dual emission mainly in polar protic
solvents.[39] The present new feature, resulting from the
charge-transfer character of the N* excited state, makes this
new fluorophore prospective for incorporation into DNA, in
which rather low polarities of internal regions can be found as
well as the local environment of the major groove is expected
to be relatively polar and comparable to methanol and there-
fore different to bulk water.[42] The incorporation of TCeU into
DNA and its further application for monitoring DNA interac-
tions is under way and will be reported in due course.
Conclusion
Figure 8. Dependence of the spectroscopic characteristics of TCeU on the
solvent polarity index ET(30). Position of the N* band (top) and logarithm of
the N*/T* ratio (bottom) as a function of ET(30). The light-gray squares, black
circles, and dark-gray triangles represent protic (MeOH, EtOH, BuOH), aprotic
neutral (CH3CN, CH2Cl2, PhCH3), and aprotic H-bond basic solvents (DMSO,
acetone, EtOAc, THF), respectively.
A new fluorescent dye based on the fusion of 3HC and uracil
moieties has been designed, synthesized, and investigated.
The dye undergoes an ESIPT reaction and exhibits a dual-band
emission in a set of solvents of varying polarity. The ratio of its
two emission bands changes as a function of the solvent polar-
ity across a broad range. The position of the N* band corre-
lates perfectly with the empirical solvent polarity scale ET(30). A
strong correlation was found also between log(N*/T*) and
ET(30) for protic and neutral aprotic solvents. Comparison with
its structural analogues allowed for the demonstration of the
electronic conjugation between 3HC and uracil heterocycles
and the charge-transfer nature of its normal excited state that
is responsible for the dependence of its N*/T* ratio on the sol-
vent polarity. The fluorophore is a prospective candidate for in-
corporation into DNA as an environment-sensitive nucleobase
mimic.
vent-dependent variation in the N*/T* intensity ratio is the dis-
tinctive feature of 3HC fluorophores.[15,35] In the case of TCeU,
the N*/T* ratio increases with the solvent polarity both in
protic and neutral aprotic solvents, showing a linear correlation
between log (IN*/IT*) and ET(30). Most importantly, the N*/T*
ratio remains relatively high in aprotic media, so that the TCeU
fluorophore is capable of distinguishing one apolar aprotic sol-
vent from another; whereas, both the parent TC (Table 2,
Figure 6) and its structural analogue 2-furyl-3-hydroxychro-
mone[20] show a uniformly low N*/T* ratio in these solvents. By
this feature, TCeU is comparable to 4’-dialkylamino-3-hydroxy-
flavones that also possess an ICT nature of their normal excited
state. However, the dependence of the N*/T* ratio of TCeU
versus ET(30) shows some upward deviation for aprotic sol-
vents that present strong H-bond-acceptor ability (basicity).
The upward influence of basic solvents on the relative intensity
of N* band was observed even at trace concentration of
a basic solvent originating from the stock solution; notably,
this was observed only for fully conjugated TCeU (Supporting
Information, Figure S1).
Experimental Section
General methods
All non-aqueous reactions involving water-sensitive reagents were
performed in oven-dried glassware under argon using dry solvents.
