A coumarin-based colorimetric fluorescent probe for hydrogen sulfide

Article abstract of DOI:10.1007/s12039-015-0788-z

A coumarin-based fluorescent probe for selective detection of hydrogen sulfide (H₂S) is presented. This 'off-on' probe exhibited high selectivity towards H₂S in aqueous solution with a detection limit of 30 nM. Notably, because of it

Full text of DOI:10.1007/s12039-015-0788-z

c
J. Chem. Sci. Vol. 127, No. 3, March 2015, pp. 359–363. ꢀ Indian Academy of Sciences.
DOI 10.1007/s12039-015-0788-z
A coumarin-based colorimetric fluorescent probe for hydrogen sulfide
YANQIU YANGa, YU LIUb, LIANG YANGa, JUN LIU a, KUN LIb,∗ and SHUNZHONG LUO a,∗
aInstitute of Nuclear Physics & Chemistry, CAEP, Mianyang, Sichuan Province, China, 621900
bCollege of Chemistry, Sichuan University, Chengdu, Sichuan Province, China, 610064
e-mail: luoshzh@caep.ac.cn; kli@scu.edu.cn
MS received 5 September 2014; revised 30 October 2014; accepted 23 December 2014
Abstract. A coumarin-based fluorescent probe for selective detection of hydrogen sulfide (H2S) is presented.
This ‘off–on’ probe exhibited high selectivity towards H2S in aqueous solution with a detection limit of 30 nM.
Notably, because of its dual nucleophilicity, the probe could avoid the interference of thiols and other sulfur
containing compounds.
Keywords. Coumarin; reactive probe; H2S probe
1. Introduction
and reported a fluorescein based probe for H2S with
high selectivity.9e Inspired by this work, we wanted
to develop a coumarin-based H2S probe with similar
mechanism.
Hydrogen sulfide (H2S) is an endogenously produced
gaseous signalling compound (gasotransmitter), along
with nitric oxide (NO) and carbon monoxide (CO),
which mediate a wide range of physiological and patho-
logical effects, such as anti-oxidation, anti-inflammation,
apoptosis, etc.1–3 Due to the important role of H2S,
developing a sensitive, rapid, and reliable detection
technique for it is in high demand and has become a
hot research area in the past few years. Many excellent
fluorescent probes for the detection of H2S have been
reported based on different strategies including H2S
mediated reduction of azide and nitro compounds,4,5
an azamacrocyclic copper(II) ion complex,6 thiolysis of
dinitrophenyl ether,7 and H2S trapping by nucleophilic
addition.8 The nucleophilic reactions of H2S toward
α, β-unsaturated acrylate methyl ester or an aldehyde
group have been widely used to design H2S-targeted
chemodosimeters. The diversity of the reaction sites is
beneficial for the design of fluorescence sensors, which
result in ON-OFF, OFF-ON or ratiometric probes.8 But
the selectivity and interference from other thiols (such
as cysteine, homocysteine and glutathione) are still a
challenge. Meanwhile, most of the probes need organic
solvents as co-solvent, which improve the solubility
of the probes or shorten the reaction time. To over-
come the problems mentioned above, two-step nucleo-
philic reaction was introduced when designing probes
for H2S, which exhibit promising future with good
anti-interference.9 More recently, Guo group exploited
2-(iodomethyl) benzoate as a new H2S trap group,
Coumarin has been largely utilized as fluorophore
in various fluorescent probes due to its large Stokes
shift, good solubility and high quantum yield.10 Previ-
ously, we reported a coumarin-based chromogenic and
ratiometric probe for hydrazine.11 As we know, when
the phenolic hydroxyl of a fluorophore is protected via
esterification, no emission of the fluorophore would
be found due to the electron-withdrawing property of
acetate group. However, the removal of ester groups
under suitable condition will lead to the fluorescence
recovery of the fluorophore. Herein, we designed a
two-step nucleophilic reaction-based H2S probe, which
employs 7-hydroxy-coumarin as fluorophore by pro-
tecting the hydroxyl via an ester group.
The fluorescent probe was generally constructed with
a 7-hydroxy-coumarin as fluorophore and 2-(iodomethyl)
benzoate as H2S trap that has two electrophilic reaction
sites. The initial nucleophilic attack of H2S would lead
to an intermediate thiol, which spontaneously under-
goes a cyclization by forming benzo[c]thiophen-1(3H)-
one and releases the fluorophore thereby eliciting the
obvious fluorescent signal (scheme 1).
2. Experimental
2.1 General
1H NMR, 13C NMR spectra were measured on a Bruker
AM400 NMR spectrometer. ESI-MS and HRMS spec-
tral data were recorded on a Finnigan LCQDECA and
∗For correspondence
359

