A chemosensor bearing sulfonyl azide moieties for selective chromo-fluorogenic hydrogen sulfide recognition in aqueous media and in living cells

Article abstract of DOI:10.1002/ejoc.201301514

A simple chemodosimeter based on a sulfonyl azide dye (1-Az), which displayed a highly selective response toward hydrogen sulfide anion in mixed aqueous media, was synthesised and characterised. Addition of hydrogen sulfide to acetonitrile/HEPES 1:1 solutions of 1-Az induced a clear colour change from red-orange to yellow, which was easily detected by the naked eye, and by an enhancement in the emission intensity. Other common anions, thiol-containing biomolecules and oxidants did not induce any noticeable colour or fluorescence modulation in the probe. The chemodosimeter also showed a good sensitivity, with limits of detection of 11.91 and 0.63 μM by using UV/Vis or fluorescence measurements, respectively. Moreover, 1-Az could be used for real-time fluorescence imaging of intracellular HS^- at micromolar concentrations. Sensitive and selective chromogenic detection of hydrogen sulfide in aqueous environments was achieved with a simple sulfonyl azide derivative. Copyright

Full text of DOI:10.1002/ejoc.201301514

FULL PAPER
DOI: 10.1002/ejoc.201301514
A Chemosensor Bearing Sulfonyl Azide Moieties for Selective
Chromo-Fluorogenic Hydrogen Sulfide Recognition in Aqueous Media and in
Living Cells
Luis E. Santos-Figueroa,[a,b,c] Cristina de laTorre,[a,b,c] Sameh El Sayed,[a,b,c]
Félix Sancenón,[a,b,c] Ramón Martínez-Máñez,*[a,b,c] Ana M. Costero,*[a,d] Salvador Gil,[a,d]
and Margarita Parra[a,d]
Keywords: Analytical methods / Sensors / Hydrogen sulfide / Sulfur / Azides
A simple chemodosimeter based on a sulfonyl azide dye (1-
Az), which displayed a highly selective response toward
hydrogen sulfide anion in mixed aqueous media, was synthe-
sised and characterised. Addition of hydrogen sulfide to
acetonitrile/HEPES 1:1 solutions of 1-Az induced a clear col-
our change from red-orange to yellow, which was easily de-
tected by the naked eye, and by an enhancement in the
emission intensity. Other common anions, thiol-containing
biomolecules and oxidants did not induce any noticeable col-
our or fluorescence modulation in the probe. The chemodosi-
meter also showed a good sensitivity, with limits of detection
of 11.91 and 0.63 μM by using UV/Vis or fluorescence mea-
surements, respectively. Moreover, 1-Az could be used for
real-time fluorescence imaging of intracellular HS– at micro-
molar concentrations.
Down’s syndrome.[13] Due to the important roles that hy-
drogen sulfide plays in environmental and in biological pro-
cesses, the development of selective detection systems for
this compound has recently become a focus of significance.