The synthetic intermediates were co-evaporated twice with tolu-
ene and dried in vacuo before use. All chemical reagents were ob-
tained from commercial sources and were used as supplied. Anhy-
drous solvents were obtained according to standard procedures.[43]
The reactions were monitored by thin-layer chromatography (TLC,
Merck silica gel 60 F254 plates) and visualized both by UV radiation
(254 and 365 nm) and by spraying with phosphomolybdic acid in
ethanol followed by a subsequent warming with a heat gun. Flash
column chromatography[44] was performed by using silica gel (40–
This deviation indicates that these aprotic solvents can
hamper the ESIPT reaction in TCeU, probably by disrupting its
intramolecular H bonding. This phenomenon is not usually ob-
served for 3HC derivatives,[15] but it is a common property of 3-
hydroxyquinolones.[40] A similar effect of basicity was also ob-
served for several other 2-thienyl-3-hydroxychromones.[25] The
effect of solvent basicity on TCeU emission confirms the fact
that unlike other 3HCs, its complex with disrupted H bonding
is probably emissive, in line with its relatively high quantum
2
63 mm). All NMR spectra (1H, 13C, D) were recorded on 200 or 500
1
Bruker Advance Spectrometers (200 or 500 MHz). H NMR (200 and
500 MHz), 13C NMR (50 and 125 MHz, recorded with complete
proton decoupling) spectra were obtained with samples dissolved
in CDCl3, CD3OD, or [D6]DMSO, with the solvent signal used as an
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internal reference: d=7.26 for CHCl3, 3.31 for CD2HOD, and
2.50 ppm for (CD3)(CD2H)S(O) for H NMR experiments, and d=77.0
wise addition of 30% aq. hydrogen peroxide solution (2 mL). The
resulting mixture was stirred 7 h at RT, then poured into cold water
(300 mL) and neutralized with diluted acetic acid. The resulting
precipitate was filtered and thoroughly washed with water and
hexane to provide as a yellow solid the target derivative 10
(1.49 g, 48%; C13H7BrO3S (323.16)). 1H NMR (200 MHz, CDCl3): d=
7.19 (d, 3J=4.0 Hz, 1H, Hb-thioph.), 7.41 (td, 3J=8.0, 4J=0.8 Hz,
1H, H6), 7.54 (d, 3J=8.4 Hz, 1H, H8), 7.71 (ddd, 3J=8.4, 3J=8.0,
1
for CDCl3, 49.0 for CD3OD, and 39.4 ppm for (CD3)2S(O) for 13C NMR
experiments.[45] Chemical shifts (d) are given in ppm to the nearest
0.01 (1H) or 0.1 ppm (13C). The coupling constants (J) are given in
Hertz (Hz). The signals are reported as follows: (s=singlet, d=dou-
blet, t=triplet, m=multiplet, br=broad). Assignments of 1H and
13C NMR signals were achieved with the help of D/H exchange,
COSY, HMQC, HMBC experiments. Regular mass spectra (MS) were
recorded on an Esquire 3000 Plus apparatus with ESI in both posi-
tive and negative mode. High-resolution mass spectrometry was
conducted with a FINIGAN MAT 95 spectrometer with EI or ESI ion-
ization techniques. Systematic flavone and nucleobase nomencla-
ture is used below for the assignments of each spectrum. All sol-
vents for absorption and fluorescence experiments were of spec-
troscopic grade. Absorption spectra were recorded on a Cary 4
spectrophotometer (Varian) using 1 cm quartz cells. Stock solutions
were prepared using dimethyl sulfoxide, except for TCeU for which
dioxane was used. The samples used for spectroscopic measure-
ments contained approximately 0.1% v/v of the stock solvent.
Fluorescence spectra were recorded on a FluoroLog 3.5 spectro-
fluorometer (Jobin Yvon, Horiba). Excitation wavelength was
360 nm. Time-resolved fluorescence measurements were per-
formed with the time-correlated, single-photon counting technique
using the excitation pulses at 315 nm provided by a pulse-picked
frequency-tripled Ti:sapphire laser (Tsunami, Spectra Physics)
pumped by a Millenia X laser (Spectra Physics). The fluorescence
decays were collected at the magic angle (54.78) of the emission
polarizer to avoid any artifact due to vertically polarized excitation
beam. The single-photon events were detected with a microchan-
nel plate photomultiplier (Hamamatsu) coupled to a pulse pream-
plifier HFAC (Becker-Hickl) and recorded on a SPC-130 board
(Becker-Hickl). Time-resolved data were analyzed by the maximum
entropy method (Pulse 5 software).[46] In all cases, the c2 values
were close to 1 and the weighted residuals as well as their auto-
correlation were distributed randomly around 0, indicating an opti-
mal fit.
3
4J=1.4 Hz, 1H, H7), 7.73 (d, J=4.0 Hz, 1H, Ha-thioph.), 8.22 ppm
3
4
(dd, J=8.0, J=1.4 Hz, 1H, H5); 13C NMR (50 MHz, [D6]DMSO): d=
112.2 (Th-Br), 117.8 (C8), 121.6 (Ca), 122.0 (C10), 122.9 (C6), 124.7
(C5), 129.3 (Cb), 131.6 (C7), 136.1 (C2-Th), 141.6 (C3), 146.5 (C2),
153.3 (C9), 176.0 ppm (C4); MS (ESI+): m/z: 344.9 [M+Na]+.
