A selective fluorescent probe for the detection of mercury (II) in aqueous media and its applications in living cells

Article abstract of DOI:10.1016/j.tetlet.2012.09.111

In this Letter we present a new probe, 2-amino-3-hydroxy-2-(hydroxymethyl) propyl 2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (PMR), which can reversibly detect mercuric ions (Hg²⁺) in HEPES buffer under physiological conditions. Possible interference with other analytes was examined. PMR displays a highly selective decrease of its fluorescence at 460 nm when it reacts with Hg²⁺. Interestingly, the probe can also be used as a fluorescent turn-on sensor for biologically relevant thiols such as glutathione and cysteine. PMR can be used to determine mercury in living cells.

Full text of DOI:10.1016/j.tetlet.2012.09.111

Tetrahedron Letters 53 (2012) 6598–6601
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A selective fluorescent probe for the detection of mercury (II) in aqueous
media and its applications in living cells
Olimpo García-Beltrán a,b,e, , Natalia Mena c,e, Tania A. Berríos d, Enrique A. Castro d, Bruce K. Cassels a,e
,
⇑
Marco T. Núñez c,e, Margarita E. Aliaga d,
⇑
a Department of Chemistry, Faculty of Sciences, University of Chile, Santiago 7800024, Chile
b Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andrés Bello, Avenida República 275, Piso 3, Santiago, Chile
c Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
d Facultad de Química, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago 6094411, Chile
e Institute for Cell Dynamics and Biotechnology, University of Chile, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter we present a new probe, 2-amino-3-hydroxy-2-(hydroxymethyl)propyl 2-(7-hydroxy-2-
oxo-2H-chromen-4-yl)acetate (PMR), which can reversibly detect mercuric ions (Hg2+) in HEPES buffer
under physiological conditions. Possible interference with other analytes was examined. PMR displays
a highly selective decrease of its fluorescence at 460 nm when it reacts with Hg2+. Interestingly, the probe
can also be used as a fluorescent turn-on sensor for biologically relevant thiols such as glutathione and
cysteine. PMR can be used to determine mercury in living cells.
Received 9 September 2012
Revised 21 September 2012
Accepted 24 September 2012
Available online 29 September 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Hg2+-selectivity
Fluorescent probe
Coumarin derivatives
Biothiols
The detection of heavy and transition metal ions is a task that is
attracting a considerable amount of attention,1 principally for bio-
logical or environmental applications. Among the heavy metals,
mercury is considered one of the most toxic and dangerous ele-
ments; specifically, in its +2 state it has a very high affinity for sulf-
hydryl groups such as those present in enzymes, other proteins and
endogenous thiols.2 Mercuric ions can easily pass through biologi-
cal membranes and cause serious damage to the central nervous
and endocrine systems.3 Thus, considerable efforts have been
made to synthesize probes that can detect these ions in a selective
and sensitive manner. Probes based on changes in fluorescence in-
duced by such ions are particularly attractive and are one of the
first choices because of the simplicity of their use and the low
detection limit of fluorescence.4
Recently, many sensitive fluorescent probes based on rhoda-
mine,5 coumarin6 or squaraine derivatives,7 as well as other fluoro-
phores,8 have been developed to detect mercury ions. It is
noteworthy that most of these only work in organic solvents,
which severely limits their potential for biological and environ-
mental applications. A few of them have been reported as water-
soluble,5a,8b,9 but the development of highly selective probes for
Hg(II) detection in aqueous solution with little or no interference
from other metal ions is still a highly desirable goal.
Herein we report a new probe, 2-amino-3-hydroxy-2-(hydroxy-
methyl)propyl
2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetate
(PMR), which is water soluble and has the desired photophysical
features. PMR was prepared through a three-step (Scheme S1;
Supplementary data) process and was characterized by 1H NMR
and 13C NMR spectroscopy (Figs. S3 (A) and (B)).
The absorption spectrum of PMR shows a maximum at 330 nm
and a shoulder near 375 nm (Fig. S4; Supplementary data), a molar
extinction coefficient (
at 460 nm (Fig. S5; Supplementary data). The quantum yield of this
probe is 0.32, with a lifetime ( ) of 5.86 ns and a Stokes shift of
8022 cmÀ1. Figure 1 shows the Hg2+ concentration-dependent
emission fluorescence spectra of PMR (20 M). When excited at
e
) of 14460 MÀ1 cmÀ1 and an emission band
s
l
330 nm, the emission fluorescence intensity at 460 nm decreases
about eightfold upon increasing the concentration of Hg2+ from 0
to 100 lM (Fig. 1). A good linear relationship between fluorescence
intensity and Hg2+ concentration was observed, with a correlation
coefficient as high as 0.9916 (Inset to Fig. 1). According to these re-
sults, the detection limit of PMR for Hg2+ is 1.1 Â 10À9 M. This result
is comparable to those reported for other probes7b,10 and suggests
the use of PMR in biological and environmental applications.
The changes observed in the fluorescence intensity of PMR after
the addition of Hg2+ ions is presumably due to the chelation of
⇑
Corresponding authors. Tel.: +56 2 978 7253; fax: +56 2 271 3888.
E-mail addresses: ojgarciab@ug.uchile.cl (O. García-Beltrán), mealiaga@uc.cl
(M.E. Aliaga).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.09.111

