2,4-dinitrophenyl ether-containing chemodosimeters for the selective and sensitive in vitro and in vivo detection of hydrogen sulfide

Article abstract of DOI:10.1080/10610278.2014.977286

Four probes (i.e. D1-D4) for the selective and sensitive fluorogenic detection of HS^- have been prepared and characterised. HEPES (10 mM, pH 7.4)-DMSO 99:1 v/v solutions of D1-D4 are essentially non-fluorescent. Changes in the emission using D1-D4 in the presence of anions (F^-, Cl^-, Br^-, I^-,N^-₃, CN^-, SCN^-, AcO^-,CO^2-₃ ,PO^2-₄,SO^2-₄, HS^- and OH^-), biothiols (GSH, Cys, Hcy, Me-Cys and lipoic acid), reducing agents (SO^2-₃ and S₂O^2-₃) and oxidants (H₂O₂) demonstrated that only HS^- is able to induce the appearance of intense emission bands in the 400-520 nm range in the four probes. The selectivity observed was ascribed to a unique hydrogen sulfide-induced hydrolysis of the 2,4-dinitrophenyl ether moiety that yielded the corresponding free highly fluorescent alcohols. The potential detection of intracellular HS^- was also studied.

Full text of DOI:10.1080/10610278.2014.977286

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2,4-dinitrophenyl ether-containing chemodosimeters
for the selective and sensitive ‘in vitro’ and ‘in vivo’
detection of hydrogen sulfide
Sameh El Sayedabc, Cristina de la Torreabc, Luis E. Santos-Figueroaabc, Ramón Martínez-
Máñezabc, Félix Sancenónabc, Mar Orzáezd, Ana M. Costeroae, Margarita Parraae & Salvador
Gilae
a Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Unidad Mixta
Universitat Politècnica de València, Universitat de València
b Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n,
46022Valencia, Spain
c CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)
Click for updates
d Laboratorio de Péptidos y Proteinas, Centro de Investigación Príncipe Felipe, Autopista El
Saler, Valencia, Spain
e Departamento de Química Orgánica, Universitat de València, Dr. Moliner 50, 46100
Burjassot, Valencia, Spain
Published online: 04 Nov 2014.
To cite this article: Sameh El Sayed, Cristina de la Torre, Luis E. Santos-Figueroa, Ramón Martínez-Máñez, Félix Sancenón,
Mar Orzáez, Ana M. Costero, Margarita Parra & Salvador Gil (2015) 2,4-dinitrophenyl ether-containing chemodosimeters for
the selective and sensitive ‘in vitro’ and ‘in vivo’ detection of hydrogen sulfide, Supramolecular Chemistry, 27:4, 244-254,
DOI: 10.1080/10610278.2014.977286
To link to this article: http://dx.doi.org/10.1080/10610278.2014.977286
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Supramolecular Chemistry, 2015
Vol. 27, No. 4, 244–254, http://dx.doi.org/10.1080/10610278.2014.977286
2,4-dinitrophenyl ether-containing chemodosimeters for the selective and sensitive ‘in vitro’ and
‘in vivo’ detection of hydrogen sulfide
a,b,c
a,b,c
Sameh El Sayeda,b,c, Cristina de la Torrea,b,c, Luis E. Santos-Figueroaa,b,c, Ramon Martınez-Man˜ez *, Felix Sancenon
,
´
´
´
´
´
Mar Orzaez , Ana M. Costero *, Margarita Parraa,e and Salvador Gila,e
d
a,e
´
a
´
`
`
Centro de Reconocimiento Molecular y Desarrollo Tecnologico (IDM), Unidad Mixta Universitat Politecnica de Valencia, Universitat
de Valencia; Departamento de Quımica, Universitat Politecnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain; CIBER de
`
b
c
`
´
`
d
´
Bioingenierıa, Biomateriales y Nanomedicina (CIBER-BBN); Laboratorio de Peptidos y Proteinas, Centro de Investigacion Prıncipe
Felipe, Autopista El Saler, Valencia, Spain; Departamento de Quımica Organica, Universitat de Valencia, Dr. Moliner 50, 46100
´
´
´
e
´
´
`
Burjassot, Valencia, Spain
(Received 15 August 2014; accepted 12 October 2014)
Four probes (i.e. D1–D4) for the selective and sensitive fluorogenic detection of HS2 have been prepared and characterised.
HEPES (10 mM, pH 7.4)–DMSO 99:1 v/v solutions of D1–D4 are essentially non-fluorescent. Changes in the emission
using D1–D4 in the presence of anions (F2, Cl2, Br2, I2, N32, CN2, SCN2, AcO2, CO322, PO422, SO242, HS2 and OH2),
biothiols (GSH, Cys, Hcy, Me–Cys and lipoic acid), reducing agents (SO232 and S2O322) and oxidants (H2O2) demonstrated
that only HS2 is able to induce the appearance of intense emission bands in the 400–520 nm range in the four probes. The
selectivity observed was ascribed to a unique hydrogen sulfide-induced hydrolysis of the 2,4-dinitrophenyl ether moiety that
yielded the corresponding free highly fluorescent alcohols. The potential detection of intracellular HS2 was also studied.