In the last years, several probes for the fluorogenic detec-
tion of hydrogen sulfide in water or in mixed organic-aque-
ous environments have been described.[14] In most cases, the
probes follow a chemodosimeter approach in which an
irreversible chemical reaction induced by hydrogen sulfide
is coupled with effective fluorescence modulations. In par-
ticular, the reduction of azide,[15] hydroxylamine[16] and
nitro[17] moieties to amine have been used as key reactions
in the development of hydrogen sulfide-selective fluorescent
probes. Moreover, the hydrogen sulfide-induced hydrolysis
of disulfide bonds,[18] dinitrophenyl ethers,[19] and cyano-
vinyl moieties,[20] linked with fluorophores have also been
used for the fluorescent recognition of this anion. Another
interesting approach is the use of demetallation reactions of
nonfluorescent CuII complexes induced by hydrogen sulfide
that resulted in a turn-on of the emission.[21] Recently, Han
and co-workers prepared several fluorescent BODIPY dyes
that were functionalised with selenoxide moieties for the de-
tection of hydrogen sulfide through HS–-promoted re-
duction of the selenoxide group to selenide, which resulted
Introduction
Hydrogen sulfide (H2S), a colourless and flammable gas
with high solubility in water and in organic solvents, is well-
known for its disagreeable “rotten eggs” smell. Hydrogen
sulfide has been traditionally studied for its known toxic-
ity,[1] its presence as a corrosive industrial waste product,[2]
and emission in the energy industry,[3] as a parameter for
geological activity,[4] as quality control of foods with high
protein content,[5] and as an important compound related
to microbial activity in anaerobic processes. Moreover, re-
cently, hydrogen sulfide has been discovered to have a role
as a gasotransmitter[6] and has relevance in a number of
biological processes, such as neurotransmission,[7] vasorel-
axation,[8] cardioprotection,[9] and anti-inflammation.[10] In
fact, some H2S-releasing drugs are currently being used to
treat these pathologies. Furthermore, the abnormal pro-
duction of H2S has been associated with diseases such as
chronic kidney disease, liver cirrhosis,[11] Alzheimer[12] and
[a] Centro de Reconocimiento Molecular y Desarrollo Tecnológico
(IDM), Unidad Mixta Universitat Politècnica de Valéncia –
Universitat de València,
Camino de Vera s/n, 46071 Valencia, Spain
E-mail: rmaez@qim.upv.es
idm.webs.upv.es
[b] Departamento de Química, Universitat Politècnica de Valéncia, in quenching of the initial emission.[22]
Camino de Vera s/n, 46022 Valencia, Spain
In addition to fluorescence changes, three of the exam-
ples mentioned above also show colour modulations in the
presence of hydrogen sulfide. For instance, Han and co-
workers prepared a cyanine derivative functionalised with
an azide moiety that showed an absorption band at 610 nm
[c] CIBER de Bioingeniería, Biomateriales
(CIBER-BBN)
y Nanomedicina
[d] Departamento de Química Orgánica, Universitat de València,
Dr. Moliner 50, 46100 Burjassot, Valencia, Spain
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301514.
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Selective Chromo-Fluorogenic Hydrogen Sulfide Recognition
(blue) in water at pH 7.4 that was shifted to 660 nm in the
presence of hydrogen sulfide.[15b] Some other examples in-
volve reduction of the nitro moiety to amino in dimethyl
sulfoxide (DMSO) in certain coumarin derivatives,[17b] the
demetallation reaction in a CuII complex,[21c] nucleophilic
addition to tricyanovinyl dyes,[22a] and nucleophilic aro-
matic substitution reactions.[23] Furthermore, some of us
have reported colorimetric sulfide-sensing based on sulfide-
induced transformation of a pyrylium group into the corre-
sponding thiopyrylium derivative.[24]
In particular, we believe that the design of chromogenic
chemodosimeters for hydrogen sulfide has a special interest,
because they offer the possibility of straightforward use of
widely available instrumentation and simple semi-quantita-
tive detection of this toxic anion with the naked eye. Such
simple systems may be used to control H2S concentrations
in target industrial processes or for environmental and geo-
logical monitoring in situ. Nevertheless, in spite of these
possible applications, relatively few examples of selective
and sensitive chromogenic probes for the detection of hy-
drogen sulfide have been reported (see above).
Bearing in mind the concepts mentioned above and fol-
lowing our interest in the development of new approaches
for optical sensing,[25] we attempted to develop a simple
probe for the detection of sulfide in mixed aqueous media.
In the design of the probe, among the possible signalling
subunits, we chose azo dyes, which have been widely used
due to their straightforward synthesis and their well-known
spectroscopic properties. In fact the simple reaction be-
tween N,N-disubstituted anilines and diazonium salts of a
great variety of aniline derivatives results in the preparation
of azo dyes with characteristic charge-transfer absorption
bands in the 400–500 nm interval and compounds that
range from pale-yellow to red-orange. Moreover, as a sig-
nalling reporter we took advantage of the strategy recently
reported by Wang and co-workers that used the accelerated
reduction of azide groups directly attached to sulfonyl moi-
eties to amine in the presence of hydrogen sulfide.[15] By
following this approach we report herein the development
of 1-Az (see Scheme 1) as a new probe for the selective col-
orimetric detection of hydrogen sulfide. The probe contains
a sulfonyl azide signalling moiety attached to an azo dye
framework.
