A turn-on coumarin-based fluorescent sensor with high selectivity for mercury ions in aqueous media

Article abstract of DOI:10.1039/b926384e

The synthesis and spectral profile of a turn-on fluorescent sensor selective for Hg²⁺ ions in aqueous media is described.

Full text of DOI:10.1039/b926384e

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A ‘‘turn-on’’ coumarin-based fluorescent sensor with high selectivity
for mercury ions in aqueous mediaw
Styliani Voutsadaki, George K. Tsikalas, Emmanuel Klontzas, George E. Froudakis and
Haralambos E. Katerinopoulos*
Received (in Cambridge, UK) 15th December 2009, Accepted 2nd March 2010
First published as an Advance Article on the web 25th March 2010
DOI: 10.1039/b926384e
The synthesis and spectral profile of a ‘‘turn-on’’ fluorescent
sensor selective for Hg2+ ions in aqueous media is described.
maxima. Moreover, depending of the degree of their
hydrophilicity, mercury probes have been used in organic
solvents, aqueous–organic systems (with the organic solvent
comprising Z 5% of the mixture), and in aqueous solutions.
One of the best combinations is that of turn-on/aqueous
sensors since their use has a number of advantages such as
(a) it reduces the chance of a false positive, observed in some
turn-off probes, (b) allows for the use of multiple probes,
selective for different ions, and (c) is applicable in the analysis
of both, aqueous environmental and biological samples.1
In this report we describe the synthesis and fluorescence
spectral profile studies of a new ratiometric fluorescent ion
probe. Structure-wise the compound consists of a monoaza-
15-crown-5 moiety, acting as an ionophore, connected via
Mercury contamination, bioaccumulation, and the resulting
alarming consequences on human health are problems of
global dimensions.1 The Environmental Protection Agency
of the USA has already set the upper limit of Hg2+ levels in
drinking water as low as 2 ppb (10 nM).2 Human health risks,
springing from mercury exposure have been the subject of
extensive investigations.3 Ethylmercury in the form of a
preservative is added to certain vaccines.4 Methyl mercurial
species are known to be involved in CNS dysfunction.5
Inorganic mercury is also neurotoxic, and attacks the immune
system.6 Endocrine-related mechanisms of mercury action
across the thyroid and adrenal systems have been detected.7
A number of hypotheses were raised about potential molecular
mechanisms for the metal genotoxicity.8 The presence of
Hg2+–Se2+ compounds in biological systems has been
suggested and their potentially key roles in alleviating the
toxicity of Hg2+ has been studied.9 Detecting and monitoring
of mercury ions in environmental and biological systems
today present a great challenge for the scientific community,
triggering a large number of related investigations that have
been recently reviewed.1,10
Hg2+ belongs to the so-called ‘‘silent ions’’ since, unlike
some biological metal ions (such as Fe2+, Mn2+ or Cu2+), it
does not have an intrinsic spectroscopic or magnetic
signal because of its 5d10 6s0 electronic configuration. The
fluorometric detection of such ions is based in the use of
small-molecule fluorescent sensors that consist of an ionophore–
chromophore system. These probes may coordinate selectively
to the target ion and respond to changes in ion levels with
changes in their fluorescence profile that may be translated to
accurate measurements of the ion concentrations.
a
thiourea linker to 7-amino-4-methylcoumarin, which
functions as the chromophore of the system (Scheme 1). The
choice of the ionophore was based in previous literature
reports using similar crown ether systems for the coordination
of heavy metals.1,10,11 The choice of the aminocoumarin
presented
a challenge. Aminocoumarins incorporate an
electron donor (amino group) conjugated to an electron
acceptor (carbonyl group). This system is expected to follow
a photoinduced charge transfer (PCT) mechanism. Upon ion
binding, a blue or a red shift is observed in the excitation
spectrum, provided that the target ion, in this case Hg2+
,
coordinates with the donor or the acceptor moiety,
respectively. Studies on N-substituted and N,N-disubstituted
7-aminocoumarins indicated that excitation of the system
leads to a twisted intramolecular charge transfer (TICT) state
and therefore, to radiationless decay.12–15 Alternatively, the
system is expected to follow a photoinduced electron transfer
(PET) mechanism where, upon excitation of the free probe, an
electron would be transferred from the crown ether to the
fluorophore diminishing fluorescence.16 Coordination of
the mercury ion with the heteroatoms of the crown ether
would cancel the electron transfer, increasing considerably
the fluorescence signal, a process followed in a turn-on sensor
system.
