A selective thioxothiazolidin-coumarin probe for Hg2+ based on its desulfurization reaction. Exploring its potential for live cell imaging

Article abstract of DOI:10.1016/j.saa.2019.117372

Sensing the most toxic heavy metal (mercury) has attracted a lot of attention in recent years due to its extreme harmfulness to both human health and the environment. Thus, we reported herein the synthesis, spectroscopic and kinetic characterization, and biological evaluation of a new thioxothiazolidin coumarin derivative (ILA92), which undergoes a desulfurization reaction induced by mercuric ions (Hg²⁺). This process is the origin of a selective sensing of Hg²⁺ ions in aqueous solution by colorimetric and fluorescent methods. Furthermore, the probe showed great potential for imaging Hg²⁺ in living cells.

Full text of DOI:10.1016/j.saa.2019.117372

Accepted Manuscript
A selective thioxothiazolidin-coumarin probe for Hg2+ based on
its desulfurization reaction. Exploring its potential for live cell
imaging
Margarita E. Aliaga, Marcela Gazitua, Andrea Rojas-Bolaños,
Marcial Fuentes-Estrada, Diego Durango, Olimpo García-Beltrán
PII:
S1386-1425(19)30762-0
DOI:
https://doi.org/10.1016/j.saa.2019.117372
Article
Number:
Reference:
117372
SAA 117372
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Revised date:
Accepted date:
9 April 2019
6 July 2019
7 July 2019
Please cite this article as: M.E. Aliaga, M. Gazitua, A. Rojas-Bolaños, et al., A selective
thioxothiazolidin-coumarin probe for Hg2+ based on its desulfurization reaction.
Exploring its potential for live cell imaging, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117372
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ACCEPTED MANUSCRIPT
A selective thioxothiazolidin-coumarin probe for Hg2+ based on its
desulfurization reaction. Exploring its potential for live cell
imaging.
Margarita E. Aliaga,a* Marcela Gazitua,a Andrea Rojas-Bolaños,b Marcial Fuentes-
Estrada, b Diego Durangoc and Olimpo García-Beltránb*
a.Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 6094411,
Chile.
b.Facultad de Ciencias Naturales y Matemáticas, Universidad de Ibagué, Carrera 22, Calle 67, Ibagué 730002,
Colombia.
c.Universidad Nacional de Colombia, Sede Medellín, Escuela de Química, Carrera 65, No. 59A-110, Medellín,
Colombia.
Abstract
Sensing the most toxic heavy metal (mercury) has attracted a lot of attention in
recent years due to its extreme harmfulness to both human health and the
environment. Thus, we reported herein the synthesis, spectroscopic and kinetic
characterization, and biological evaluation of a new thioxothiazolidin coumarin
derivative (ILA92), which undergoes a desulfurization reaction induced by mercuric
ions (Hg2+). This process is the origin of a selective sensing of Hg2+ ions in
aqueous solution by colorimetric and fluorescent methods. Furthermore, the probe
showed great potential for imaging Hg2+ in living cells.
Keywords:
derivative; naked eye sensor; colorimetric fluorescent dye.
Desulfurization reaction; Hg2+ ions; thioxothiazolidin coumarin