3-Methoxy-2-(thiophen-2-yl)-chromen-4-one (4): A solution of
4.5m KOH (2 mL), 18-crown-6 (5 mol%, 25 mg), and dimethyl sul-
fate (370 mL, 3.86 mmol) were added to a stirred suspension of 2
(472 mg, 1.93 mmol) in CH2Cl2 (8 mL). The reaction mixture
became homogenous in 5 min and was stirred at RT for 1 h. After
quenching by addition of H2O (10 mL), the organic layer was ex-
tracted with CH2Cl2 (3ꢃ), dried over MgSO4, filtered, and the vola-
tiles were removed in vacuo. The residue was purified by flash
chromatography on silica gel eluted with cyclohexane/EtOAc mix-
ture (10:1!4:1, v/v) to provide the desired compound 4 as
1
a yellow solid (483 mg, 97%; C14H10O3S (258.29)). H NMR (200 MHz,
CDCl3): d=4.06 (s, 3H, CH3), 7.19 (dd, 3J=4.0, 5.0 Hz, 1H, Hb-
thioph.), 7.36 (td, 3J=8.0, 4J=1.0 Hz, H6), 7.50 (dd, 3J=8.4, 4J=
3
4
1.0 Hz, 1H, H8), 7.60 (dd, J=5.0, J=1.2 Hz, 1H, Hg-thioph.), 7.65
(td, 3J=8.4, 4J=1.6 Hz, 1H, H7), 7.94 (dd, 3J=4.0, 4J=1.2 Hz, 1H,
Ha-thioph.), 8.22 ppm (dd, 3J=8.0, 4J=1.6 Hz, 1H, H5); 13C NMR
(50 MHz, CDCl3): d=59.6 (OMe), 117.7 (C8), 124.2 (C10), 124.6 (C6),
125.6 (C5), 127.4 (Cb), 129.5 (Ca), 131.5 (Cg), 131.7 (C2-Th), 133.3
(C7), 138.6 (C3), 151.5 (C2), 154.7 (C9), 174.0 ppm (C4); HRMS
(ESI+): m/z calcd for C14H11O3S: 259.0423 [M+H]+; found 259.0424.
3-Methoxy-2-(5-bromothiophen-2-yl)-chromen-4-one (11): A solu-
tion of 4.5m aq. KOH (5 mL), 18-crown-6 (5 mol%, 65 mg), and di-
methyl sulfate (930 mL, 9.84 mmol) were added to a stirred suspen-
sion of 10 (1.59 g, 4.92 mmol) in CH2Cl2 (35 mL). The reaction mix-
ture became homogenous in 5 min and was stirred at RT for 1 h.
After quenching by addition of H2O (40 mL), the organic layer was
extracted with CH2Cl2 (3ꢃ), dried over MgSO4, filtered and the vola-
tiles were removed in vacuo. The residue was purified by flash
chromatography on silica gel eluted with cyclohexane/EtOAc mix-
ture (10:1!4:1, v/v) to provide the desired compound 11 as
Synthesis
3-Hydroxy-2-(thiophen-2-yl)-chromen-4-one (2): A 5n NaOH solu-
tion (6 mL) was added drop-wise to a stirred solution of o-hydroxy-
acetophenone 7 (1.16 mL, 9.63 mmol) and 2-thiophenecarbalde-
hyde 8 (900 mL, 9.63 mmol) in ethanol (50 mL). The reaction mix-
ture was stirred 48 h at RT before the drop-wise addition of 30%
aq. hydrogen peroxide solution (2 mL). The resulting mixture was
stirred 7 h at RT, then poured into cold water (300 mL) and neutral-
ized with diluted acetic acid. The resulting precipitate was filtered
and thoroughly washed with water and hexane to provide the
target derivative 2 (1.17 g, 50%; C13H8O3S (244.27)) as a yellow
solid. 1H NMR (200 MHz, [D6]DMSO): d=7.30 (dd, 3J=5.0, 3J=
1
a yellow solid (1.30 g, 78%; C14H9BrO3S (337.19)). H NMR (200 MHz,
CDCl3): d=4.07 (s, 3H, CH3), 7.16 (d, 3J=3.8 Hz, 1H, Hb-thioph.),
7.38 (td, 3J=8.0 Hz, 4J=1.0 Hz, H6), 7.49 (dd, 3J=8.4, 4J=1.0 Hz,
3
1H, H8), 7.62–7.79 (m, 1H, H7), 7.66 (d, J=3.8 Hz, 1H, Ha-thioph.),
8.22 ppm (dd, 3J=8.0, 4J=1.6 Hz, 1H, H5); 13C NMR (50 MHz,
CDCl3): d=59.7 (OMe), 117.7 (C8), 119.8 (Th-Br), 124.2 (C10), 124.7
(C6), 125.7 (C5), 129.2 (Ca), 130.2 (Cb), 132.7 (C2-Th), 133.5 (C7),
138.4 (C3), 150.2 (C2), 154.7 (C9), 173.8 ppm (C4); HRMS (ESI+): m/z
calcd for C14H10BrO3S: 336.9529 [M+H]+; found 336.9531.