O. García-Beltrán et al. / Tetrahedron Letters 53 (2012) 6598–6601
6599
reaction conditions (10
lM PMR and 100–800
lM HgCl2 in HEPES
buffer) and an ionic strength of 0.2 M (maintained with NaNO3) at
30 °C. The time-dependent response of PMR to mercury ions was
monitored using fluorescence spectroscopy (Fig. 2A). The observed
rate constants (kobs) were found to vary from 1.4 Â 10À3 sÀ1 to
3.0 Â 10À3 sÀ1 for 100
lM and 800 l
M Hg2+, respectively, as shown
in Figure 2B.
In order to evaluate the selectivity of the PMR probe, different
metal ions (Hg2+, Fe3+, Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+, Mg2+
,
Ni2+, Pb2+ or Cd2+) were tested. Among these ions, only Hg2+ signif-
icantly affected the fluorescence intensity of PMR (Fig. 3).
To further check the practical applicability of the PMR probe as
a Hg2+-selective fluorescent sensor, we carried out competitive
experiments in the presence of 20
tal ions at 200 M. As shown in Figure 4, these experiments dem-
onstrate that none of the selected ions interfere to any obvious
extent with the detection of Hg2+
l
M Hg2+, mixed with other me-
l
Figure 1. Fluorescence emission spectra (kex = 330 nm) of PMR (20
addition of HgCl2 (0–200 M) in 20 mM HEPES buffer, pH 7.4. Inset shows the
relationship between fluorescence intensity and Hg2+ concentration (2–60
M).
lM) upon
.
l
As is well known, reversibility is an important feature of excel-
lent chemical probes. Thus, experiments were conducted by
adding glutathione (GSH) to examine the reversibility of the
PMR-Hg2+ interaction ( Fig. 5A). This figure clearly shows that
the fluorescence intensity of a solution containing PMR and
Hg2+ increases with the addition of GSH. The latter can be ex-
plained considering the high affinity of the sulfhydryl group of
l
these ions by the oxygen (hydroxyl and coumarin carbonyl) and
nitrogen atoms of the probe. Typically, when heavy metals such
as Hg2+ are chelated by heteroatoms in close proximity to a fluoro-
phore, they quench the fluorescence by enhanced spin-orbit
coupling.11
GSH towards Hg2+ 12 thus, this biothiol can remove the Hg2+ ions
;
The stability constant Ka of the PMR-Hg2+ interaction was
determined using the Benesi-Hildebrand equation, which gives a
value of 1.29 Â 104 MÀ1 (Fig. S6; Supplementary data). The binding
stoichiometry of the PMR-Hg2+ complex was determined from the
Job plot (Fig. S7; Supplementary data). Maximum emission inten-
sity was observed when the mole fraction of Hg2+ was close to
0.5, which indicates the formation of a 1:1 complex between
bound to the probe and release the latter, leading to the recovery
of the fluorescence quenched by Hg2+ (Fig. 5A). Similar results
were obtained when cysteine (Cys), another biothiol, was used in-
stead of GSH (not shown). We also observed this behaviour when
the test was performed in SH-SY5Y human neuroblastoma cells.
PMR permeates into the living cells and is thus suited for fluores-
cence imaging of Hg2+ in such systems. Thus, cultured SH-SY5Y
PMR and Hg2+ with a total concentration of 100
lM. This was fur-
cells were incubated with 5
ature, and a significant PMR fluorescence was detected in the
interior of the cells (Fig. 5B). However, when 10 M HgCl2 was in-
lM PMR for 20 min at room temper-
ther confirmed by the appearance of a peak at m/z 524.0482
assignable to [PMR+Hg(II)]+ in the ESI spectrum (Fig. S8; Supple-
mentary data). A possible binding mode between PMR and Hg2+
is proposed in Scheme 1. This was tested by 1H NMR spectroscopy
of the probe in the absence and presence of Hg2+. Upon addition of
Hg2+, the recognizable CH2 resonances of the Tris moiety (d 4.10
and 3.78) undergo slight downfield shifts (between d 4.20 and
3.92, respectively, with considerable broadening). The OH proton
resonance (d 4.82) is shifted upfield (d 4.40) and a signal appears
at d 3.10, assigned to the NH2 protons which in the PMR spectrum
are obscured by the H2O signal in standard conditions (Fig. S9;Sup-
plementary data). Surprisingly, however, the coumarin signals are
practically unchanged.
l
cluded in the incubation medium for another 3 h, a loss of fluores-
cence from the intracellular area was evident ( Fig. 5C).
Interestingly, an increment of fluorescence was observed when
the cells previously exposed to HgCl2 were treated with 20 mM
N-acetylcysteine (NAC) (Fig. 5D). This result is consistent with
that presented in Figure 5(A) and is based on the ability of NAC
to increase GSH levels.
In conclusion, we have developed a new, highly sensitive and
selective ‘turn-off’ fluorescent probe for mercuric ion detection in
an aqueous environment. The method proved to be simple,
selective and sensitive. Most importantly, this probe was success-
fully applied to the imaging of Hg2+ ions in cells.
In addition, kinetic studies of the response of PMR to Hg2+ were
performed using a spectrofluorimeter under pseudo-first order
Figure 2A. Kinetic profile for the interaction between PMR (10
(600 M). Results are expressed as Relative Fluorescence Units (RFU) at
kem = 460 nm.
l
M) and Hg2+
l
Scheme 1. Possible binding mode of PMR with Hg2+
.

