A "reactive" ratiometric fluorescent probe for mercury species

Article abstract of DOI:10.1021/ol2011693

A ratiometric fluorescent probe for mercury species is developed based on the metal-promoted hydrolysis of a vinyl ether derivative of 2-(benzothiazol-2-yl)phenol in a buffer solution. The probe responds selectively to mercury species over various other m

Full text of DOI:10.1021/ol2011693

ORGANIC
LETTERS
2011
Vol. 13, No. 13
3422–3425
A “Reactive” Ratiometric Fluorescent
Probe for Mercury Species
Mithun Santra, Basab Roy, and Kyo Han Ahn*
Department of Chemistry and Center for Electro-Photo Behaviors in Advanced
Molecular Systems, POSTECH, San 31 Hyoja-dong, Pohang,
790-784 Republic of Korea
ahn@postech.ac.kr
Received May 2, 2011
ABSTRACT
A ratiometric fluorescent probe for mercury species is developed based on the metal-promoted hydrolysis of a vinyl ether derivative of
2-(benzothiazol-2-yl)phenol in a buffer solution. The probe responds selectively to mercury species over various other metal ions with a marked
fluorescence change from blue to cyan through the excited state intramolecular proton transfer (ESIPT) process. The fluorescence titration is
complete with 0.5 equiv of HgCl2, which indicates that the probe also responds to organomercury species, RHgCl.
The widespread contamination of pollutants such as
highly poisonous mercury species could jeopardize our
ecosystem, imposing a great threat to human health. In
particular, organomercury species (typically methylmer-
cury, CH3HgX, X = Cl, AcOꢀ, etc.) are much more
virulent than inorganic mercury(II), as they readily accu-
mulate in various organs as well as cross the bloodꢀbrain
barrier to cause damage in the central nervous system.1,2
Ingestion of methylmercury-contaminated fish and grain
triggered the epidemics in Japan and in Iraq, respectively.3
Therefore, there is an impetus to develop versatile analy-
tical tools for the detection and quantification of mercury
species including methylmercury. Among the existing ana-
lytical methods, those based on fluorometry attract con-
siderable attention owing to their favorable features of
operational simplicity and cost-effectiveness, in addition
to the high sensitivity and selectivity. A number of fluo-
rescent molecular probes based on the coordination of
heteroatom-based ligands to mercury(II) ions have been
reported in recent years, enabling easy detection of mer-
cury ions with high sensitivity.4 These fluorescent probes,
however, suffer from incomplete selectivity over the com-
peting metal ions and, in most cases, show a lack of
sensitivity toward organomercury species. To overcome
these limitations, recently we devised a novel reaction-
based sensing approach that does not involve the metal ion
coordination by heteroatom-based ligands but relies on a
chemical reaction specific to the mercury species.5 We have
found that the “reactive” fluorescent probe based on the
(4) (a) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443. (b)
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Descalzo, A. B.; Martınez-Manez, R.; Radeglia, R.; Rurack, K.; Soto, J.
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J. Am. Chem. Soc. 2003, 125, 14270. (d) Guo, X.; Qian, X.; Jia, L. J. Am.
Chem. Soc. 2004, 126, 2272. (e) Caballero, A.; Martınez, R.; Lloveras,
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V.; Ratera, I.; Vidal-Gancedo, J.; Wurst, K.; Tarraga, A.; Molina, P.;
Veciana, J. J. Am. Chem. Soc. 2005, 127, 15666. (f) Yoon, S.; Albers,
A. E.; Wong, A. P.; Chang, C. J. J. Am. Chem. Soc. 2005, 127, 16030. (g)
Zhu, X.-J.; Fu, S.-T.; Wong, W.-K.; Guo, J.-P.; Wong, W.-Y. Angew.