3
4
3.8 Hz, 1H, Hb-thioph.), 7.46 (td, J=8.0, J=1.2 Hz, 1H, H6), 7.72
(dd, 3J=8.4, 4J=1.2 Hz, 1H, H8), 7.73–7.84 (m, 1H, H7), 7.91 (dd,
3J=5.0, 4J=1.2 Hz, 1H, Hg-thioph.), 7.97 (dd, 3J=3.8, 4J=1.2 Hz,
1H, Ha-thioph.), 8.10 (dd, J=8.0, 1.2 Hz, 1H, H5), 10.28 ppm (s, 1H,
OH); 13C NMR (50 MHz, [D6]DMSO): d=118.0 (C8), 121.7 (C10), 124.5
(C6), 124.7 (C5), 127.7 (Cb), 128.3 (Ca), 130.9 (Cg), 132.3 (C2-Th),
133.6 (C7), 136.6 (C3), 143.1 (C2), 154.1 (C9), 171.9 ppm (C4); MS
(ESI+): m/z: 267.0 [M+Na]+.
3-Benzyloxycarbonyloxy-2-(5-bromothiophen-2-yl)-chromen-4-
one (12): A solution of 4.5m aq. KOH (9 mL), 18-crown-6 (5 mol%,
43 mg), and benzyl chloroformate (700 mL, 4.88 mmol) were added
to a stirred suspension of 10 (1.05 g, 3.25 mmol) in CH2Cl2 (13 mL).
The reaction mixture became homogenous in 5 min and was
stirred at RT for 1 h. After quenching by addition of H2O (10 mL),
the organic layer was extracted with CH2Cl2 (3ꢃ), dried over
MgSO4, filtered, and the volatiles were removed in vacuo. The resi-
due was purified by flash chromatography on silica gel eluted with
cyclohexane/EtOAc mixture (9:1!7:3, v/v) to provide the desired
compound 12 as white crystals (1.20 g, 81%; C21H13BrO5S (457.29)).
2-(5-Bromothiophen-2-yl)-3-hydroxy-chromen-4-one (10): A 5n
NaOH solution (6 mL) was added drop-wise to a stirred solution of
o-hydroxyacetophenone 7 (1.16 mL, 9.63 mmol) and 5-bromo-2-thi-
ophenecarbaldehyde 9 (1.14 mL, 9.63 mmol) in ethanol (50 mL)
was. The reaction mixture was stirred 48 h at RT before a drop-
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Rf =0.53 (cyclohexane/EtOAc=7:3); 1H NMR (200 MHz, CDCl3): d=
(763 mg, 68%; C6H7IN2O2 (266.04)) as an amorphous white solid.
1H NMR (200 MHz, CDCl3): d=3.41 (s, 3H, N3CH3), 3.42 (s, 3H,
N1CH3), 7.64 ppm (s, 1H, H6); 13C NMR (50 MHz, CDCl3): d=29.4
(N3CH3), 37.2 (N1CH3), 67.1 (C5), 147.3 (C6), 151.4 (C2), 160.4 ppm
(C4); MS (ESI+): m/z: 288.9 [M+Na]+.