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O. García-Beltrán et al. / Tetrahedron Letters 53 (2012) 6598–6601
Figure 2B. Plot of kobs vs concentration of Hg2+ ions for the interaction of PMR with
Hg2+ at pH 7.4.
Figure 5. (A) Effect on the PMR fluorescence of the addition of Hg2+ (20
then GSH (200 M) to the solution. (B) The basal fluorescence of cells incubated
with the probe. (C) Decrease in fluorescence after treating the cells with 10
lM), and
l
l
M
HgCl2 for 3 h. (D) Change in cellular fluorescence of cells previously exposed to
HgCl2 after treatment with 20 mM NAC for 4 h.
Acknowledgments
This work was supported by the Millennium Scientific Initiative
(Grant P05-001-F) and by Fondecyt Grant #11090115. The authors
are grateful to Prof. Frank H. Quina for the use of spectroscopic
facilities in the Institute of Chemistry and the USP Research Con-
sortium for Photochemical Technology (NAP-PhotoTech), Univer-
sity of São Paulo, São Paulo, Brazil.
Supplementary data
Figure 3. Fluorescence spectra of PMR (20
different metal salts (Hg2+, Fe3+, Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+, Mg2+, Ni2+, Pb2+ or
Cd2+; 200
M) in 20 mM HEPES buffer, pH 7.4.
lM) alone and in the presence of several
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.09.
111.
l
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Figure 4. Competitive selectivity of the PMR probe (2.0
(20
M) in the presence of other metal ions: Hg2+, Fe3+, Fe2+, Co2+, Cu2+, Ca2+, Zn2+
Mn2+, Mg2+, Ni2+, Pb2+ or Cd2+ (200
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l
M) towards Hg2+ ions
l
,
l
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Hg2+ in the absence and in the presence of other metal ions, respectively. The dark
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.

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