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1. Introduction
The detection and quantification of heavy metal ions have emerged as a
research area due to the prominent impact of these ions in biologically and
environmentally important matrices [1-8]. In particular, mercury (Hg) is one of the
most highly toxic heavy metals [9,10]. Thus, sensing the metal has attracted a lot
of attention in recent years [11,12] due to its extreme harmfulness to both human
health and the ecosystem. In fact, according to the established agreements in the
Minamata Convention [10], it is very important to perform measurements of
mercury-compounds emissions to both land and water, and monitor their
regulation.
Typically, traditional mercury species detection methods include atomic
absorption/emission spectrometry [4], hyper Rayleigh scattering (HRS) [13],
ultrasensitive stripping voltammetry, [14] inductively coupled plasma mass
spectrometry (ICP-MS) [15], among others. However, all these methods have
various limitations because they not only require sophisticated instrumentations but
also are time-consuming. Therefore, it is still a great challenge to develop simple,
fast, and low-cost methods to detect mercury species sensitively and selectively.
Thus, between various reported methods for the determination of such heavy-
metal ions, colorimetric and fluorescent probes arise as optimal tools [1-3,16-24].
The latter is because of their inherent cheapness, operational simplicity and high
stability and sensitivity. In this context, attractive sensors for the recognition of Hg2+
have been reported. For example, some fluorescent chemosensor for the detection
of such ions are based on their coordination to heteroatom containing ligands
[23a].
Other specific probes reported act via hydrolysis [23b] or desulfurization [24]
reaction induced by the metal. Moreover, other derivatives have induced dual
fluorogenic signalling responses toward Hg2+ and Fe3+ ions depending upon the
nature of substituent, [25] as well as a function of solvent medium [26]. Prompted
by these observations, we explored sensitive and selective chemosensors for the
recognition of mercuric ions over other metal ions, in particular, iron. Thus, in this
work we present a new sensor (Z)-2-(5-((7-(diethylamino)-2-oxo-2H-chromen-3-

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yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)ethanesulfonic acid (ILA92) see
Scheme 1, which has been designed to incorporate a diethylaminocoumarin
fragment with a rhodanine unit, in which the thiocarbonyl group would be would be
in particulary in charge of recognizing the Hg2+ ions. We speculate that mercuric
ions may promote a reaction of the sulphur-containing group. The latter may
influence the π-electron delocalization of the system and, consequently change the
absorption properties of dye.
2. Material and Methods
All analytes were purchased from Sigma-Aldrich and Merck and were used as
received. The metal salts used were: Hg2+ (HgCl2); Na+ (NaCl); K+ (KCl); Ca2+
(CaCl2), Mg2+ (MgCl2); Zn2+ (ZnCl2); Fe3+ (FeNH4(SO4)2·12H2O); Fe2+ (Fe-
(NH4)2(SO4)2·6H2O); Cu2+ (CuCl2) and Ag+ (AgCl). Unless indicated otherwise, all
solutions employed in this study were prepared in HEPES-DMSO (99/1 v/v; pH
7.0).
1H- and 13C-NMR spectra were recorded on a Bruker multidimensional 400 MHz
spectrometer, using the solvent or the TMS (Trimethylsilane) signal as an internal
standard. All chemical shifts are reported in the standard notation of parts per
million.
HRMS-ESI experiments were executed using a Thermo Scientific Exactive Plus
Orbitrap spectrometer with a constant nebulizer temperature of 250 °C. The
experiments were carried out in negative ion mode, with a scan range of m/z
400.000-1000.000 with resolution 140.000. The samples were infused directly into
the ESI source, via a syringe pump, at flow rates of 5 µL min-1, through the
instrument’s injection valve.
Absorption spectra were obtained by means of an Agilent Cary 60. The emission
spectra were recorded on an Agilent Cary Eclipse fluorometer. Zeiss 710 confocal
microscopy was used. The confocal microscopy at 63X amplification.
2.1.Synthesis of 7-diethylaminocoumarin (2)
4-Diethylaminosalicylaldehyde (1) (1.93 g, 10 mmol), diethyl malonate (3.2 g,
20 mmol), piperidine (1.0 mL) and one drop of AcOH, were combined in absolute