Keywords: hydrogen sulfide; fluorescence; dinitrophenyl ether; in vivo detection; chemodosimeter
Introduction
hydrogen sulfide and exogenous administration of hydro-
gen sulfide has been demonstrated to exert protective
effects in many pathologies. For example, hydrogen sulfide
has been shown to relax vascular smooth muscle, induce
vasodilation of isolated blood vessels and reduce blood
pressure (13). Hydrogen sulfide has proved also to inhibit
leukocyte adherence in mesenteric microcirculation in
vascular inflammation in rats, suggesting that hydrogen
sulfide is a potent anti-inflammatory molecule (14).
Additionally, it has become evident that hydrogen sulfide
is a potent antioxidant and, under chronic conditions, can
up-regulate antioxidant defense (15).
Hydrogen sulfide (H2S), a colourless and flammable gas
with high solubility in water and in organic solvents, is well
known for its unpleasant ‘rotten eggs’ smell. Hydrogen
sulfide has been traditionally studied for its toxicity (1), its
presence as a corrosive industrial refuse and emission in the
energy industry (2), as parameter for geological activity
(3), as quality control of foods and as an important
compound related with the presence of microbial activity in
anaerobic environments (4). Moreover, this simple
molecule has been recently recognised to play a relevant
role in living systems. For instance, hydrogen sulfide has
been documented to be the third gaseous transmitter (the
other two being nitric oxide and carbon monoxide) (5–7).
The production of H2S in mammalian systems has been
attributed to at least three endogenous enzymes, i.e.
cystathionine b-synthase (CBS), cystathionine g-lyase
(CSE), and 3-mercaptopyruvate sulfurtransferase (MPST)
(8–11). These enzymes use cysteine or cysteine derivatives
as substrates and convert them into hydrogen sulfide in
different organs and tissues. In addition to these enzymatic
pathways, there are also a range of simple chemical events
that may liberate hydrogen sulfide from the intracellular
pool of ‘labile’ sulfur such as the so-called ‘sulfane pool’
(compounds containing sulfur atoms bound only to other
sulfur atoms) (12). The production of endogenous
However, hydrogen sulfide exact mechanism of action
and production in living systems is still under active
investigation. For example, it is known that hydrogen
sulfide can react readily with methemoglobin to form
sulfhemoglobin, which act as the metabolic sink for
H2S. As a potential reductant, hydrogen sulfide is likely to
be consumed by endogenous oxidant species such as
hydrogen peroxide, superoxide, peroxynitrite, etc. This
process is significant and it provides a mechanism by
which the concentration of hydrogen sulfide changes.
Besides, abnormal levels of hydrogen sulfide have been
associated with various diseases such as chronic kidney
disease, liver cirrhosis (16), Alzheimer (17), and Down
syndrome (18).
*Corresponding authors. Emails: rmaez@qim.upv.es; Ana.Costero@uv.es
q 2014 Taylor & Francis

Supramolecular Chemistry
245
Due to the important roles that hydrogen sulfide plays
in environmental and in biological processes, the
development of selective detection systems for this
compound has recently become a focus of importance.
In particular, it was a major challenge to clarify the
complex contributions of H2S in certain diseases by
finding methods for selective tracking of this small
molecule within living systems. Current techniques for
hydrogen sulfide detection, such as colorimetry (19–21),
electrochemical assays (22), gas chromatography (23) and
metal-induced sulfide precipitation (24), show some
shortcomings typically involving destruction of the sample
and/or poor selectivity.
group. Finally, Liu and Feng prepared an ESIPT (excited
state intramolecular proton transfer) probe functionalised
with a 2,4-dinitrophenyl ether moiety for the rapid
chromo-fluorogenic recognition of hydrogen sulfide (61).
The earlier described facts demonstrate that the design of
new fluorogenic probes for hydrogen sulfide is a timely
field of interest. Such simple systems may be used to
control H2S concentrations in target industrial processes,
for in situ environmental, geological monitoring and
detection of hydrogen sulfide levels in living systems.
Nevertheless, in spite of these possible applications, there
are still relatively few examples of optical probes for the
selective and sensitive detection of hydrogen sulfide (vide
ante).
In addition to the aforementioned classical method-
ologies, fluorescence is an attractive highly sensitive
technique for studying biomolecule distribution in living
cells. For instance, very recently, fluorescent probes for
monitoring redox cycles in vivo have been described (25).
In this area, several fluorescent probes, designed taking
into account supramolecular chemistry concepts, for the
selective recognition of H2S or HS2 have been described
(26). Most of the fluorescent probes reported are based on
the hydrogen sulfide-induced reduction of azides or nitro
moieties, electronically connected with a fluorophore, to
amines (27–36). This reduction induced perturbations in
the electronic structure of the signalling unit that is
reflected in emission changes. Another important group of
probes is constructed under a displacement paradigm and
is based on non-fluorescent Cu(II) complexes (37–46).
In these systems, the hydrogen sulfide-induced demetalla-
tion of the probes induced marked emission enhance-
ments. Other sulfide-selective fluorescent probes used
Michael type addition reactions coupled with emission
changes (47–50). Also, cyclisation processes that yielded
fluorescent compounds have recently been used for the
design of fluorescent probes for sulfide anion (51–53).