Chem., Int. Ed. 2006, 45, 3150. (h) Yoon, S.; Miller, E. W.; He, Q.; Do,
P. H.; Chang, C. J. Angew. Chem., Int. Ed. 2007, 46, 6658.
(5) (a) Santra, M.; Ryu, D.; Chatterjee, A.; Ko, S. K.; Shin, I.; Ahn,
K. H. Chem. Commun. 2009, 2115. (b) Yang, Y. K.; Yook, K. J.; Tae,
J. S. J. Am. Chem. Soc. 2005, 127, 16760. (c) Song, F.; Watanabe, S.;
Floreancig, P. E.; Koide, K. J. Am. Chem. Soc. 2008, 130, 16460. For a
recent review, see: (d) Jun, M. E.; Roy, B.; Ahn, K. H. Chem. Commun.
DOI: 10.1039/c1cc00014d.
(1) (a) ATSDR 1999. Toxicological Profile for Mercury; Atlanta, GA,
U.S. Department of Health and Human Services. (b) ATSDR 2005.
ToxProfiles: Mercury; Atlanta, GA, U.S. Department of Health and Human
Services.
(2) (a) Jitaru, P.; Adams, F. J. Phys. IV 2004, 121, 185. (b) Clarkson,
T. W.; Magos, L. Off. Rev. Toxicol. 2006, 36, 609.
(3) Bakir, F.; Damluji, S. F.; Amin-Zaki, L.; Murtadha, M.; Khalidi,
A.; Al-Rawi, N. Y.; Tikriti, S.; Dhahir, H. I.; Clarkson, T. W.; Smith,
J. C.; Doherty, R. A. Science 1973, 181, 230.
(6) Koide and co-workers later reported a work of mercury sensing
with exactly the same probe as ours (ref 5a): Ando, S.; Koide, K. J. Am.
Chem. Soc. 2011, 133, 2556.
r
10.1021/ol2011693
Published on Web 06/06/2011
2011 American Chemical Society

hydrolysis of an aryl vinyl ether promoted by mercury
species,6 so-called the oxymercuration reaction, can pro-
vide two notable features that overcome the existing
challenges: the reaction is specific toward mercury(II) ions;
more importantly, the reaction also proceeds by methyl-
mercury species. The reactive probe also emits “turn-on”
fluorescence toward the mercury species.
evaluated vinyl ethers 1ꢀ6 with respect to their reactivity
toward HgCl2, their solubility in aqueous media, and their
photophysical properties. The results can be summarized
as follows: The hydrolysis reaction becomes faster when
electron-donating groups are substituted at the phenol
ring, in particular at the para position with respect to the
hydroxyl group; otherwise, the hydrolysis becomes slower,
and thus the corresponding vinyl ethers are not suitable for
the purpose of sensing. For example, vinyl ethers 1ꢀ3
reacted faster with HgCl2 than the meta derivatives 4 and 5
as well as the nonsubstituted analogue 6. Also, among the
para analogues, vinyl ether 1 undergoes the hydrolysis
reaction faster than 2 and 3; the hydrolysis is complete
within 30 min in the case of 1 (10 μM, 2 equiv with respect
to[HgCl2]); however, ittakes more than1 h inthe cases of 2
and 3 (Figures S1ꢀS5). In addition, the vinyl ethers
substituted with the ꢀNHCOPh or ꢀNHCOCF3 group,
for example, 2 and 3, required 20ꢀ30% of CH3CN (by
volume) to dissolve them in water, whereas only ∼1% of
CH3CN is sufficient to dissolve 1 in a phosphate buffer
solution.
Our continuing efforts to develop versatile molecular
probes with useful features for bioimaging, environmental
monitoring, or ready quantification of mercury species led
us to investigate a ratiometric version of the vinyl ether
probe. Such a ratiometric fluorescent probe would offer an
advantage over the intensity-based probes such as less
sensitivity to the errors associated with the probe concen-
tration, photobleaching, instrument’s sensitivity, and en-
vironmental effects.7 Various strategies have been adopted
forthe design of ratiometric fluorescent probes.8 A handful
of ratiometric fluorescent probes for mercury species,
however, have been reported so far.9 As our approach
of the vinyl ether hydrolysis by mercury ions has proven
to be effective,5a a ratiometric fluorescent probe based
on the hydrolysis reaction would provide us with a
valuable tool for the detection of the toxic species, with
an added improvement over the potential errors. Re-
ported here are preliminary results from our efforts
in this endeavor.