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EtOH (60 mL) and refluxed for 6 h. All volatiles were evaporated under reduced
pressure, and then concentrated, HCl (20 mL) and glacial acetic acid (20 mL) were
added to cyclize, hydrolyze and decarboxylate the intermediate ester while the
reaction was continued at reflux temperature for 24 hours. This solution was cooled
to room temperature and poured into ice water (100 mL). NaOH solution (40%)
was added dropwise to adjust the pH to 5, and a grey precipitate formed
immediately. After stirring for 1 h, the mixture was filtered, washed with water, and
dried to yield product 7-(diethylamino)-2H-chromen-2-one (2) (1.78 g, 8.2 mmol) in
82.0 % yield. 1H-NMR (DMSO-d6) δ 7.50 (d, J=8.0 Hz, 1H), 7.21 (d, J=8.0 Hz, 1H),
6.53 (dd, J=12.0 Hz, J=2.0 Hz, 1H), 6.44 (d, J=2.0 Hz, 1H); 5.99 (d, J=8.0 Hz. 1H),
13
3.42 (q, J=8.0 Hz, 4H), 1.17 (t, J=8.0 Hz, 6H). C NMR (DMSO-d6): 162.7, 157.1,
151.1, 144.1, 129.2, 109.5, 109.1, 108.5, 97.9, 45.2, 12.8.
2.2. Synthesis of 7-diethylaminocoumarin-3-aldehyde (3)
Freshly distilled DMF (6.5 mL) was added dropwise to POCl3 (6.5 mL) at 20-
50 °C under N2 atmosphere and stirred for 30 minutes yielding a red solution. The
solution was added with 7-diethylaminocoumarin (2) (4.50 g, 20.7 mmol) in 30 mL
DMF to afford a scarlet suspension. The mixture was stirred at 60 °C for 24 h and
then poured into ice water (300 mL). NaOH solution was added to adjust the pH to
5.2, generating an abundant precipitate. The crude product was filtered thoroughly
washed with water, dried and recrystallized in absolute ethanol to give 3 (2.80 g,
1
11.4 mmol) in 55.2% yield. H NMR (DMSO-d6) δ 10.05 (s, 1H), 8.18 (s, 1H), 7.36
(d, J=8.0 Hz, 1H), 6.59 (dd, J=8.0 Hz, J=2.0 Hz, 1H), 6.42 (d, J=2.0 Hz, 1H), 3.44
(q, J=8.0 Hz, 4H), 1.22 (t, J=8.0 Hz, 6H). 13C NMR (DMSO-d6): 188.2, 162.2,
159.3, 153.9, 145.7, 132.9, 114.6, 110.6, 108.6, 96.5, 45.7, 12.8 [27].
2.3. Synthesis of (Z)-2-(5-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)ethanesulfonic acid
2-(4-oxo-2-thioxotetrahydrothiophen-3-yl)ethanesulfonic acid (1 mmol) and
appropriate 7-diethylaminocoumarin-3-aldehyde (1 mmol) were dissolved in
methanol (5 mL). The solution was refluxed for 2-18 h in the presence of a small
amount of piperidine as a catalyst [28-29]. After the reaction was completed

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(monitored by TLC), the mixture was cooled, the precipitate was filtered and
crystallized from ethanol to produce corresponding products (ILA92). (0.350 g, 75
1
%). H NMR (DMSO-d6) δ 8.13 (s, 1H), 7.59 (d, J=8.0 Hz, 1H), 7.57 (s, 1H), 6.79
(d, J=8.0 Hz, 1H), 6.58 (s, 1H), 4.27 (m, 2H), 3.49 (q, J=8.0 Hz, 4H), 2.77 (m, 2H),
1.15 (t, J=8.0 Hz, 6H). 13C NMR (DMSO-d6): 194.2, 167.3, 160.6, 157.1, 152.9,
147.4, 131.9, 129.2, 120.1, 111.9, 110.8, 109.1, 96.7, 47.7, 44.9, 41.5, 12.8.
2.4. High-resolution mass spectrometry (HRMS-ESI) studies
High-resolution mass spectra (HRMS-ESI) were extracted from high
resolution mass spectrometer Exative™ Plus Orbitrap, Thermo Fisher Scientific.
The analysis for the reaction products was performed with the following scan
parameters: resolution 140.000; AGC target 1e6; inject time 200. HESI source was
heath gas flow 8; auxiliary gas flow rate 3; sweep gas flow rate 0; capillary
temperature 250°C, S-lens RF 100; Heater temperature 100°C, at mode positive
with spray voltage 2.8 kV.
2.5. Nuclear magnetic resonance (NMR) studies
13C NMR spectra were obtained at 25°C on a Bruker Advance 400 MHz
spectrometer using TMS as an internal standard. The NMR spectra were
processed with MestreNova software v9.0. All solutions were prepared by mixing
appropriate volumes of stock solutions of ILA92 in DMSO-d6 and D2O.
2.6. Kinetic measurements
These measurements were performed spectrophotometrically (diode array)
in the 300-700 nm range, by following the disappearance of ILA92 (at 530 nm) or
the formation of product at 485 nm, by means of a Hewlett-Packard 8453
instrument. The reactions were carried out in water, at 25 ºC. At least a 10-fold
excess of mercuric ions over the substrate (ILA92) was employed. Pseudo-first-
order rate coefficients (kobsd) were found for all reactions. They were obtained by
means of the kinetic software of the spectrophotometer at the wavelength where
the greatest absorbance changes were observed.
2.7. Determining the quantum yield of emission