In other examples, hydrolysis reactions coupled with
emission changes have been also used for the preparation
of other hydrogen sulfide-sensitive probes (54–56).
Finally, selenium containing BOPIPY dyes have also
been used for the reversible detection of sulfide in solution
and in living cells (57,58). Among probes that used
hydrolysis reactions, as far as we know, only three probes
containing 2,4-dinitrophenyl ethers have been described
and used for the selective and sensitive recognition of HS2
anion. In particular, Cao et al. (59) prepared a NIR dye
functionalised with a 2,4-dinitrophenyl ether moiety. PBS
(pH 7.0)–ethanol 9:1 v/v solutions containing CTAB and
this probe were non-fluorescent, whereas addition of
increasing quantities of HS2 anion induced a progressive
appearance of an emission band. This emission enhance-
ment was ascribed to a thiolysis of the 2,4-dinitrophenyl
ether moiety. The same sensing mechanism was used by
Liu et al. (60) who prepared a 1,8-naphthalimide
fluorophore equipped with a 2,4-dinitrophenyl ether
Bearing in mind the aforementioned concepts and
following our interest in the development of new
approaches for optical sensing (62), we show herein a
family of chemodosimeters (D1–D4) composed by
selected fluorophores functionalised with 2,4-dinitrophe-
nyl ether moieties for the detection of hydrogen sulfide.
The synthesis and sensing features of probe D3 were
recently published by us in a short communication (63).
These dosimeters were easily synthesised through
nucleophilic aromatic substitution reactions with 1-fluro-
2,4-dintrobenzene with different fluorophores. The four
probes displayed sensing features in buffered aqueous
solution containing 1% of DMSO and gave marked
emission enhancements only in the presence of HS2
anion, whereas other biothiols were unable to induce
emission changes. The probes showed excellent sensi-
tivities, with limits of detection in the 0.05–0.30 mM
range, and some of the chemosensors were used for the
fluorescence detection of both exogenous and endogenous
HS2 in living cells.
Results and discussion
Synthesis and characterisations of the probes
The path of synthesis and the structures of probes D1–D4
are shown in Scheme 1. D1–D4 are 2,4-dinitrophenyl
ether derivatives which are easily obtained in good yield
(65–88%) through an aromatic nucleophilic substitution
reaction between 1-fluoro-2,4-dinitrobenzene (1) and
alcohols 1c, 2a, 3a and 4b. Compounds 2a and 3a are
commercially available. Alcohol 1c was prepared through
the base-catalysed condensation between 4-hydroxyben-
zaldehyde (1a) and 4-picoline (1b), whereas alcohol 4b
was obtained through methylation of fluorescein (4a) with
methyliodide.
1
All new probes were characterised by H, 13C NMR
and HRMS. At this respect, the more indicative signals in
1
the H MNR spectra of dosimeter D1 in DMSO-D6 were
the doublets centred at 7.81 and 7.32 ppm attributed to the
protons of the trans double bond. Moreover, the protons of
the 2,4-dinitrophenyl ether moiety appeared at 8.91 (1H,

246
S. El Sayed et al.
OH
OH
NO2
NO2
N
1b
N
1c
O
KOH
H
O
D1
1a
N
NO2
Et3N
HO
O
OH
OH
F
2a
NO2
NO2
D2
O
O
Et3N
1
O
NO2
OH
4a
CH3I
NaOH
Et3N
3a
OH
O2N
O
NO2
N
O
O
N
D3
Et3N
O
4b
NO2
O
NO2
O
O
O
O
O
D4
Scheme 1. Synthesis and chemical structure of chemodosimeters D1–D4.
d), 8.47 (1H, dd) and 7.57 (1H, d) ppm. On changing to
chemodosimeter D2, the most characteristic signals in the
1H NMR (CDCl3) spectrum were also those of the 2,4-
dinitrophenyl ether moiety centred at 8.97, 8.41 and
7.39 ppm. Nearly the same pattern was observed for D3 in
which the dinitrophenyl ether protons were located at 8.93
(1H, d), 8.18 (1H, dd) and 7.83 (1H, d) ppm. Dealing with
fluorescein derivative D4, the signals ascribed to the
dinitrophenyl ether appeared at nearly the same chemical
shift than those observed for D1–D3, whereas the
methoxy moiety appeared as singlet at 3.76 ppm (in
DMSO-D6).
simple sensing mechanism was used, very recently, in the
preparation of selective and sensitive chemodosimeters for
HS2 anion in water and in cellular media (vide ante) (59–
61). The high selectivity obtained for HS2 versus thiols
(vide infra) relies on the fact that H2S is a small molecule
with a pKa of 6.9, while typical cellular-free thiols (i.e.
GSH and Cys) have pKa values of ca. 8.5 (65). Taking into
account these differences, at physiological pH, the
thiolysis of 2,4-dinitrophenyl ethers is chemoselective
for HS2 over GSH, Cys and other biologically relevant
thiols.