3
5.37 (s, 2H, CH2), 7.16 (d, J=4.0 Hz, 1H, Hb-thioph.), 7.34–7.50 (m,
3
4
3
6H, H6 and Cbz), 7.52 (dd, J=8.4, J=0.6 Hz, 1H, H8), 7.65 (d, J=
4.0 Hz, 1H, Ha-thioph.), 7.72 (td, 3J=8.4, 4J=1.6 Hz, 1H, H7),
8.25 ppm (dd, 3J=8.0, 4J=1.6 Hz, 1H, H5); 13C NMR (50 MHz,
CDCl3): d=71.3 (OC(O)CH2Ph), 117.8 (C8), 120.4 (Th-Br), 123.6 (C10),
125.4 (C6), 126.1 (C5), 128.4 (o-CPh), 128.7 (m-CPh), 128.8 (p-CPh),
131.0 (Cb), 131.0 (Ca), 131.5 (C2-Th), 132.0 (C3), 134.1 (C7), 134.5
(i-CPh), 150.3 (C2), 151.8 (OC(O)CH2Ph), 155.0 (C9), 171.3 ppm (C4);
HRMS (ESI+): m/z calcd for C21H14BrO5S: 456.9740 [M+H]+; found:
456.9732.
5-(Trimethylsilyl)ethynyl-1,3-N,N-dimethyluracil
(15):
[PdCl2-
(PPh3)2] (6 mol%, 89 mg) and CuI (6 mol%, 24 mg) were added to
a stirred solution of alkenyl iodide 14 (544 mg, 2.0 mmol), trime-
thylsilylacetylene (441 mL, 3.0 mmol), and DIPEA (1.80 mL,
10.0 mmol) in a 2:1 THF/DMF mixture (10 mL) under argon. The re-
action mixture was warmed to 558C for 4 h. Then, the volatiles
were removed in vacuo and the residue was purified by flash chro-
matography on silica gel eluted with toluene/EtOAc (10:1!4:1,
v/v) to provide the TMS-alkyne derivative 15 (390 mg, 82%;
C11H16N2O2Si (236.34)) as a white foam. 1H NMR (200 MHz, CDCl3):
d=0.17 (s, 9H, SiMe3), 3.30 (s, 3H, N3CH3), 3.38 (s, 3H, N1CH3),
7.49 ppm (s, 1H, H6); 13C NMR (50 MHz, CDCl3): d=ꢀ0.3 (SiMe3),
28.1 (N3CH3), 37.2 (N1CH3), 95.6 (-CꢂC-TMS), 98.7 (-CꢂC-TMS), 98.7
(C5), 146.2 (C6), 150.7 (C2), 161.5 ppm (C4); MS (ESI+): m/z: 259.1
[M+Na]+.
3-Hydroxy-2-(5-((trimethylsilyl)ethynyl)thiophen-2-yl)-chromen-
4-one (3): [PdCl2(PPh3)2] (3 mol%, 20 mg) and CuI (3 mol%, 5 mg)
were added to a stirred solution of aryl bromide 10 (323 mg,
1 mmol), trimethylsilylacetylene (210 mL, 1.5 mmol) and N,N-diiso-
propylethylamine (DIPEA; 280 mL, 2 mmol) in THF (6 mL) under
argon. The mixture was heated at reflux for 1 h, then allowed to
cool down to RT. The volatiles were removed in vacuo and the resi-
due was purified by flash chromatography on silica gel eluted with
CH2Cl2/MeOH (99:1!97:3, v/v) to provide the coupled derivative 3
as a yellow solid (142 mg, 41%; C18H16O3SSi (340.47)). 1H NMR
5-Ethynyl-1,3-N,N-dimethyluracil (16): TBAF (1m in THF, 931 mL,
0.93 mmol) was added drop-wise to a stirred solution of 15
(200 mg, 0.85 mmol) in THF (150 mL). The reaction mixture was
stirred at RT for 1 h. Then, the volatiles were removed in vacuo
and the residue was purified by flash chromatography on silica gel
eluted with toluene/EtOAc (10:1!3:1, v/v) to provide the terminal
alkyne 16 (126 mg, 91%; C8H8N2O2 (164.16)) as a white solid.
1H NMR (500 MHz, CDCl3): d=3.17 (s, 1H, (-CꢂCH), 3.37 (s, 3H,
N3CH3), 3.43 (s, 3H, N1CH3), 7.50 ppm (s, 1H, H6); 13C NMR
(125 MHz, CDCl3): d=28.4 (N3CH3), 37.4 (N1CH3), 74.9 (-CꢂCH), 81.6
(-CꢂCH), 98.0 (C5), 146.4 (C6), 150.9 (C2), 161.8 ppm (C4); MS
(ESI+): m/z: 187.0 [M+Na]+.