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Fluorescence quantum yield of ILA92 was measured using a solution of
Rhodamine 101 in ethanol as standard (Φs = 1). All values were corrected
considering the solvent refraction index. Quantum yield was calculated using Eq.
(1), where the subscripts x and s denote sample and standard, respectively, Ф is
the quantum yield, η is the refractive index, and Grad is the slope from the plot of
integrated fluorescence intensity vs. absorbance.
Φ푥
Eq. (1)
퐺푟푎
= Φ푠 (
퐺푟푎
2.8. Detection limit
The detection of limit (LOD) was calculated based on absorbance and
fluorescence titrations. To determine the S/N ratio, the absorbance (at 525 nm) or
emission intensity of ILA92 with Hg2+ was measured three times and the standard
deviation of calibration curve was determined. The detection limit was calculated
with the equation: LOD = 3σb/m, where σb is the standard deviation of calibration
curve and m is the slope of the plot of absorbance or fluorescence intensity versus
analyte concentration.
2.9. Cell culture and fluorescence imaging
Human neuroblastoma SH-SY5Y cells (CRL-2266, American Type Culture
Collection, Rockville, MD) were cultured in MEM-F12 medium supplemented with
10% FBS, non-essential amino acids, antibiotic-antimycotic mixture, and 20 mM,
pH 7.0 HEPES buffer. The medium was replaced every 2 days. Cells were
exposed to 2 μM ILA92 for 30 min and washed. Then, the basal fluorescence was
taken after the cells were treated with 30 μM HgCl2 for 120 min. Changes were
observed by epifluorescence microscopy, 63× objective [23b].
3. Results and discussion
The proposed compound ILA92 was synthesized in four steps, as shown in
Scheme 1. Firstly, 4-(diethylamino)-2-hydroxybenzaldehyde (1) was condensed
with diethyl malonate in a Knoevenagel condensation, cyclized and decarboxylated
in one step to afford 7-(diethylamino)-2H-chromen-2-one (2). Subsequently, the