In a first step, we tested the fluorescent response of the
probes D1–D4 (5 mM) in HEPES (10 mM, pH 7.4)–DMSO
99:1 v/v solution upon addition of excess (10 equivalents)
of selected anions (F2, Cl2, Br2, I2, N23 , CN2, SCN2,
AcO2, CO232, PO242, SO242, HS2 and OH2), biothiols
(GSH, Cys, Hcy, Me–Cys and lipoic acid), reducing agents
(SO322 and S2O322) and oxidants (H2O2). As it can be seen
in Figure 1, the four dosimeters were practically non-
fluorescent; however, addition of HS2 induced a very
remarkable fluorescent ‘turn-on’ response, whereas
addition of the other chemicals tested induced negligible
changes in the emission behaviour. Besides, the fluorescent
behavior of the four probes in the presence of GSH 6 mM
(GSH concentrations in cells are in the 1–10 mM range)
Sensing features of the probes
The four synthesised probes contain a chromo-fluorogenic
subunit functionalised with a sulfide-sensitive 2,4-
dinitrophenyl ether moiety. It is well known that the
dinitrophenyl group has been often used for tyrosine
protection in peptide synthesis (64). The removal of this
protective group is carried out using thiols under basic
conditions. In our prepared probes, the dinitrophenyl ether
was electronically coupled with the fluorophore and the
rupture of the ether bond was expected to induce changes
in the emission spectra of the fluorogenic subunit. This

Supramolecular Chemistry
247
Figure 1. (Colour online) Emission intensity of D1 (at 515 nm upon excitation at 390 nm), D2 (at 415 nm upon excitation at 350 nm), D3
(at 516 nm upon excitation at 450 nm) and D4 (at 515 nm upon excitation at 450 nm) dosimeters (5 mM) in HEPES (10 mM, pH 7.4)–
DMSO 99:1 v/v solutions upon addition of 10 equivalents of anions, biothiols, reducing agents and oxidants (after 60, 70, 30 and 50 min of
the addition for D1, D2, D3 and D4, respectively). The temperature was set at 258C.
was also tested. As could be seen in Figure 2, GSH was
unable to induce a remarkable fluorescence enhancement of
the four probes even at mM concentrations.
solution of dosimeter D1 induced the appearance of an
emission band at 515 nm (lex ¼ 390 nm) that reached its
maximum intensity after 60 min (15-fold enhancement)
(see Figure 3). For D2, addition of HS2 induced a 46-fold
enhancement of the emission intensity at 415 nm
(lex ¼ 350 nm) after 70 min (Figure 4).
More in detail, addition of 10 equivalents of HS2
anion to HEPES (10 mM, pH 7.4)–DMSO 99:1 v/v
D2
1400
D1
1000
800
600
400
200
0
1200
D3
1000
800
600
400
200
0
D4
450
500
550
λ/nm
600
650
Figure 2. Emission intensity of D1 (at 515 nm upon excitation
at 390 nm), D2 (at 415 nm upon excitation at 350 nm), D3 (at
516 nm upon excitation at 450 nm) and D4 (at 515 nm upon
excitation at 450 nm) dosimeters (5 mM) in HEPES (10 mM, pH
7.4)–DMSO 99:1 v/v solutions alone (white bars) and upon
addition of 50 mM GSH (light grey bars), 6 mM GSH (dark grey
bars) and 50 mM HS2 (black bars). The temperature was set at
258C.
Figure 3. Fluorescence enhancement of dosimeter D1 (5 mM)
in HEPES (10 mM, pH 7.4)–DMSO 99:1 v/v solution upon
addition of increasing quantities of HS2 anion (from 0 to 10
equivalents) after 60 min of the addition (lex ¼ 390 nm). The
temperature was set at 258C.

248
S. El Sayed et al.
After assessing the highly selective turn-on response of
1600
1400
1200
1000
800
600
400
200
0
D1–D4 to hydrogen sulfide, the sensitivity of the probes
was studied from titration profiles with HS2 obtained in
HEPES–DMSO 99:1 v/v solutions. From these studies, a
limit of detection (LOD) of 0.20 mM for HS2 was
determinedforD1. Nearlythesameresponse andsensitivity
was obtained with probe D2 (LOD ¼ 0.29 mM). A more
sensitive response was found on changing to dosimeters D3
and D4. In these cases, the titration profiles obtained
allowed to calculate LOD of 0.06 and 0.05 mM for D3
and D4, respectively. In all cases, the probes displayed
LODs below the hydrogen sulfide concentration required
to elicit physiological responses (10–1000 mM) (66).
The selective emission enhancements observed for
D1–D4 in the presence of HS2 is ascribed in all cases to
an HS2-induced hydrolysis of the 2,4-dintrophenyl ether
groups that results in the release of the highly fluorescent
alcohols 1c, 2a, 3a and 4b. In order to corroborate this
hypothesis, probes D1–D4 were dissolved in ethanol and
excess of Na2S was added. The reactions were stirred at
room temperature for 3 h and then the solvent was
eliminated in a rotary evaporator. The crudes obtained
375
400
425
450
λ\nm
475
500
525
550
Figure 4. Fluorescence enhancement of dosimeter D2 (5 mM)
in HEPES (10 mM, pH 7.4)–DMSO 99:1 v/v solution upon
addition of increasing quantities of HS2 anion (from 0 to 10
equivalents) after 70 min of the addition (lex ¼ 350 nm). The
temperature was set at 258C.