3
(500 MHz, [D6]DMSO): d=0.27 (s, 9H, SiMe3), 7.47 (dd, J=8.0 Hz,
3J=7.5 Hz, H6), 7.50 (d, 3J=4.0 Hz, 1H, Hb-thioph.), 7.72 (d, 3J=
3
3
4
8.5 Hz, 1H, H8), 7.81 (ddd, J=8.5, J=7.5, J=1.5 Hz, 1H, H7), 7.85
(d, 3J=4.0 Hz, 1H, Ha-thioph.), 8.10 (dd, 3J=8.0, 4J=1.5 Hz, 1H,
H5); 10.59 ppm (brs, 1H, OH); 13C NMR (125 MHz, [D6]DMSO): d=
ꢀ0.4 (SiMe3), 97.2 (-CꢂC-TMS), 101.9 (-CꢂC-TMS), 118.0 (C8), 121.7
(C10), 124.5 (C6), 124.8 (C5), 125.3 (C2-Th), 127.8 (Ca), 133.2 (Cb),
133.5 (Th-CꢂC), 133.7 (C7), 137.5 (C3), 141.9 (C2), 154.1 (C9),
172.0 ppm (C4); HRMS (ESI+): m/z calcd for C18H17O3SSi: 341.0662
[M+H]+; found: 341.0657.
3-Methoxy-2-(5-((trimethylsilyl)ethynyl)thiophen-2-yl)-chromen-
4-one (5): [PdCl2(PPh3)2] (4 mol%, 45 mg) and CuI (4 mol%, 12 mg)
were added to a stirred solution of aryl bromide 11 (536 mg,
1.6 mmol), trimethylsilylacetylene (340 mL, 2.4 mmol), and DIPEA
(430 mL, 3.2 mmol) in THF (10 mL) under argon. The mixture was
heated at reflux for 2 h, then allowed to cool down to RT. The vola-
tiles were removed in vacuo and the residue was purified by flash
chromatography on silica gel eluted with cyclohexane/EtOAc mix-
ture (9:1!5:1, v/v) to provide the coupled derivative 5 as yellow
solid (554 mg, 98%; C19H18O3SSi (354.49)). 1H NMR (500 MHz,
5-[5-(3-Benzyloxycarbonyloxy-4-oxo-chromen-2-yl)-thiophen-2-
yl-ethynyl]-1,3-N,N-dimethyluracil (17): [PdCl2(PPh3)2] (5 mol%,
19 mg) and CuI (5 mol%, 5 mg) were added to a stirred solution of
aryl bromide 12 (236 mg, 0.50 mmol), 16 (92 mg, 0.55 mmol), and
DIPEA (450 mL, 2.51 mmol) in THF (5 mL) under argon. The mixture
was heated at reflux for 5 h, then allowed to cool down to RT. The
volatiles were removed in vacuo and the residue was purified by
flash chromatography on silica gel eluted with CH2Cl2/Et2O (9:1!
6:1, v/v) to provide the coupled derivative 17 as an orange solid
3
1
CDCl3): d=0.28 (s, 9H, SiMe3), 4.09 (s, 3H, CH3), 7.29 (d, J=4.0 Hz,
(160 mg, 59%; C29H20N2O7S (540.5)). H NMR (500 MHz, CDCl3): d=
1H, Hb-thioph.), 7.39 (dd, 3J=8.0, 3J=7.0 Hz, H6), 7.50 (d, 3J=
3.39 (s, 3H, N3CH3), 3.47 (s, 3H, N1CH3), 5.38 (s, 2H, OC(O)OCH2Ph),
3
3
4
3
3
8.0 Hz, 1H, H8), 7.67 (ddd, J=8.0, J=7.0, J=1.2 Hz, 1H, H7), 7.79
(d, 3J=4.0 Hz, 1H, Ha-thioph.), 8.24 ppm (dd, 3J=8.0, 4J=1.2 Hz,
1H, H5); 13C NMR (125 MHz, CDCl3): d=ꢀ0.2 (SiMe3), 59.8 (OMe),
96.9 (-CꢂC-TMS), 102.7 (-CꢂC-TMS), 117.7 (C8), 124.3 (C10), 124.8
(C6), 125.8 (C5), 128.9 (C2-Th), 129.0 (Ca), 132.3 (Th-CꢂC), 132.6
(Cb), 133.5 (C7), 139.0 (C3), 150.5 (C2), 154.8 (C9), 173.9 ppm (C4);
HRMS (ESI+): m/z calcd for C19H19O3SSi: 355.0819 [M+H]+; found
355.0815.