Results and Discussion
Probe 1-Az was prepared by following a slight modifica-
tion of previously reported procedures.[26] In particular,
commercially available dabsyl chloride (1-Cl) was dissolved
in acetone and then an aqueous concentrated solution of
sodium azide was carefully added with constant stirring at
0 °C (see Scheme 1). The structure of the 1-Az probe was
fully confirmed by 1H and 13C NMR, IR and HRMS
analyses (see the Supporting Information for full details).
1H NMR spectra of 1-Az showed the typical doublets
centred at δ = 6.88 and 7.87 ppm ascribed to the 1,4 disub-
stituted benzene ring bearing the N,N-dimethylamine moi-
ety and other AB system with signals centred at δ = 8.01
and 8.15 ppm, ascribed to the 1,4-disubstituted benzene
ring bearing the sulfonyl azide group. Moreover, the pro-
tons of the N,N-dimethylamine moiety appeared as a singlet
centred at δ = 3.12 ppm. HRMS-EI measurements con-
firmed the structure of the final product (m/z calcd. for
C14H15N6O2S+: 331.0972; found: 331.0969).
MeCN/HEPES (30 mm, pH 7.5; 1:1 v/v) solutions of 1-
Az (1.0ϫ10–5 m) presented an intense CT absorption band
(centred at 478 nm) due to the presence of a N,N-dimeth-
ylamino donor moiety and an electron-acceptor sulfonyl az-
ide group (see Figure 1). The UV/Vis behaviour of 1-Az was
tested in the presence of 10 equiv. of selected anions (i.e.,
–
–
–
HS–, Cl–, Br–, I–, F–, OH–, AcO–, CN–, N3 , NO3 , HCO3 ,
2–
SO42–, S2O32–, HPO4 and citrate), other thiol-containing
derivatives [i.e., l-cysteine (l-Cys), dl-homocysteine (DL-
Hcy), and glutathione (GSH)] and certain oxidants (i.e.,
H2O2). As can be seen in Figure 1, only hydrogen sulfide
induced a hypsochromic shift of 28 nm (from 478 to
450 nm) of the visible band of 1-Az together with a moder-
ate hypochromic effect that was reflected in a colour modu-
lation from orange-red to yellow (see Figure 2). This ob-
served change in colour (that was practically instantaneous)
Figure 1. UV/Vis spectra of 1-Az (1.0ϫ10–5 m) in MeCN/HEPES
(30 mm, pH 7.5; 1:1 v/v) upon addition of 10 equiv. of HS–, Cl–,
Br–, I–, F–, OH–, AcO–, CN–, N3 , NO3 , HCO3 , SO42–, S2O3
,
–
–
–
2–
Scheme 1. Synthesis of chemodosimenter 1-Az and its reaction
with hydrogen sulfide to yield 1-Am.
HPO42–, citrate, l-Cys, dl-Hcy, GSH or H2O2.
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R. Martínez-Máñez, A. M. Costero et al.
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was ascribed to hydrogen sulfide-induced reduction of the was found that the same chromogenic response of 1-Az
sulfonyl azide moiety in 1-Az to a sulfonamide (see struc- took place upon addition of HS– alone or HS– in the pres-
ture 1-Am in Scheme 1). The reduced electron-acceptor ence of 10 equiv. of the above studied species (see Figure 4).
character of the sulfonamide moiety, when compared with
the initial sulfonyl azide, accounts for the observed hyp-
sochromic shift.