Nearly the same enhancement (32-fold) in the intensity
at 515 nm (lex ¼ 450 nm) was obtained upon addition of
HS2 anion to probe D4 (Figure 5). In this case, the
response was faster (when compared with D1 and D2) as
the maximum enhancement was observed after 30 min
upon HS2 addition. Besides, dosimeter D3 gives the larger
response; in this case, the addition of HS2 anion induced a
remarkable 344-fold enhancement in the emission band at
516 nm (lex ¼ 450 nm) after 50 min (see Figure 6). The
subtle differences in response time and in emission
enhancement upon addition of HS2 anion to the four
dosimeters could be ascribed to the different electronic
properties of the probes although in all cases a clear highly
selective turn-on response was observed.
1
were purified by silica column chromatography and H
and 13C confirmed the presence of the alcohols 1c, 2a, 3a
and 4b (for D1, D2, D3 and D4 respectively) as main
products of the reaction.
The sensing features of D1–D4 probes were quite
similar to those reported for analogous fluorophores
functionalised with 2,4-dinitrophenylethers (see Table 1).
In particular, reaction times when using hydrolysis of 2,4-
dinitrophenylether as sensing mechanism are in the range
of minutes, whereas the detection limits are in the 0.05–
0.29 mM interval. Table 1 also shows the typical sensing
parameters for probes for hydrogen sulfide when using
1200
1100
1000
900
800
700
600
500
400
300
200
100
1000
800
600
400
200
0
0
480 500 520 540 560 580 600 620 640
500
550
600
λ/nm
λ/nm
Figure 5. Fluorescence enhancement of dosimeter D4 (5 mM)
in HEPES (10 mM, pH 7.4)–DMSO 99:1 v/v solution upon
addition of increasing quantities of HS2 anion (from 0 to 10
equivalents) after 30 min of the addition (lex ¼ 450 nm). The
temperature was set at 258C.
Figure 6. Fluorescence enhancement of dosimeter D3 (5 mM)
in HEPES (10 mM, pH 7.4)–DMSO 99:1 v/v solution upon
addition of increasing quantities of HS2 anion (from 0 to 10
equivalents) after 50 min of the addition (lex ¼ 450 nm). The
temperature was set at 258C.

Supramolecular Chemistry
249
Table 1. Mechanism used in the development of fluorogenic probes for hydrogen sulfide.
Mechanism
Time
Limit of detection
References
Hydrolysis of 2,4-dinitrophenylether (D1–D4)
Hydrolysis of 2,4-dinitrophenylether
Demetallation of Cu(II) complexes
Michael addition reactions
Other hydrolysis reactions
Reduction reactions
30–70 min
5–20 min
Few seconds to 5 min
2–60 min
0.05–0.29 mM
0.05–0.48 mM
0.01–16 mM
0.12–1 mM
0.05–9 mM
0.01–5 mM
This paper
59–61
37–46
47–50
54–56
27–36
57, 58
30–60 min
3–120 min
5 min
Oxidation of Se-containing fluorophores
Cyclisation reactions
Not reported
0.1–100 mM
2–50 min
51–53
different sensing mechanisms. From the data given in
Table 1, it is apparent that the best response, in terms of
reaction time, is obtained with probes in which the sensing
mechanism is related with demetallation of Cu(II)
complexes (from few seconds to minutes). As seen also
in Table 1, lower limits of detection for hydrogen sulfide
are obtained when demetallation processes and reduction
reactions are used as transduction mechanisms.
Detection of HS2 in HeLa cells
Once the biocompatibility of the prepared probes was
assessed, and with the aim to test the dosimeters in highly
competitive environments, we prospectively used probes
D1–D4 for the fluorescence imaging of sulfide in living
cells. Of the four probes tested, only D1 and D3 gave
remarkable results due to the low solubility of D2 and D4
at the concentrations required in the confocal microscopy
studies.
In a typical experiment, HeLa cells were incubated in
DMEM supplemented with 10% fetal bovine serum.
To conduct fluorescence microscopy studies, HeLa cells
were seeded in 24 mM glass coverslips in 6-well plates and
were allowed to settle for 24 h. Cells were treated with the
probes in PBS–DMSO 99:1 v/v at final concentrations of
10 and 30 mM, for D1 and D3 respectively. After 30 min,
the medium was removed and solutions of different
concentrations of NaHS in PBS were added (0, 100, 200
and 500 mM) and cells were incubated for another 10
minutes.
Cell viability assays
The selective emission enhancement of D1–D4 in the
presence of HS2 and the poor response observed upon the
addition of biothiols (GSH, Cys and Hcy) strongly suggest
that these probes could be used for HS2 imaging in living
cells. Based on these observations, the cytotoxicity of D1–
D4 was first evaluated. For this purpose, HeLa cells were
treated with probes D1–D4 at different concentrations (5,
10, 20 and 50 mM) over a 24-h period and cell viability
was determined by using a WST-1 assay. The obtained
results are shown in Figure 7. As seen, the four dosimeters
were essentially non-toxic in the range of concentrations
tested.