7.27 (d, J=4.0 Hz, 1H, Hb-thioph.), 7.37 (d, J=7.0 Hz, 1H, p-HPh),
3
3
7.41 (t, J=7.0 Hz, 2H, m-HPh), 7.42 (m, 1H, H6’), 7.48 (s, J=7.0 Hz,
2H, o-HPh), 7.53 (d, J=8.0 Hz, 1H, H8’), 7.57 (s, 1H, H6), 7.71 (ddd,
3
3J=8.0 Hz, J=7.0 Hz, J=1.8 Hz, 1H, H7’), 7.76 (d, J=4.0 Hz, 1H,
Ha-thioph.), 8.23 ppm (dd, 3J=7.8, 4J=1.8 Hz, H5’); 13C NMR
(125 MHz, CDCl3): d=28.4 (N3CH3), 37.5 (N1CH3), 71.3 (OC(O)CH2Ph),
85.6 (Th-CꢂC-), 88.5 (Th-CꢂC-), 98.3 (C5), 117.9 (C8’), 123.6 (C10’),
125.3 (C6’), 126.0 (C5’), 128.3 (o-CPh), 128.7 (m-CPh), 128.7 (p-CPh),
129.2 (C2’-Th-), 130.7 (Ca), 131.6 (Th-CꢂC-), 131.9 (C3’), 132.8 (Cb),
134.1 (C7’), 134.5 (i-CPh), 145.9 (C6), 150.6 (C2’), 150.7 (C2), 151.8
(OC(O)CH2Ph), 155.0 (C9’), 161.1 (C4), 171.3 ppm (C4’); HRMS (ESI+):
m/z calcd for C29H20N2NaO7S: 563.0883 [M+Na]+; found: 563.0872.
3
4
3
5-Iodo-1,3-N,N-dimethyluracil (14): Dimethyl sulfate (870 mL,
9.2 mmol) was added drop-wise to a stirring slurry of 5-iodouracil
13 (1.00 g, 4.2 mmol) and NaOH (497 mg, 10.5 mmol) in H2O (7 mL)
at 08C. The mixture was heated under reflux for 2 h, cooled to RT,
extracted with CH2Cl2 (3ꢃ), dried over MgSO4, filtered, and reduced
in vacuo. The resulting insoluble solid after the evaporation was
washed several times with cyclohexane and subsequently with tol-
uene, ether, EtOAc, and cold CH2Cl2. For each solvent, the solid par-
ticles were allowed to settle and the supernatant washings were
taken out carefully with a syringe with a fine needle. The washings
were collected and evaporated and the residual solid was washed
as before. All the residues were combined and dried to obtain 14
5-[5-(3-Hydroxy-4-oxo-chromen-2-yl)-thiophen-2-yl-ethynyl]-1,3-
N,N-dimethyluracil (1): A solution of 33% aq. NH4OH (870 mL,
22.2 mmol) was added drop-wise to a stirred solution of 17
(30 mg, 55.5 mmol) in CH2Cl2 (7 mL). The reaction mixture was
stirred at room temperature for 1 h. The resulting insoluble solid
after the evaporation was washed several times with cyclohexane
and subsequently with toluene, ether, EtOAc, and CH2Cl2. For each
solvent, the solution was centrifuged for settling solid particles and
Chem. Eur. J. 2014, 20, 1998 – 2009
www.chemeurj.org
2007
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
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the supernatant washings were carefully taken out with a syringe
with a fine needle. The washings were collected and evaporated
and the residual solid was washed as before. All the residues were
combined and dried to obtain 1 (18 mg, 81%; C21H14N2O5S (406.4))
1
as a bright-yellow solid. H NMR (500 MHz, CDCl3): d=3.42 (s, 3H,
3
N3CH3), 3.48 (s, 3H, N1CH3), 6.94 (brs, 1H, OH), 7.36 (d, J=4.0 Hz,
1H, Hb-thioph.), 7.42 (ddd, 3J=7.0, 3J=8.0, 4J=1.5 Hz, 1H, H’6),