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compound was formylated (Vilsmeier-Haack) to obtain 7-(diethylamino)-2-oxo-2H-
chromene-3-carbaldehyde (3) [27], which was condensed with 2-(4-oxo-2-
thioxotetrahydrothiophen-3-yl) ethanesulfonic acid to yield the proposed compound
(Z)-2-(5-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)ethanesulfonic acid (ILA92). The chemical structure of
1
precursors and the final product ILA92 were well characterized by using H and
13C NMR spectroscopy (see Supporting Information Figures S1-S4). In addition, for
the characterization of ILA92, high-resolution mass spectrometry (HRMS-ESI) was
employed (Figure S5).
Scheme 1. Synthetic route to ILA92. Reagents and conditions: a) Diethyl malonate, piperidine,
reflux, 6h; b) HCl, AcOH, reflux, 24 h; c) POCl3, DMF, 60 °C, 24 h; d) 3, 2-(4-oxo-2-
thioxotetrahydrothiophen-3-yl)ethanesulfonic acid, MeOH, piperidine, 10-12 h.
Compound ILA92 was also examined by UV−Vis absorption, in HEPES-DMSO
(99/1 v/v; pH 7.0). The absorption spectrum of compound ILA92 (5 µM) in such a
medium exhibited a strong band at 530 nm (Figure S6; SI) and a molar extinction
coefficient (ε) of 148518 M-1 cm-1 (Figure S7; SI). Regarding the fluorescent
properties of ILA92, its emission spectrum shows an emission band at 645 nm
(Figure S8A; SI). Compound ILA92 has a quantum yield of 0.049 (using
Rhodamine 101 as standard, see Figure S9) and a Stokes shift of 110 nm.
The selectivity of compound ILA92 was tested in the presence of a series of
different metal ions of biological and environmental interest, such as Hg2+, Na+, K+,
Ca2+, Mg2+, Zn2+, Fe3+, Fe2+, Cu2+ and Ag+ (Figure S8B; SI). As shown in Figure 1,
relevant absorbance changes at 530 nm were observed for this compound only in
the presence of solutions containing Hg2+ ions. It is important to mention that ferric
ions, a typical interferent [25], produced slight variations only in spectral properties

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of ILA92. Moreover, the variation of absorbance (ratio A/A0) associated with the
ILA92-Hg2+ system at 530 nm in the presence and absence of coexisting metal
ions under competitive conditions ([ILA92]:[Hg2+]:[Mn+] = 1:10:20) exhibited no
significant differences in their responses by being compared between them.
Results show a ratio that is in the range between 0.23 and 0.34 for [Hg2+]:[Fe3+]
and [Hg2+]:[ Ag+], respectively, which implies that ILA92 depicts a high selectivity
for mercuric ions even in the presence of other competitive metal ions.
Thus, we can establish that the absorption changes of ILA92 are highly specific for
Hg2+ and minor interference from this metal ion occurs, in the same experimental
conditions.
Fig. 1. Selectivity and competition assays. The ratio of the absorption at 530 nm (A/A0) of ILA92 (2
μM) upon addition of 10 equiv. of different metal ions (including 1: Hg2+, 2: Na+, 3: K+, 4: Ca2+, 5:
Mg2+, 6: Zn2+, 7: Fe3+, 8: Fe2+, 9: Cu2+, 10: Ag+). In the case of competition experiments (orange
line dashes) the absorbance ratio was assessed under competitive conditions ([ILA92]:[Hg2+]:[Mn+]
= 1:10:20). Each set of data was obtained 2h after the addition of different metal ions in in HEPES-
DMSO (99/1 v/v; pH 7.0) at 25 °C.
In fact, upon addition of five equivalent of Hg2+ ions to the solution of ILA92, the
absorption band at 530 nm slightly decreased and a new band (shifted 50 nm)
time-dependent was formed at 485 nm (Figure S10; SI), with a colour change from
dark to light pink, evident through the naked eye (Insert Figure S10; SI).
Furthermore, more marked changes were observed in the fluorescence spectra of
the dye after 120 minutes of incubation in presence of 10 eq. of mercuric ions
(Figure 2). As shown in the figure, the fluorescence associated to the dye
increases and the maximum emissive wavelength shifts from 630 nm to 580 nm (0-
120 minutes).
Fig. 2. Changes in the emission spectra of ILA92 (2×10-6 M) upon addition of HgCl2 (1×10-4 M)
during 120 minutes. Each spectrum was acquired in HEPES-DMSO (99/1 v/v; pH 7.0) at 25 °C.