Figure 4. Absorbance at 450 nm of 1-Az (1.0ϫ10–5 m) in MeCN/
Figure 2. Photograph of MeCN/HEPES (30 mm, pH 7.5; 1:1 v/v)
HEPES (30 mm, pH 7.5; 1:1 v/v) upon addition of 10 equiv. of HS–
solutions of 1-Az upon addition of 10 equiv. of target chemicals.
alone and in the presence of 10 equiv. of HS– and 10 equiv. of se-
From left to right and top to bottom: blank, HS–, Cl–, Br–, I–, F–,
lected anions, oxidants and biological thiols.
–
–
–
OH–, AcO–, CN–, N3 , NO3 , HCO3 , SO42–, S2O32–, HPO42–, cit-
rate, l-Cys, dl-Hcy, GSH and H2O2.
Having assessed the selective chromogenic response of 1-
Az toward HS– anions, the sensitivity of the probe was
studied. In particular, the changes in the visible band
centred at 478 nm, of MeCN/HEPES (30 mm, pH 7.5; 1:1
v/v) solutions of 1-Az (1.0ϫ10–5 m) was monitored upon
addition of HS– anions. The addition of increasing quanti-
ties of HS– anions induced a progressive hypsochromic shift
of the visible band in 1-Az (see the Supporting Infor-
mation). From typical calibration curves, a remarkable de-
tection limit of 11.91Ϯ0.09 μm (δ = 0.4 ppm) was calcu-
lated by using a simple conventional UV/Vis spectropho-
tometer. Furthermore, the highly selective response of 1-Az
to HS– anions was also studied by monitoring the emission
change. Thus, 1-Az (MeCN/HEPES, 1:1 v/v solutions) was
weakly emissive (quantum yield of 0.036), showing a broad
emission band centred at 557 nm with a shoulder at
ca. 520 nm (upon excitation at 478 nm). Solutions of 1-Az
were tested in the presence of several anions (i.e., HS–, Cl–,
The mechanism of the chromogenic response was fully
corroborated by the synthesis and isolation of compound
1-Am upon reaction of 1-Az with sodium sulfide in aceto-
nitrile (see the Supporting Information for full synthetic de-
1
tails). Figure 3 shows the H NMR spectra of the aromatic
part of chemodosimeter 1-Az compared to that of sulfon-
amide 1-Am obtained upon reaction of 1-Az with HS–. Re-
duction of the sulfonyl azide moiety in 1-Az induced upfield
shifts of the signals centred at 8.01 (Hc) and 8.15 (Hd) ppm
to 7.90 and 7.96 ppm, respectively, which is indicative of the
formation of sulfonamide moieties (see Scheme 1 for proton
assignation). Furthermore, the protons of the sulfonamide
group appeared as a broad singlet centred at δ = 7.42 ppm.
HRMS (EI) measurements confirmed the formation of 1-
Am (m/z calcd. for C14H17N4O2S+ 305.1072; found:
305.1068).
–
–
–
2–
Br–, I–, F–, OH–, AcO–, CN–, N3 , NO3 , HCO3 , SO4
,
2–
S2O32–, HPO4 and citrate), thiol-containing derivatives
(i.e., l-Cys, dl-Hcy and GSH) and H2O2. Only HS– in-
duced a moderate emission enhancement at 557 nm (quan-
tum yield of 0.048). From the corresponding calibration
curves
a remarkable detection limit for sulfide of
0.63Ϯ0.05 μm (δ = 0.02 ppm) was determined (see the Sup-
porting Information). The sensitivity of the system was suf-
ficient to determine levels of 1-Az below the concentration
of H2S required to elicit physiological responses (10–
1000 μm).[27]
1
Figure 3. H NMR spectra of the aromatic protons of chemodosi-
Further experiments were carried out in the presence of
higher concentrations of l-Cys, dl-Hcy and GSH. In par-
meter 1-Az and of 1-Am (both at 1.0ϫ10–3 m) in CD3CN.