7.56 (d, 3J=8.0 Hz, 1H, H8’), 7.57 (s, 1H, H6), 7.71 (ddd, 3J=7.0,
3J=8.0, 4J=1.5 Hz, 1H, H7’), 7.88 (d, 3J=4.0 Hz, 1H, Ha-thioph.),
3
4
8.23 ppm (dd, J=8.0 Hz, J=1.5 Hz, 1H, H5’); 13C NMR (125 MHz,
CDCl3): d=28.5 (N3CH3), 37.5 (N1CH3), 86.3 (Th-CꢂC-), 87.5
(Th-CꢂC-), 98.8 (C5), 118.1 (C8’), 121.0 (C10’), 124.7 (C6’), 125.5 (C5’),
126.8 (C2’-Th-), 129.1 (Ca), 133.1 (C7’), 133.8 (Cb), 134.0 (C3’), 136.7
(Th-CꢂC-), 141.6 (C2’), 145.5 (C6), 150.8 (C2), 155.1 (C9’), 161.3 (C4),
172.4 ppm (C4’); HRMS (ESI+): m/z calcd for C21H15N2O5S: 407.0696
[M+H]+; found: 407.0683.
5-[5-(3-Methoxy-4-oxo-chromen-2-yl)-thiophen-2-yl-ethynyl]-1,3-
N,N-dimethyluracil (6): [PdCl2(PPh3)2] (6 mol%, 16 mg) and CuI
(6 mol%, 4 mg) were added to a stirred solution of aryl bromide
11 (125 mg, 0.35 mmol), 16 (65 mg, 0.39 mmol), and DIPEA
(415 mL, 1.76 mmol) in THF (4 mL) under argon. The mixture was
heated at reflux for 5 h, then allowed to cool down to RT. The re-
sulting insoluble solid after the evaporation was washed several
times with cyclohexane and subsequently with toluene, ether,
EtOAc, and CH2Cl2. For each solvent, the solution was centrifuged
for settling solid particles and the supernatant washings were care-
fully taken out with a syringe with a fine needle. The washings
were collected and evaporated and the residual solid was washed
as before. All the residues were combined and dried to obtain 6
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3
3.99 (s, 3H, OCH3), 7.50 (d, J=4.0 Hz, 1H, Hb-thioph.), 7.50 (ddd,
3
4
3
3J=7.5, J=8.0, J=1.0 Hz, 1H, H6’), 7.77 (d, J=7.5 Hz, 1H, H8’),
3
4
3
7.84 (td, J=7.5, J=1.5 Hz, 1H, H7’), 7.98 (d, J=4.0 Hz, 1H, Ha-
thioph.), 8.09 (dd, 3J=8.0, 4J=1.5 Hz, 1H, H5’), 8.36 ppm (s, 1H,
H6); 13C NMR (125 MHz, [D6]DMSO): d=27.8 (N3CH3), 36.8 (N1CH3),
59.4 (OCH3), 84.1 (Th-CꢂC-), 90.0 (Th-CꢂC-), 95.2 (C5), 118.2 (C8’),
123.6 (C10’), 124.8 (C6’), 125.1 (C5’), 130.0 (Ca), 131.3 (C2’-Th-),
132.1 (C7’), 134.1 (Cb), 138.2 (C3’), 148.8 (C6), 149.5 (Th-CꢂC-), 150.4
(C2’), 154.3 (C9’), 160.9 (C2), 172.7 (C4), 186.8 ppm (C4’); HRMS
(ESI+): m/z calcd for C22H16N2NaO5S: 443.0672 [M+Na]+; found:
443.0670.
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Acknowledgements
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We thank the ANR (ANR-12-BS08-0003-02) and the FRM
(DCM20111223038) for financial support and a grant for N.B.
We thank the French Embassy in Ukraine for the Master 2
grant to I.A.K. We are grateful to Moses Moustakim for critical
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Keywords: charge transfer · fluorescent probes · nucleobases ·
solvatochromism · time-resolved spectroscopy
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