The results are shown in Figures 8 (for D1) and 9 (for
D3). The control experiment (HeLa cells without
dosimeters) and the cells incubated with D1 and D3
showed no fluorescence, whereas a clear and marked
enhancement in intracellular emission was found in the
HS2-treated cells. The emission enhancement observed
was clearly dependent of the amount of NaHS added. The
enhancement in the intracellular fluorescence intensity
with D1 was quantified by a standard image analysis and
the results are shown in Figure 8(i). A similar response
(not shown) was observed for D3.
Conclusions
In summary, here we reported the synthesis and sensing
features of four compounds (D1–D4) as fluorescent turn-
on probes for hydrogen sulfide. These chemodosimeters
were based in different fluorophores (i.e. styryl pyridine,
naphthalene, quinoline and fluorescein for D1, D2, D3 and
D4, respectively) functionalised with a 2,4-dinitrophenyl
ether moiety. This chemical modification made the final
probes poorly emissive. Probes D1–D4 were easy to
Figure 7. Cell viability assays. Hela cells were treated with
dosimeters D1 (black), D2 (dark grey), D3 (grey) and D4 (white)
at concentrations of 5, 10, 20 and 50 mM during 24 h.

250
S. El Sayed et al.
Figure 8. (Colour online) Detection of HS2 levels in living cells using dosimeter D1. (a, e) Transmitted light and fluorescence images of
untreated HeLa cells. (b, f) Hela cells incubated with D1 for 90 min. (c, g) HeLa cells incubated with D1 for 30 min at 378C and then with
200 mM Na2S for 60 min. (d, h) Hela cells incubated for 30 min with D1 and 500 mM Na2S for other 60 min. (i) Average If/I0 intensity ratios
after addition of 200 and 500 mM of Na2S in PBS buffer. The excitation and emission wavelengths were 450–470 nm and 510–530 nm.
Representative fluorescence images from replicate experiments (n ¼ 3) are shown. Error bars are ^ SD from replicate experiments(n ¼ 3).
prepare and were able to selectively detect the HS2 anion
in HEPES (10 mM, pH 7.4)–DMSO 99:1 v/v solutions via
a remarkable turn-on emission. The emission enhance-
ments observed were due to a selective sulfide-induced
hydrolysis of the 2,4-dinitrophenyl ether moiety that
released the free fluorophores. D1–D4 can selectively and
sensitively detected HS2 anion in water over other anions,
biothiols and common oxidants. The titration profiles
obtained upon addition of increasing quantities of HS2
anion to aqueous solutions of the four dosimeters allowed
Figure 9. (Colour online) Detection of HS2 levels in living cells using probe D3. HeLa cells were incubated with D3 (30 mM) for 30 min
at 378C in DMEM. Transmitted light and fluorescence images were captured for (a) Hela cells, (b) Hela cells incubated with D3, and Hela
cells incubated with D3 in the presence of HS2 at concentrations of (c) 200 mM and (d) 500 mM. The range of excitation was 330–380 nm
and emission was monitored for wavelengths exceeding 420 nm.

Supramolecular Chemistry
251
us to determine LODs of 0.20, 0.29, 0.06 and 0.05 mM for
D1, D2, D3 and D4 respectively. Besides, the four
dosimeters showed remarkably low sensitivities below the
hydrogen sulfide concentration required to elicit physio-
logical responses. From cell viability assays, it was found
that the four probes were essentially non-toxic. Moreover,
real-time fluorescence imaging measurements confirmed
that probes D1 and D3 can be used to detect intracellular
HS2 at micromolar concentrations.
the crude reaction and the mixture was refluxed for 30 min.
Then, the acetone was evaporated and the residue taken up
in 5% hydrochloric acid (10 mL). A precipitate appeared
that was filtered, washed with water and then suspended in
5% sodium hydroxide (15 mL) and stirred for 15 min.
After that, the solid was filtered, washed with water and
purified by silica column chromatography using hexane–
acetone 1:1 v/v as eluent. Dosimeter D1 was isolated as
yellow solid (310 mg, 0.85 mMol, 74% yield).
1H NMR (400 MHz, DMSO) d 8.91 (d, J ¼ 2.8 Hz, 1H),
8.56 (d, J ¼ 6.0 Hz, 2H), 8.47 (dd, J ¼ 9.3, 2.8 Hz, 1H),
7.81 (d, J ¼ 8.7 Hz, 2H), 7.57 (dd, J ¼ 4.9, 3.4 Hz, 3H),
7.34–7.25 (m, 4H).
Experimental section
Materials and methods
13C NMR (101 MHz, CDCl3) d 154.7, 153.9, 150.1,
144.2, 141.6, 139.6, 134.3, 131.8, 129.7, 129.4, 126.6,
122.0, 121.0, 120.7, 119.7.
UV–visible spectra were recorded with a JASCO V-650
Spectrophotometer. Fluorescence measurements were car-
ried out in a JASCO FP-8500 Spectrophotometer (Jasco
HR-MS-EI m/z: calcd for C19H13N3O5 363.0855;
found: 364.0944 (M þ Hþ).