The response observed was highly selective and, from all ticular, it is known that common physiological concentra-
the species tested (see above), only HS– induced shifts of tions of biothiols are in the range of 1–20 mm for GSH,
the visible band. Moreover, in competitive experiments it 0.14–0.37 mm for Cys, and 0.004–0.018 mm for Hcy. Taking
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Selective Chromo-Fluorogenic Hydrogen Sulfide Recognition
into account these data, the chromo-fluorogenic response showed no fluorescence, whereas cells incubated with 1-Az
of 1-Az (1.0ϫ10–5 m) in MeCN/HEPES (30 mm, pH 7.5; showed a very weak fluorescence (ascribed to the endoge-
1:1 v/v) solutions in the presence of 0.2, 0.3 and 0.4 mm of nous HS–). In contrast, a marked enhancement in intracel-
l-Cys, 0.5, 1, 5, 10 and 20 mm of GSH, and 0.2 mm of d,l- lular emission was observed in the HS–-treated cells, clearly
Hcy was studied. Neither l-Cys, GSH nor d,l-Hcy (in the indicating the possible use of 1-Az to detect hydrogen sulf-
range of concentrations tested) induced changes in either ide in complex media. However, HeLa cells treated with 10
the absorption or the emission bands of 1-Az.
and 40 μm of HS– showed no emission enhancement.
Finally, the stability of probe 1-Az in the presence of
water-containing mixtures and air was studied. It was found
that the intensity of the absorption and emission bands at
480 and 557 nm, respectively, of MeCN/HEPES solutions
(1:1, v/v) of the probe (measured in open cuvettes to evalu-
ate possible degradation by air) remained unchanged for at
least 12 h.
The selective emission enhancement of 1-Az in the pres-
ence of HS– and the negligible response observed upon the
addition of biothiols (GSH, l-Cys and dl-Hcy) at physio-
logical concentrations strongly suggest that the probe can
be used for HS– imaging in living cells. Based on these ob-
servations, the cytotoxicity of 1-Az was first evaluated. In
this respect, HeLa cells were treated with 1-Az at different
concentrations over a 24-hour period and cell viability was
determined by a WST-1 assay. The obtained results are
shown in Figure 5. As seen, probe 1-Az is essentially non-
toxic in the range of concentrations tested (10–100 μm).
Figure 6. Confocal fluorescence images of HeLa cells: (a) cells un-
treated; (b) cells incubated with 1-Az (30 μm); (c), (d), (e) and (f)
cells incubated with 1-Az (30 μm) in the presence of HS– (60, 80,
200 and 500 μm for c, d, e and f respectively).
Moreover, Figure 7 shows the ratio of average fluores-
cence after 10 min as compared to that found in the absence
of the probe (FI/FI0) for the fluorescence images of cells
Figure 5. Cell viability test of different concentrations of 1-Az at
24 h in HeLa cells using WST-1 assay.
In a second step, to verify the feasibility of the developed
probe to detect HS– in highly competitive environments, we
used 1-Az for the fluorescence imaging of sulfide in living
cells. HeLa cells were incubated in DMEM supplemented
with 10% fetal bovine serum. To conduct fluorescence mi-
croscopy 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 1-Az in DMSO at a final con-
centration of 30 μm. After 20 min at 37 °C, the medium was
removed and the cells were washed with PBS. Solutions of
different concentrations of NaHS (0, 60, 80, 200 and
500 μm) in PBS were added and the cells were incubated for
a further 30 min period. The results are shown in Figure 6.
Figure 7. Average FI/FI0 ratios from the fluorescence images of
HeLa cells incubated with 1-Az after the addition of 0, 200 and
500 μm of NaHS. Error bars are ϮSD from replicate experiments
The control experiment (HeLa cells without probe 1-Az) (n = 3).
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R. Martínez-Máñez, A. M. Costero et al.