2-(Benzothiazol-2-yl)phenol and its derivatives, upon
irradiation, generate the excited-state intramolecular pro-
ton transfer (ESIPT) tautomers (the keto forms), which
fluoresce more strongly and at longer wavelength com-
pared to the phenol forms. Recently O-functionalized
2-(benzothiazol-2-yl)phenols were developed as the ratio-
metric fluorescent probes for anions such as Fꢀ and
phosphatases.8dꢀg In these examples, the O-functionalized
compounds are converted to the starting phenols by
analyte-promoted/-catalyzed reactions, with the conver-
sions offering ratiometric responses because 2-(benzo-
thiazol-2-yl)phenols and their O-functionalized derivatives
fluoresce at different wavelengths.
Figure 1. Vinyl ethers 1ꢀ6 and their devinylated products evalu-
ated. The maximum emission wavelengths were obtained by
excitation at the corresponding maximum absorbance wavelengths.
We designed vinyl ethers 1ꢀ6 as potential ratio-
metric probes for mercury species (Figure 1). These vinyl
ethers are expected to undergo the mercury-promoted
oxymercuration followed by hydrolysis to generate
the corresponding 2-(benzothiazol-2-yl)phenols. We have
The emission data for the vinyl ethers and the correspond-
ing phenols also show that the emission wavelengths are
affected by the substituents. From these results, vinyl ether 1
was chosen as an optimal probe for mercury species as it
shows the desired reactivity, solubility, and photophysical
properties compared with the others. The synthesis and
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2008, 49, 2960. (d) Yang, X.-F.; Qi, H.; Wang, L.; Su, Z.; Wang, G.
Talanta 2009, 80, 92. (e) Kim, I. T.; Kang, J. H.; Han, G.; Chung, J. S.;
Kim, Y. Chem. Commun. 2009, 5895. (f) Hu, R.; Feng, J.; Hu, D.; Wang,
S.; Li, S.; Li, Y.; Yang, G. Angew. Chem., Int. Ed. 2010, 49, 4915. (g)
Chen, H. W.; Xing, Y.; Pang, Y. Org. Lett. 2011, 13, 1362.
Scheme 1. Hydrolysis of Probe 1 by Mercury Species
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8025. (b) Yuan, M.; Li, Y.; Li, J.; Li, C.; Liu, X.; Lv, J.; Xu, J.; Liu, H.;
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Jiang, J.; Tian, H. Sens. Actuators B 2009, 140, 162.
Org. Lett., Vol. 13, No. 13, 2011
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characterization of vinyl ethers 1ꢀ6 are given in the Sup-
porting Information.
keto form (at 500 nm), whereas the emission from the enol
form (at 420 nm) is very weak, as seen from the emission
spectrum at the saturation stage (Figure 2b). The ratio of the
two emission bands thus correlates with the mole ratio of the
remaining probe 1 and the hydrolyzed product 7.10
Probe 1 showed only blue fluorescence as the hydroxyl
group was vinylated, whereas it readily underwent HgCl2-
promoted hydrolysis and consequently emitted cyan fluor-
escence responsible for the keto form generated through the
ESIPT (Scheme 1). The changes in the UV/vis absorption
and fluorescence spectra depending on the reaction time
were recorded for a 2:1 mixture of 1 (10 μM) and HgCl2 in a
phosphate buffer solution (PBS: phosphate buffered saline,
pH 7.4) containing ∼1% CH3CN by volume at room
temperature (Figure 2). Both the absorption and emission
spectra showed gradual changes as the vinyl ether under-
went mercury ion-promoted hydrolysis to generate phenol
7. The absorption maximum at 295 nm gradually decreased
while the absorption at 330 nm increased. When the
mixture was excited at the isosbestic point (318 nm) of
the absorption spectra, the emission peak at 420 nm was
much higher than that at 500 nm (Figure S1). Thus, a better
ratiometric feature in the spectra was obtained at an
excitation wavelength of 365 nm.