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To view the ability of mercuric ions to modify the fluorescence spectra of dye,
Insert to Figure 2 depicts the effect of increasing concentrations of these ions on
the emission bands associated with the dye.
Regarding the disappearance of the UV-vis band at 530 nm and the formation of a
new band at 485 nm, it could be attributed to the interaction of mercuric ions (soft
acid) with the thiocarbonyl group (sulphur atom, as a soft base) leading to a
chemical reaction. In fact, as mentioned above, it is well known that thiocarbonyl
containing compounds can undergo desulfurization reaction in the presence of
mercury salts [30-31]. Recently, some authors have reported that a blue-shift was
observed when a specific mercury-promoted desulfurization reaction of
thiocoumarin to coumarin occurs [31]. Considering these pieces of evidence and
the chemical structure of compound ILA92, we propose that it is thiocarbonyl
moiety, in the presence of mercuric ions, that turns more electrophile. Thus, ILA92
would be more easily attacked by water, leading to its conversion into the carbonyl
analogue. As a result, the spectroscopic changes described in the UV-Vis of the
probe could be associated with conversion. Thus, the clear isosbestic point at 505
nm observed in the absorption spectra of ILA92 (Figure S8; SI). The latter
indicates the reaction of ILA92 with Hg2+ produces a single new component.
Concerning the kinetic studies, mercuric ions solutions were used in excess (10
equiv.) over the dye ILA92 to ensure first-order conditions, (details see Supporting
Information Table S1; SI). The first-order rate constants kobsd were obtained from
the exponential decays of the absorbance of the dye or the absorption associated
with the product formation (Insert Fig. 3).
Fig. 3. Dependence of kobsd against [Hg2+], in HEPES-DMSO (99/1 v/v; pH 7.0), for the reaction of
ILA92 at 25 °C. Inset: Kinetic profile of disappearance of ILA92 (2×10-6 M) at 530nm and formation
of product at 485nm.

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As depicted in Figure 3, plot of kobsd in contrast to [Hg2+] at constant pH is non-
linear (saturation kinetic). The kinetic behavior of the desulfurization reaction is
consistent with the mechanism described in Scheme 2.
Scheme 2. Proposed mechanism for the studied reaction between mercuric ions and ILA92.
Applying the steady-state conditions to the intermediate in Scheme 2, yields to eq.
2:
2+
[
2
]
푘 × 푘 퐻푔
1
푘표푏푠푑
=
Eq. (2)
2+
[
+푘 퐻푔
2
]
푘
−1
Two limiting situations can arise: at low concentrations of the metal a first-order
kinetic model is established, k–1 >> k2 [Hg2+]; In this case, eq. 2 reduces to eq. 3,
where K1 = k1/k–1.
2+
[
]
푘표푏푠푑 = 퐾1 × 푘2 퐻푔
Eq. (3)
At high concentrations of the metal, they do not affect the kinetic rate, thus a zero
order occurs due to that. k–1 << k2 [Hg2+]; therefore, eq. 2 simplifies to eq. 4.
푘표푏푠푑 = 푘1
Eq. (4)
Thus, the dye is able to interact with mercuric ions and produce a different kinetic
response until concentration reaches 1×10-4 M.
On the other hand, the quantitative signaling behavior of ILA92 for Hg2+ ions was
assessed in aqueous solution using UV-vis titration (Figure S11; SI). In fact, the
absorbance at 530 nm decreased as the concentration of Hg2+ increased, up to
500 µM; By using this concentration, dependent absorption change the detection
limit for the determination of Hg2 as it was estimated to be 15.6×10−6 M
(Absorbance) and 15.1×10−6 M (Fluorescence) (Figure S11; SI). These results
demonstrated that ILA92 can detect the submillimolar concentration range of the
analyte, being the LOD value comparable with other recognized sensitives mercury
probes [18].