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ture and the reaction was stirred for 3 h. Dichloromethane
(200 mL) was added and the organic phase was washed with water
(2ϫ100 mL), sodium hydrogen carbonate 5% solution (100 mL),
and again with water (2ϫ100 mL). The organic phase was then
dried with anhydrous magnesium sulfate, filtered, and concentrated
in a rotary evaporator. The solid residue was suspended in aceto-
nitrile (2 mL) and filtered. The final product 1-Az (280 mg,
0.85 mmol, 39%) was isolated as an orange-red solid. 1H NMR
(400 MHz, [D6]DMSO): δ = 3.12 (s, 6 H), 6.88 (d, J = 6.5 Hz, 2
H), 7.87 (d, J = 6.5 Hz, 2 H), 8.01 (d, J = 6.0 Hz, 2 H), 8.15 (d, J
= 6.0 Hz, 2 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 40.2,
111.7, 122.9, 125.9, 128.9, 136.5, 142.7, 153.5, 156.2 ppm. HRMS
(EI): m/z calcd. for C14H15N6O2S+ 331.0972; found 331.0969.
after the addition of 0, 200 and 500 μm NaHS. A clearly
enhanced fluorescence ratio is observed, which related well
with the increment in HS– concentration.
Conclusions
We have prepared a simple chemodosimeter based on a
sulfonyl azide dye (1-Az) that displayed a highly selective
response toward hydrogen sulfide anions in mixed aqueous
media. In the presence of this anion a clear colour change
from red-orange to yellow, which was easily detected by the
naked eye, and an enhancement of emission intensity were
observed. Other common anions, thiol-containing biomole-
cules, and oxidants did not induce any noticeable colour or
fluorescence modulation in the probe. The chemodosimeter
also showed a good sensitivity, with limits of detection of
11.91 and of 0.63 μm by using UV/Vis or fluorescence mea-
surements, respectively. The prepared probe is easy to syn-
thesise and allows an efficient, fast and low-cost detection
of hydrogen sulfide anions in mixed aqueous solutions.
Moreover, real-time fluorescence imaging measurements
confirm that probe 1-Az can be used to detect intracellular
HS– at micromolar concentrations. Similar designs using
other available dyes for the straightforward signalling of
HS– are being investigated in our laboratory.
Synthesis of 1-Am: Sodium sulfide (45 mg, 0.58 mmol) dissolved in
water (450 μL) was added into a solution of 1-Az (40.7 mg,
0.12 mmol) in acetonitrile (15 mL). The reaction was stirred for
6 h at room temperature, then the solvents were evaporated under
reduced pressure to give 1-Am (29.3 mg, 0.096 mmol, 78%) as an
orange-yellow solid. 1H NMR (400 MHz, [D6]DMSO): δ = 3.10 (s,
6 H), 6.87 (d, J = 7.0 Hz, 2 H), 7.42 (br. s, 2 H), 7.84 (d, J = 7.0 Hz,
2 H), 7.90 (d, J = 6.0 Hz, 2 H), 7.96 (d, J = 6.0 Hz, 2 H) ppm. 13C
NMR (100 MHz, [D6]DMSO): δ = 40.1, 112.1, 122.5, 125.8, 127.4,
143.0, 144.5, 153.5, 154.6 ppm. HRMS (EI): m/z calcd. for
C14H17N4O2S+ 306.1072; found 306.1068.
Quantum Yield Measurements: The quantum yield of 1-Az (f =
0.036) and 1-Am (f = 0.048) were calculated by using an acid solu-
tion of quinine sulfate (0.5 n in H2SO4) as reference and by the
known comparative method following equation:
Experimental Section
Chemicals: Sodium azide, dabsyl chloride (1-Cl), l-cysteine (Cys),
d,l-homocysteine (Hcy), glutathione (GSH), and hydrogen perox-
ide were purchased from Sigma–Aldrich. Analytical-grade solvents,
sodium hydrogen carbonate, and anhydrous magnesium sulfate
were purchased from Scharlau (Barcelona, Spain). For the chromo-
fluorogenic studies, tetrabutylammonium or sodium salts of hydro-
gen sulfide, fluoride, chloride, bromide, iodide, hydroxide, acetate,
cyanide, azide, nitrate, hydrogen carbonate, sulfate, thiosulfate, hy-
drogen phosphate and citrate were used. For cell culture experi-
ments, cell proliferation Reagent WST-1 was obtained from Roche
Applied Science. DMEM with l-glutamine, piruvate and Fetal Bo-
vine Serum (FBS), trypan blue solution (0.4%) cell culture grade
and trypsin were purchased from Gibco-Invitrogen.
where f is the quantum yield, I is the integrated of fluorescent in-
tensity, n is the refractive index, and A is the absorbance intensity.