Next, we evaluated the fluorescence behavior of probe 1
toward other typical metal species, the results of which are
summarized in Figure 3. The fluorescence data were
obtained in the PBS buffer (pH = 7.4) for a 2:1 mixture
of probe 1 (10 μM) and each of the metal species. Probe 1
showed a distinct ratiometric behavior only toward Hg2þ
species among the various metal ions examined. The probe
solution, emitting blue fluorescence (λmax = 420 nm) in the
absence of metal ions, showed only little or small changes
in that peak uponaddition ofothermetal ions(Cr2þ, Ca2þ
Mg2þ, Co2þ, Mn2þ, Ni2þ, Pb2þ, Zn2þ, Ba2þ, Cd2þ, Al3þ
Cu2þ, Fe3þ, Fe2þ, and Agþ).
,
,
Figure 3. (a) Fluorescence changes observed after 1 h for a 2:1
mixture of 1 (10 μM) and each of metal species (Mg2þ, Ca2þ
,
,
Ba2þ, Zn2þ, Co2þ, Ni2þ, Pb2þ, Agþ, Al3þ, Fe3þ, Cd2þ, Cu2þ
Cr2þ, Mn2þ, Fe2þ, Au3þ, and Hg2þ; as their chloride salts except
for AgNO3) in PBS buffer (pH 7.4) containing ∼1% CH3CN. (b)
A bar graph showing the relative fluorescence intensity of the ions
(from A to Q, which denote the ions in the same order as that in (a))
in comparison with probe 1 (the peak height at 500 nm).
In contrast, in the case of Hg2þ, the probe’s emission at
420 nm disappeared while the emission from the keto form
of 7 (λmax = 500 nm) appeared, indicating the hydrolysis
occurs only in the presence of Hg2þ species. Also probe 1
showed the same behavior toward the Hg2þ species even
in the presence of all the other metal ions, showing
little fluorescence interference from these metal species
(Figure S7). Thus, vinyl ether 1 is a ratiometric probe
specific for Hg2þ species. The fluorescence titration of
probe 1 with an increasing amount of HgCl2 (from 0
to 1.0 equiv) showed a saturation behavior at 0.5 equiv
(Figure 4a) in the PBS buffer (pH 7.4). The result indicates
that the probe also reacts with the organomercury inter-
mediate 8 generated in the oxymercuration process
(Scheme 2).
Figure 2. Time-dependent (a) UV/visspectra and (b) fluorescence
spectra measured for a 2:1 mixture of probe 1 (10.0 μM) and HgCl2
in PBS buffer (pH 7.4) containing ∼1% CH3CN. Inset: a plot of
the fluorescence intensity change (based on the peak heights at the
maxima) depending on time.
(10) (a) Li, S. Y.; Wang, Q.; Qian, Y.; Wang, S. Q.; Li, Y.; Yang,
G. Q. J. Phys. Chem. A 2007, 111, 11793. (b) Sun, W. H.; Li, S. Y.; Hu,
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R. W.; Blayney, M. B.; Hochberg, F.; Toribara, T. Y.; Cernichiari, E.;
Clarkson, T. N. Engl. J. Med. 1998, 338, 1672. (b) Methylmercury. No.
101 of Environmental health criteria. Geneva: World Health Organiza-
tion, 1990.