1
analıtica, Madrid, Spain). H and 13C NMR spectra were
´
acquired in a BRUKER ADVANCE III (400MHZ) (Bruker,
Billerica, MA, USA), where mass spectra were carried out in
a TRIPLETOF T5600 (ABSciex, Framingham, USA)
spectrometer. The chemicals 4-picoline, 2-hydroxynaphtha-
lene, 8-hydroxyquinoline, fluorescein, iodomethane, 1-
fluoro-2,4-dinitrobenzene, 4-hydroxybenzaldehyde (98%),
acetic anhydride (98%), hexadecyltrimethylammonium
bromide (CTABr) and sodium bicarbonate were purchased
from Sigma-Aldrich (Madrid, Spain). Triethylamine (98%)
was purchased from J.T. Baker. Analytical-grade solvents,
potassium hydroxide, sodium sulfate and hydrochloric acid
(37%) were purchased from Scharlau (Barcelona, Spain).
Synthesis of 2-(2,4-dinitrophenoxy)naphthalene (D2)
Dosimeter D2 was prepared by the same experimental
procedure used for D1. The final product was isolated as
pale yellow solid (725 mg, 2.33 mMol, 88 % yield).
1H NMR (400 MHz, CDCl3) d 8.97 (d, J ¼ 2.7 Hz,
1H), 8.41 (dd, J ¼ 9.3, 2.7 Hz, 1H), 8.09 (d, J ¼ 8.9 Hz,
1H), 8.04–8.00 (m, 1H), 7.96–7.92 (m, 1H), 7.67 (ddd,
J ¼ 9.4, 4.2, 2.3 Hz, 3H), 7.40–7.37 (m, 1H), 7.18 (s, 1H).
13C NMR (101 MHz, CDCl3) d 156.3, 151.2, 141.6,
139.6, 134.2, 131.7, 131.2, 128.9, 128.1, 127.6, 126.5,
122.2, 119.7, 118.8, 117.6.
HR-MS-EI m/z: calcd for C16H10N2O5 310.0590;
found: 310.0817 (Mþ).
Synthesis of 4-((E)-2-(pyridin-4-yl)vinyl)phenol (1c)
4-hydroxybenzaldehyde (1a, 500 mg, 4.1 mMol) and 4-
picoline (1b, 0.4 mL, 4.1 mMol) were refluxed in acetic
anhydride (5 mL) for 24 h. After cooling to room
temperature, a precipitate appeared. This was collected
and recrystallised from ethanol. Then, the solid was
dissolved in ethanol–water (20 mL, 1:1 v/v) containing
potassium hydroxide (400 mg, 7.1 mMol) and refluxed for
6 h. Afterwards, the crude reaction was neutralised with
diluted hydrochloric acid. The product 1c precipitated and
was isolated as a yellow solid after filtration (749 mg,
3.8 mMol, 85% yield).
Synthesis of 8-(2,4-dinitrohydroxy)quinoline (D3)
Dosimeter D3 was prepared by the same experimental
procedure used for D1. The final product was isolated as
pale yellow solid (703 mg, 2.25 mMol, 85 % yield).
1H NMR (400 MHz, CDCl3) d 8.93 (d, J ¼ 2.8 Hz,
1H), 8.79 (dd, J ¼ 4.2, 1.7 Hz, 1H), 8.25 (dd, J ¼ 8.4,
1.7 Hz, 1H), 8.18 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.83 (dd,
J ¼ 7.9, 1.7 Hz, 1H), 7.65–7.56 (m, 2H), 7.47 (dd,
J ¼ 8.4, 4.2 Hz, 1H), 6.79 (d, J ¼ 9.3 Hz, 1H).
1H NMR (400 MHz, DMSO) d 12.10 (s, 1H), 10.79 (d,
J ¼ 5.6 Hz, 2H), 9.75 (dd, J ¼ 21.4, 11.6 Hz, 5H), 9.29 (d,
J ¼ 16.4 Hz, 1H), 9.10 (d, J ¼ 8.5 Hz, 2H).
13C NMR (101MHz, CDCl3) d 157.4, 151.0, 149.4,
141.4, 140.6, 138.9, 136.4, 130.3, 128.7, 126.8, 122.4, 120.9.
HR-MS-EI m/z: calcd for C15H10N3O5 311.0542;
found: 312.0628 (M þ Hþ).
Synthesis of 4-(4-(2,4-dinitrophenoxy)styryl)pyridine
(D1)
Synthesis of 3-hydroxy-6-methoxy-3H-spiro
[isobenzofuran-1,9-xanthen]-3-one (4b)
1c (200 mg, 1.17 mMol) was dissolved in acetone (5 mL)
and then triethyl amine (0.5 mL, 3.6 mMol) was added.
After that, 1-fluoro-2,4-dinitrobenzene (217.7 mg,
1.17 mMol) dissolved in acetone (5 mL) was added to
Fluorescein (4a, 2 g, 6.18 mMol) was suspended in
methanol (50 mL) and NaOH (160 mg, 4 mMol) was
then added. The final mixture was stirred until complete

252
S. El Sayed et al.
dissolution of fluorescein. After evaporation of methanol,
the disodium salt (2.0 g, 5.32 mMol) was suspended in
DMF (50 mL) containing methyl iodide (332 mL,
5.32 mMol) and the reaction was then stirred at room
temperature for 48 h. After this time, the crude was diluted
with 5% NaHCO3 (50 mL) and extracted with diethyl ether
(2 £ 25 mL). The organic phase was washed with brine
(20 mL), dried over Na2SO4 and evaporated to give an
orange precipitate. This solid was dissolved in methanol
(30 mL), 2M NaOH (10 mL) was added and the mixture
was stirred at room temperature for 2 h. After evaporation
of methanol, the crude was diluted with water (20 mL) and
impurities were extracted with diethyl ether (2 £ 10 mL).