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Yanqiu Yang et al
= 1:4) of the crude product over silica gel gave a
colourless powder. Yield: 3.63 g (47.7%).
2.4 Synthesis of probe 1
To a solution of DCC (0.206 g, 1 mmol) in CH2Cl2
(10 mL) cooled at 0◦C was added 2-iodomethylbenzoic
acid (0.262 g, 1 mmol) followed by DMAP (4-dimethy-
laminopyridine, 0.012 g, 0.1 mmol) and 7-hydroxy-3-
methyl-coumarin (0.176 g, 1 mmol). The mixture was
stirred at room temperature for 6 h and then filtered,
washed with CH2Cl2. The filtrate was concentrated to
afford the crude product, then purified by silica gel
column chromatography (PE: EA = 20:1) to afford
a white solid (0.15 g, 36%). 1HNMR (400 MHz,
CDCl3)δ(ppm): 8.21 (dd, 1H, J = 8.0 Hz, 1.0 Hz), 7.57
(s, 2H), 7.527–7.507 (m,2H), 7.473–7.432 (m, 1H),
7.30–7.295 (m, 1H), 7.252 (dd, 1H,J = 8.0 Hz, 2.0
Hz), 4.987 (s, 2H), 2.261–2,258 (s, 3H). 13C NMR
(100 MHz, CDCl3)δ(ppm):164.5, 161.8, 153.8, 152.0,
142.4, 138.7, 133.7, 132.1, 131.5, 128.3, 127.7,
126.9, 125.4, 118.5, 117.6, 110.3, 17.2, 3.4. HRMS:
m/zC18H13INaO+4 Calcd 442.9756, found 442.9758.
Scheme 1. The proposed sensing mechanism of probe 1
for H2S.
a BrukerDaltonics Bio TOF mass spectrometer, respec-
tively. Fluorescence emission spectra were obtained
using FluoroMax-4Spectrofluorophotometer (HORIBA
JobinYvon) at 298 K. Unless otherwise noted, materi-
als were obtained from commercial suppliers and were
used without further purification. All the solvents were
dried according to the standard methods prior to use. All
the solvents were either HPLC or spectroscopic grade
in the optical spectroscopic studies.
2.2 Synthesis of 2-iodomethylbenzoic acid9e
2.5 Determination of the fluorescence quantum yield
In this paper, the fluorescence quantum yields of
probe 1 in the presence and absence of H2S were deter-
mined in PBS buffer (10 mM, pH 7.4, containing 1 mM
CTAB), using quinine sulfate (ꢀ = 0.542) as standard.
The fluorescence quantum yield was calculated using
the following equation:
A solution of phthalide (1.34 g, 10 mmol) and iodo-
trimethylsilane (3.00 g, 15 mmol) in CH2Cl2 (15 mL)
was refluxed for 3 h, cooled to room temperature,
quenched with water (10 mL) and the precipitate was
filtered off, washed with water to give a white solid (2.4
g, 90%). The crude product was used directly without
further purification.
ꢀx = ꢀs(AsFx/AxFs)
(1)
where, Ax and As are the absorbance of the sample and
the reference, respectively, at the same excitation wave-
length, Fx and Fs are the corresponding relative inte-
grated fluorescence intensities. Absorbance of sample
and reference at their respective excitation wavelengths
was controlled to be lower than 0.05.
2.3 Synthesis of 7-hydroxy-3-methyl-coumarin12
2, 4-dihydroxybenzaldehyde (6.0 g, 43.2 mmol), sodium
propionate (9.0 g, 93.6 mmol) and propionic anhydride
(15 mL, 117 mmol) were placed in a 100 mL round-
bottom flask. Triethylamine (6 mL, 43.2 mmol) was
then added via the addition funnel, and the reaction
mixture was heated to reflux for 6 h. After the reaction,
water (30 mL) was poured and the resulting pink solid
3. Results and Discussion
was collected by filtration and washed with cold water. 3-methyl-coumarin-7-yl 2-(iodomethyl)benzoate (probe 1)
Column chromatography (ethyl acetate: petroleumether could be easily prepared by the reaction of 7-hydroxy-
Scheme 2. Synthesis of probe 1.