The mercury ion-promoted hydrolysis of probe 1 was
complete within 30 min under the given conditions
(Figure 2b). As known for other ESIPT compounds, phenol
7 itself shows a strong emission band originated from the
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Org. Lett., Vol. 13, No. 13, 2011

Scheme 2. A Mechanism for the Hydrolysis of Probe 1 by HgCl2
Figure 4. (a) Fluorescence intensity changes of probe 1 (10 μM)
with respect to the equivalents of HgCl2, expressed as the peak
heights at 420 and 500 nm, respectively; taken after 30 min of
each addition. (b) A plot of fluorescence intensity (the peak
height at 500 nm) for a 2:1 mixture of probe 1 and HgCl2 for
the concentration range from 0.1 to 1.0 μM, taken after 30 min of
each addition. Both probe 1 and HgCl2 were dissolved in PBS buffer
(pH 7.4) containing ∼1% CH3CN. Excited at 365 nm.
ions in the PBS buffer solution (Figures S6, S9). We
obtained a linear relationship between the fluorescence
intensity at 500 nm and the concentration of mercury
ions (Figure 4b). From the data, a detection limit of 20
ppb was obtained, one order of magnitude lower in
value in comparison with that of the previous fluor-
escein-based vinyl ether,5a which precludes its applica-
tion to detect mercury ions down to the very low
concentration limit required for drinking water (2 ppb).14
For such a purpose, a further study is necessary to
improve the fluorescence quantum yield of the fluor-
ophore, 2-(benzothiazol-2-yl)phenol and probe 1 (ΦF ≈
0.2).15 The present probe may be used for other situa-
tions where the potential errors from detection condi-
tions become a concern.
In summary, we have devised a ratiometric fluorescent
probe for mercury species based on the metal-promoted
hydrolysis of vinyl ether. The probe, an optimized vinyl
ether derivative of 2-(benzothiazol-2-yl)phenol, only
responds to mercury species and not to various other
metal ions, showing a marked fluorescence change from
blue to cyan (∼80 nm bathochromic shift) through
ESIPT. The reaction stoichiometry indicates that the
probe can also be used for the detection of methylmer-
cury species.
A case of a fatal accident in handling organomercury
in a laboratory11a and other warnings11b deterred us from
carrying out further experiments with methylmercury
species.12 However, the 2:1 stoichiometry in the above
fluorescence titration of 1 with HgCl2 indicates that
methylmercury, a typical organomercury species, may also
be detected by the probe.
The hydrolysis reactivity of such vinyl ether compounds
by organomercury species seems to be dependent on the
aryl moiety of the vinyl ether compounds. A vinyl ether
derived from a coumarin precursor developed by us
previously13 showed reactivity only toward inorganic mer-
cury and not toward organomercury species.
We investigated a set of fluorescence studies at different
pH’s from 4 to 9. In the absence of HgCl2, the fluorescence
intensity of probe 1 at pH 4 remained nearly constant even
after 24 h (Figure S8). As the pH of the solution increased
from acidic to basic (pH = 9), the fluorescence change
became slow (Figure S10). These observations support
that the cleavage of the hemiacetal intermediate produced
by the oxymercuration is facilitated by acid; hence, the
overall hydrolysis rate becomes slow at higher pH. These
observations corroborate our previous report.5a
Acknowledgment. This work was supported by grants
from the EPB Center (R11-2008-052-01001).
Supporting Information Available. Experimental pro-
cedure, spectral data for all new compounds, and some
optical spectra of probe 1. This material is available free
of charge via the Internet at http://pubs.acs.org.
The fluorescence intensity of the probe solution gradu-
ally increased with the increasing concentration of mercury
(12) Warnings: Handling of mercury species has been carried out in a
well-ventilated fume hood because diorganomercury species, R2Hg,
generates according to the reaction mechanism in Scheme 2, and all
the resulting wastes including titrated solutions are carefully disposed
following our institution’s guidelines for toxic wastes.
(14) Mercury Update: Impact of Fish Advisories. EPA Fact Sheet
EPA-823-F-01-011; EPA, Office of Water: Washington, DC, 2001.
(15) Kirkbright, F. G.; Spillane, E. M. D.; Anthony, K.; Brown,
G. R.; Hepworth, D. J.; Hodgson, W. K.; West, A. M. Anal. Chem. 1984,
56, 1644.
(13) Cho, Y.-S.; Ahn, K. H. Tetrahedron Lett. 2010, 51, 3852.
Org. Lett., Vol. 13, No. 13, 2011
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