Depending on the impact of Hg2+ levels to the spectral
changes of the free probe, mercury fluorescent probes have
been subdivided into ‘‘turn-on’’ (enhancement of fluorescence
in the presence of Hg2+), ‘‘turn-off’’ (quenching of fluorescence
by Hg2+), and ratiometric probes, the latter group exhibiting a
mercury-induced shift in the excitation and/or emission
Probe 3 was synthesized in only two steps, via an isothio-
cyanate intermediate, in 97% overall yield (Scheme 1). The
synthesis of 4-methyl-7-[(2-thioxoethenylidene)amino]-1H-
isochromen-1-one (2) was accomplished easily by using two
equivalents of base, excess of CSCl2, dichloromethane as
the solvent, and heating at 35–40 1C. It is important that the
excess of thiophosgene is fully removed (caution!), after the
reaction is completed, to avoid reaction with the monoaza-
macrocycle in the next step. Nucleophilic attack of the
Department of Chemistry, University of Crete, Voutes Campus, Crete,
71003 Heraklion, Greece. E-mail: kater@chemistry.uoc.gr;
Fax: +30 2810 545001; Tel: +30 2810 545026
w Electronic supplementary information (ESI) available: Experimental
section, 1H, 13C NMR, and all fluorescence spectra not shown in the
text. See DOI: 10.1039/b926384e
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Scheme 1 Synthesis of fluorescent Hg2+ sensors 3.
ionophore at intermediate 2 was carried out very rapidly, at
room temperature, and almost quantitatively (see ESIw).
Fluorescence studies of probe 3 revealed the profile of a
photoinduced electron transfer (PET) indicator with a high
response to the presence of Hg2+ ions. The ion-free form of
this PET probe exhibited close to zero fluorescence intensity,
while addition of increasing concentrations of Hg2+ ions up to
80 mM resulted in a twenty nine-fold increase in fluorescence
intensity, with no shifting in the excitation and emission
maxima (Fig. 1). Spectral studies of probe 3 were performed
in nanopure water at pH = 7.0 and its Kd value for Hg2+ ions
was calculated17 to be 13.1 mM, while lmax of excitation and
emission spectra were detected at 341 nm and 407 nm,
respectively (Table 1).
Ion competition study of probe 3 revealed its selectivity for
Hg2+ in the presence of a host of competitive ions, including
heavy metals (Fig. 2). Specifically, in the first set of bars, the
first bar (indicated as: free) represents the ratio of fluorescence
intensities (Fbound ꢁ Ffree)/Ffree, which is zero, while the
second bar (indicated as Hg(II)) represents the fluorescence
Fig. 1 Excitation (top) and emission (bottom) spectra of sensor 3 in
solutions of increasing Hg2+ concentrations in nanopure water. The
emission and excitation wavelengths were set at 407 and 341 nm,
respectively.
ratio of the probe after the addition of 60 mM Hg2+
.
Table
1 Spectral properties of fluorescent indicator 3, in
Hg2+ solutions
The degree of selectivity of indicator 3 for Hg2+ is depicted
in the next set of measurements. The third bar (Zn(II)) shows
the probe’s fluorescence ratio in the presence of Zn2+ ions. It
has a very low value, indicating that Zn2+ ion binding is very
week, as compared to that of Hg2+. The fourth bar (Zn–Hg)
represents the probe’s fluorescence ratio in the presence of
both Zn2+ and Hg2+ ions: mercury ions do bind to 3 with the
same affinity as before, independently of the presence of zinc
or other competitive ions.