The aqueous phase was acidified (pH 2.0) by the addition
of concentrated HCl and then extracted with diethyl ether
(3 £ 15 mL), washed with brine (25 mL) and dried over
Na2SO4. After evaporation of the diethyl ether, a brown
solid was obtained. The final product was purified by silica
column chromatography using diethyl ether–hexane 2:1
v/v as eluent. Compound 4b was isolated as brown solid
(831.2 mg, 2.4 mMol, 45% yield).
to settle for 24 h. Dosimeters D1, D2, D3 and D4 were
added to the cells at final concentrations of 10, 20, 30 and
50 mM. After 23 h, WST-1 (7 mL of a 50 mg/mL solution)
was added to each well. Cells were further incubated for
1 h (a total of 24 h of incubation was therefore studied) and
then shacked thoroughly for 1 min on a shaker, and the
absorbance was measured at 450 nm against a background
control as blank using a microplate ELISA reader. The
reference wavelength is 690 nm.
Live confocal microscopy
Hela cells were seeded in 24-mm glass coverslips in 6-well
plates at a seeding density of 1025 cells/well. After 24 h,
cells were treated with D1 and D3 at a final concentration
of 10 and 30 mM, respectively. After 30 min, the medium
was removed to eliminate compounds and washed with
PBS. Then a solution of Na2S in PBS was added at a final
concentrations of 0, 100, 200, 500 mM and cells were
incubated for 1 h at 378C. After that, slides were washed
twice with PBS to remove traces of the dosimeters. Then
slides were visualised under a confocal. Confocal
microscopy studies were performed by confocal
microscopy service. The images were acquired with a
Leica TCS SP2 AOBS (Leica Microsystems Heidelberg
GmbH, Mannheim, Germany) inverted laser scanning
confocal microscope using oil objectives: 63X Plan-
Apochromat-Lambda Blue 1.4 N.A. The excitation
wavelengths were 450 nm (argon laser) for both dosi-
meters. Two-dimensional pseudo-colour images (255
colour levels) were gathered with a size of 1024 £ 1024
pixels and Airy 1 pinhole diameter. All confocal images
were acquired using the same settings and the distribution
of fluorescence was analysed using the Image J Software.
Identical experiments were done three times to obtain
reproducible results.
Synthesis of 3-(2,4-dinitrophenoxy)-6-methoxy-3H-spiro
[isobenzofuran-1,9-xanthen]-3-one (D4)
Dosimeter D4 was prepared by the same experimental
procedure used for D1. The final product was isolated as
brown solid (162 mg, 0.315 mMol, 65 % yield).
1H NMR (400 MHz, DMSO) d 8.84 (d, J ¼ 2.8 Hz,
1H), 8.58 (d, J ¼ 3.1 Hz, 1H), 8.45 (dd, J ¼ 9.2, 2.6 Hz,
1H), 8.01 (d, J ¼ 7.6 Hz, 1H), 7.90–7.77 (m, 1H), 7.73
(t, J ¼ 7.3 Hz, 1H), 7.37 (d, J ¼ 9.2 Hz, 1H), 7.22 (dd,
J ¼ 9.8, 5.1 Hz, 1H), 6.92 (ddd, J ¼ 20.1, 13.2, 5.6 Hz,
1H), 6.74 (dd, J ¼ 8.9, 2.5 Hz, 1H), 6.67 (d, J ¼ 8.8 Hz,
1H), 6.45 (d, J ¼ 9.7 Hz, 1H), 3.76 (s, 1H).
13C NMR (101 MHz, DMSO) d 158.3, 155.0, 144.7,
141.2, 140.9, 139.1, 138.7, 138.5, 136.6, 129.7, 127.9,
126.6, 126.0, 125.8, 125.6, 124.8, 123.0, 121.7, 120.3,
117.8, 114.8, 115.8, 113.0, 110.1, 107.8, 56.1, 18.6.
HR-MS-EI m/z: calcd for C27H16N2O9 512.0856;
found: 513.0935 (M þ Hþ).
Supplemental data
Supplemental data for this article are available online.
Funding
Cell culture conditions
Financial support from the Spanish Government (Project
MAT2012-38429-C04-01) and the Generalitat Valencia (Project
PROMETEO/2009/016) is gratefully acknowledged. S.E. is
grateful to the Generalitat Valenciana for his Santiago Grisolia
fellow. L.E.S.F. also thanks the Carolina Foundation and
UPNFM-Honduras for his doctoral grant.
The HeLa human cervix adenocarcinoma cells were
purchased from the German Resource Centre for
Biological Materials (DSMZ) and were grown in DEM
supplemented with 10% FBS. Cells were maintained at
378C in an atmosphere of 5% CO2 and 95% air and
underwent passage twice in a week.
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