The subscript R refers to the reference fluorophore (quinine sulf-
ate).
Limit of Detection by UV/Vis Spectroscopy: Changes in the visible
band centred at 478 nm, of MeCN-HEPES (30 mm, pH 7.5; 1:1
v/v) solutions of 1-Az (1.0ϫ10–5 m), were monitored upon addition
of increasing amounts of HS– anions. Addition of increasing quan-
tities of HS– anions induced a progressive hypsochromic shift of
the visible band. From the typical titration profile (representation
of absorbance at 478 nm vs. log[HS–]), a detection limit of
1.2ϫ10–5 m (δ = 0.4 ppm) was calculated by using a conventional
UV/Vis spectrophotometer. The experiments were carried out three
times to calculate the standard deviation.
All studies with 1-Az and hydrogen sulfide were performed by
using freshly prepared MeCN/HEPES (30 mm, pH 7.5; 1:1 v/v) so-
dium hydrogen sulfide solutions.
General Techniques: UV/Vis spectra were recorded with a JASCO
V-650 Spectrophotometer. Fluorescence measurements were car-
ried out with a JASCO FP-8500 spectrophotometer. 1H and
13C NMR spectra were acquired with a BRUKER AVANCE III
(400 MHz) instrument, and mass spectra were recorded with a
TRIPLETOF T5600 (ABSciex, USA) spectrometer. Cell viability
measurements were carried out with a Wallac 1420 Workstation.
Limit of Detection by Fluorescence: Changes in the emission band
centred at 557 nm (upon excitation at 478 nm) of MeCN-HEPES
(30 mm, pH 7.5; 1:1 v/v) solutions of 1-Az (1.0ϫ10–5 m) were moni-
tored upon addition of increasing amounts of HS– anions. This
addition induced a progressive enhancement of the emission band
at 478 nm. From the calibration curve (representation of the emis-
sion intensity at 557 nm vs. log[HS–]), a detection limit for HS– of
6.0ϫ10–7 m (δ = 0.02 ppm) was calculated. The experiments were
carried out three times to calculate the standard deviation.
Synthesis of 1-Az: Dabsyl chloride (1-Cl; 710 mg, 2.19 mmol) was
dissolved in acetone (10 mL) and cooled to 0 °C with an ice bath.
A solution of sodium azide (142 mg, 2.19 mmol) in water (2 mL)
was then slowly added dropwise under vigorous stirring. The crude
mixture was stirred for 2 h at 0 °C and then for 4 h at room tem-
perature. Finally, to achieve complete transformation of the sulf-
onyl chloride into sulfonyl azide, sodium azide (32 mg, 0.1 mmol)
dissolved in water (500 μL) was added dropwise at room tempera-
Cell Culture Conditions: HeLa human cervix adenocarcinoma cells
were purchased from the German Resource Centre for Biological
Materials (DSMZ) and were grown in DMEM supplemented with
10% FBS. Cells were maintained at 37 °C in an atmosphere of 5%
carbon dioxide and 95% air and underwent passage twice a week.
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Supporting Information (see footnote on the first page of this arti-
1
cle): H NMR, 13C NMR and IR spectra of 1-Az and 1-Am, UV/
Vis changes of 1-Az in the presence of selected anions, UV/Vis
changes of 1-Az upon addition of increasing quantities of HS– and
fluorescence titration of 1-Az with HS–.
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Received: October 5, 2013
Published Online: January 28, 2014
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