Free
Hg2+-bound
exc/nm lemis/nm F18
lexc/nm
l
emis/nm
F
l
Indicator
Kd/mM
0.43 13.1
3
341
407
0.015 341
407
As mentioned before, the mode of binding of Hg2+ ions
onto the probe is a crucial factor determining the spectral
profile of the mercury sensor that was synthesized. Indeed,
N-substituted and N,N-disubstituted 7-aminocoumarins
undergo charge transfer upon excitation leading to a TICT
state and therefore, to radiationless decay. The question here is
if the nitrogen of a thioamide (such as the 7-aminocoumarin
nitrogen in 3) is able to participate in a charge transfer process
given that its free electron pair is delocalized within the
thiocarbonyl system of the thiourea linker. As indicated in
Fig. 1, the excitation spectrum does not show the wavelength
Fig. 2 Ion competition study for dye 3.
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Notes and references
z DFT calculations were carried out using Becke-Perdew86 (BP86)
functional,21,22 the Resolution-of-the-Identity (RI) approximation23
for computing the electronic Coulombic energy and the def2-SVP24,25
basis set for all atoms (default ECPs were included for Hg) and the
corresponding auxiliary basis sets26 for the RI method as implemented
in the Turbomole 5.9.027 Program Package. The molecular system was
optimized without any constraints during the optimization procedure.
1 E. M. Nolan and S. J. Lippard, Chem. Rev., 2008, 108, 3443.
2 Mercury Update: Impact of Fish Advisories. EPA Fact Sheet
EPA-823-F-01-011; EPA, Office of Water: Washington, DC, 2001.
3 B. L. Vallee and K. H. Falchuk, Physiol. Rev., 1993, 73, 79;
J. E. Fergusson, in The Heavy Elements: Chemistry, Environmental
Impact and Health Effects, Pergamon Press, Oxford, 1990;
P. B. Hammond, and E. C. Foulkes, in Metal Ions in Biology,
H. Siegel, ed. Marcel Dekker, New York, vol. 20, 1986,
pp. 177–182.
4 T. W. Clarkson and L. Magos, Crit. Rev. Toxicol., 2006, 36, 609.
5 T. W. Clarkson, L. Magos and G. J. Myers, N. Engl. J. Med., 2003,
349, 1731; G. Shanker, L. A. Mutkus, S. J. Walker and
M. Aschner, Mol. Brain Res., 2002, 106, 1.
Fig. 3 DFT geometry optimization for the 3–Hg2+ complex. Atoms
are represented in colours; mercury: blue, sulfur: yellow, nitrogen:
pink, oxygen: red, carbon: grey, hydrogen: white.
shift expected in a PCT process. In our systems, containing an
ionophore with heteroatoms, it is known that an alternative
mechanism involves a photoinduced electron transfer (PET)
from the ionophore to the excited chromophore, thus
quenching the fluorescence process in the free probe. When
the metal binds onto the ionophore this process is cancelled
and an enhancement of fluorescence intensity is observed
with increasing metal concentrations. Literature data19 on
compounds containing (a) the 7-aminocoumarin nitrogen as
amide, and (b) a monoaza crown ether, show that upon
photoexcitation the predominant effect is that of the electron
transfer (PET) from the crown ether to the excited fluoro-
phore. In another study20 the authors indicated that this
electron transfer may take place from a 15-crown-5 to a
chromophore group. To verify our proposed interaction of
Hg2+ ions with the heteroatoms in sensor 3 we performed first
principles calculations in the DFT level of theory for the
Hg2+–3 complex.z
6 E. K. Silbergeld, I. A. Silva and J. F. Nyland, Toxicol. Appl.
Pharmacol., 2005, 207, 282.
7 S. W. Tan, J. C. Meiller and K. R. Mahaffey, Crit. Rev. Toxicol.,
2009, 39, 228.