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Evidence for the proposed desulfurization reaction [30-33] was supported by 13C
NMR spectra of ILA92 after its reaction with Hg2+. As depicted in the partial 13C
NMR spectrum in Figure 4, upon addition of Hg2+, the NC=S carbon atom C2’
(rhodanine fragment) was clearly shifted from =194.7 to 166.0 ppm (NHC=O
carbon of product).
Fig 4. 13C NMR (DMSO-d6) spectrum of probe ILA92 alone (A) and in the presence of mercuric
ions (B).
In addition, to confirm the mechanism for the reaction between ILA92 and Hg2+
HRMS-ESI analyses were carried out. As shown in Figure 5, peaks of m/z 467.04
and 451.06 were observed after the addition of one equivalent of HgCl2 to the
solution containing ILA92. These peaks are attributed to the presence of reagent
(probe ILA92) and of the desulfurization reaction product ILO92, respectively.
Fig. 5. HRMS-ES of probe ILA92 in the presence of HgCl2 (1 eq.).
Therefore, we confirm that the thiocarbonyl group of ILA92 is readily transformed
to its carbonyl function by reaction with Hg2+ ions and the liberated sulphur atom
form a stable species of HgS with Hg2+ ions (Scheme 3).
Scheme 3. Desulfurization reaction proposed between compound ILA92 and mercuric ions.
To explore the potential of the probe ILA92 as a tool for live cell imaging of
mercuric ions, we used an in vitro model system (SHSY5Y neuroblastoma cells)
and investigated the effects of ILA92 in the absence and presence of mercuric
ions. In addition, it was observed under the microscope that SH-SY5Y cells in the
presence of ILA92 (5 M) did not induce structural modifications during the whole

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trial. It demonstrates that, at the concentrations used it does not present cytotoxic
effects.
Thus, considering this control experiment (Fig. 3), the SH-SY5Y cells were first
treated with ILA92 (2 µM) for 20 min, and subsequently a solution containing Hg2+
(30 µM) was added and incubated for another 30 min. As shown in Figure 6A, the
cells incubated with only ILA92 emitted fluorescence. However, upon the addition
of Hg2+ , the fluorescence emission showed an increase of the emission. These
results are in accordance with the changes of the emission spectra obtained in
aqueous solutions, which demonstrates that the probe ILA92 is capable of
detecting Hg2+ in both aqueous solutions and biological samples. And when
compared to those reported in the literature [34-36], it has good behavior in
biological matrices.
Fig. 6. (A) Basal fluorescence of SH-SY5Y cells lines incubated with the probe ILA92 (2 µM). (B)
Increase in fluorescence after treating the SH-SY5Y cells lines with 30 µM HgCl2 for 30 min.
4. Conclusions
We have reported the synthesis, kinetic study and biological application of a
coumarin derivative compound ILA92 that selectively recognizes Hg2+ ions over a

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series of other metal ions. The detection limits obtained were (~ 10−7 M). The
sensing is performed colorimetrically and fluorometrically and it is based on a
desulfurization reaction. Fluorescence imaging for Hg2+ in living cells shows that
sensor ILA92 could be potentially used for further biological applications.
Acknowledgment
This work was supported by COLCIENCIAS Grant #130774559056 Colombia and
FONDECYT Grant #1170753.
Conflicts of interest
There are no conflicts to declare.
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Graphical Abstract
Desulfurization reaction of a thioxothiazolidin coumarin derivative induced by mercuric ions. Exploring
its potential for live cell imaging
Margarita E. Aliaga,a Marcela Gazitua,a Andrea Rojas-Bolaños,b Marcial Fuentes-Estrada,b Diego Durangoc and Olimpo García-
Beltránb*

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Highligths
 A new thioxothiazolidin coumarin derivative (ILA92) is designed for specific
sensing of mercury.
 The sensing of mercuric ions is performed colorimetrically and
fluorometrically and it is based on the desulfurization reaction of ILA92.
 The detection limit of sensing was obtained 2.34×10−6 M (Absorbance) and
4.50×10−6 M (Fluorescence).
 The ILA92 can successfully use for sensing Hg2+ in aqueous medium and
living cells.