8 M. E. Crespo-Lo
´
pez, G. L. Macedo, S. I. D. Pereira, G. P. F.
o-Diniz, J. L. M. d. Nascimento and
Arrifano, D. L. W. Picanc¸
A. M. Herculano, Pharmacol. Res., 2009, 60, 212.
9 M. A. K. Khan and F. Wang, Environ. Toxicol. Chem., 2009, 281, 567.
10 I. W. Zhu, Y. Xu and X. Qian, Prog. Chem., 2007, 19, 1229; Leray
and B. Valeur, Eur. J. Inorg. Chem., 2009, 3525; D. W. Domaille,
E. L. Que and C. J. Chang, Nat. Chem. Biol., 2008, 4, 168.
11 F. A. Khan, K. Parasuraman and K. K. Sadhu, Chem. Commun.,
2009, 2399.
12 W. Rettig and R. Lapouyade, in Topics in Fluorescence Spectro-
scopy, ed. J. R. Lakowicz, Plenum Press, New York, vol. 4, 1994,
pp. 109–150.
13 B. Bangar Raju, J. Phys. Chem. A, 1997, 101, 981.
14 G. Chu and F. Yangbo, J. Chem. Soc., Faraday Trans. 1, 1987, 83,
2533.
15 J. E. T. Corrie, V. R. N. Munasinghe and W. Rettig, J. Heterocycl.
Chem., 2000, 37, 1447.
16 H. E. Katerinopoulos, Curr. Pharm. Des., 2004, 10, 3835.
17 G. Grynkiewicz, M. Poenie and R. Y. Tsien, J. Biol. Chem., 1985,
260, 3350.
18 Reported quantum yields are based on anthracene, F = 0.27 in
ethanol: W. H. Melhuish, J. Phys. Chem., 1961, 65, 229; A value of
F = 0.36 has also been reported: I. B. Berlman, Handbook of
Fluorescence Spectra of Aromatic Molecules, Academic Press,
New York 1971. Using the latter value, the quantum yield for the
Hg2+-bound sensor was calculated as F = 0.53.
The structure obtained is illustrated in Fig. 3. Apparently,
mercury, being a thiophile, coordinates with the sulfur atom
interacting at the same time with elements of the macrocycle
system. This spatial arrangement prevents the Hg2+ ion from
coordinating with the aminocoumarin nitrogen, therefore
depleting the PCT effect and initiating the PET process.
In conclusion, a water soluble, fluorescent ion probe with
high selectivity for Hg2+ ions was designed and synthesized
via a short and high yield synthetic procedure. The probe
belongs to the turn-on class of sensors, functioning via the
photoinduced electron transfer process, thus providing a dye
that is a potential tool for the detection and quantification of
mercury in environmental and biological samples.
19 P. Kele, J. Orbulescu, T. L. Calhoun, R. E. Gawley and
R. M. Leblanc, Tetrahedron Lett., 2002, 43, 4413.
20 K. Kubo, S. Sakaguchi and T. Sakurai, Talanta, 1999, 49, 735.
21 A. D. Becke, Phys. Rev. A: At., Mol., Opt. Phys., 1988, 38, 3098.
22 J. P. Perdew, Phys. Rev. B: Condens. Matter, 1986, 33, 8822.
This work is part of the 03ED375 research project,
implemented within the framework of the ‘‘Reinforcement
Programme of Human Research Manpower’’ (PENED) and
co-financed by the National and Community Funds
(25% from the Greek Ministry of Development-GSRT and
75% from E.U.-European Social Fund). We thank
Mr T. Stergiannakos for technical expertise, Professor
S. Pergantis and JEOL Inc. for mass spectra recording.
¨
23 K. Eichkorn, O. Treutler, H. Ohm, M. Haser and R. Ahlrichs,
¨
Chem. Phys. Lett., 1995, 242, 652.
24 A. Schafer, H. Horn and R. Ahlrichs, J. Chem. Phys., 1992, 97, 2571.
¨
25 F. Weigend and R. Ahlrichs, Phys. Chem. Chem. Phys., 2005, 7,
3297.
26 F. Weigend, Phys. Chem. Chem. Phys., 2006, 8, 1057.
27 R. Ahlrichs, M. Bar, M. Haser, H. Horn and C. Kolmel, Chem.
¨
Phys. Lett., 1989, 162, 165.
¨
¨
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This journal is The Royal Society of Chemistry 2010
3294 | Chem. Commun., 2010, 46, 3292–3294