Colorimetric fluorescent probe for hydrogen sulfide
3-methyl-coumarin with 2-(iodomethyl)benzoic acid in
361
CH2Cl2 at room temperature in a yield of 36%
(scheme 2). Its structure was further confirmed by
1H NMR, 13C NMR and HRMS. 7-hydroxy-3-methyl-
coumarin with 2-(iodomethyl) benzoic acid were syn-
thesized according to the literature.9e,12
Firstly, we investigated the properties of probe 1
in PBS buffer. However, because of the poor solu-
bility of probe 1, only 10-fold fluorescence intensity
enhancement was observed in the presence of large
quantity H2S (100 eq). Considering that the addition
of excessive organic solvent might restrict the practical
application of the probes, we envisioned that a ratio-
nally chosen micelle system could be substituted for
the DMSO-PBS mixture. In the past decade, surfactants
have been explored for modifying and improving the
sensing capability of fluorescent probes.13 To explore
the optimum condition of fluorescent turn-on probe for
H2S, cetyl trimethyl ammonium bromide (CTAB) was
utilized to improve the solubility. We first evaluated the
effect of CTAB concentration on the fluorescence of the
probe. As shown in figure S1, the thiolysis reaction is
restricted without CTAB, and upon increasing CTAB
concentration, the reaction becomes fast and more than
60-fold fluorescence enhancement was achieved. The
reaction is complete within 1 min using 1 mM CTAB
(figure S1). The pH value also could affect the process
(figure S2). The result showed that the action of the
probe is rapid and highly sensitive selective for detection
of hydrogen sulfide under optimum condition (10 mM
PBS, pH 7.4, containing 1 mM CTAB). Acidic condi-
tion inhibits ionization of hydrogen sulfide, while probe
is easy to hydrolyze under alkaline condition.
Then the selectivity of the probe was tested. As
expected, probe 1 exhibited high selectivity towards H2S
over other biologically relevant reactive sulfur (RSS),
oxygen (ROS) and reactive nitrogen species (RNS)
under the same conditions. As shown in figure 1, most
anions, ROS and RNS showed absolutely no change in
the emission intensity compared with probe 1. Other
abundant thiols in cells, such as glutathione and cystein,
due to the absence of the dual nucleophilicity, they
could not perform a second nucleophilic addition and
exhibited limited fluorescence responses (100 eq. GSH
or Cys). Additionally, a blue colour change was also
observed in the presence of H2S under UV light
(365 nm). These results proved that probe 1 displayed
excellent selectivity for H2S over other abundant repre-
sentative anions.
Figure 1. Fluorescence response of probe 1(5 μM) upon
addition of various species in PBS buffer (10 mM, pH 7.4,
containing 1 mM CTAB). λex = 350 nm, λem = 466 nm.
fluorescence enhancement. The fluorescence quantum
yield (ꢀ) of probe 1 was determined as 0.04 in the
absence of H2S, and improved to 0.66 after incubation
10 min in the presence of H2S.
More information on the optical responses of probe
1 towards H2S was then studied using UV-Vis spec-
trum (figure S4). Probe 1 showed a moderately strong
maximum absorption peak centred at 306 nm and the
molar extinction coefficient values were determined as
1.6 ×104M−1·cm−1. Upon addition of H2S, this peak
gradually decreased and red shifted to a new absorption
band centred at 314 nm, which could be attributed to the
removal of 2-iodomethylbenzoic acid group from probe
1. To further confirm our proposed mechanism, HRMS
was used to investigate the reaction process. After the
reaction, the peak at 199.0368 was assigned as [M +
Na]+ (calc. 199.0371, figure S5).
Subsequently, the quantitative analytical behaviour
of probe 1 for the analysis of H2S was examined by
fluorescence titration. As displayed in figure 2, upon
gradual addition of H2S to the probe, the emission
intensity at around 466 nm increased significantly,
while no change was found when 5 eq. H2S or more was
added. A good linear relationship was achieved with the
H2S concentration from 0 to 25 μM (figure S6), and the
limit of detection (LOD) was calculated as 30 nM based
on S/N= 3.
To further confirm the selectivity of this fluorescent
probe for hydrogen sulphide, the competition exper-
iments were performed in the presence of various
anions. As displayed in figure 3, a similar fluorescence
The time-dependent fluorescence spectra were then change was observed upon addition of 5 eq. H2S in
examined in the presence and absence of H2S the presence of various anions (100 eq.), such as SO23−,
(figure S3). The results indicated that the process could SO24−, SCN−, F−, Cl−, Br−, I−, PO33−, HPO42−, AcO−,
be completed within 15 min with approximately 65-fold HCO−3 , N3−, C2O42−. Meanwhile, probe 1 displayed

362
Yanqiu Yang et al
Figure 2. Fluorescence spectra of probe 1 (5 μM) upon addition of H2S (0–
100 μM) in PBS buffer (10 mM, pH 7.4,containing 1 mM CTAB). λex = 350
nm, λem = 466 nm.
and explored through a two-step nucleophilic reaction.
The probe exhibited high selectively towards H2S and
good anti-interference towards many anions. The sens-
ing of H2S was rapid and finished within 15 min with
detection limit as 30 nM.
Supplementary Information
1H NMR, 13C NMR and ESI-Mass spectra of probe and
figures S1–S6 are available at www.ias.ac.in/chemsci.
Acknowledgement
This work was financially supported by the National
Science Foundation of China (91026022) and China
Post-doctoral Science Foundation funded project
(2014M560718).
Figure 3. Fluorescence response of probe 1 (5 μM) upon
addition of various species in the presence of sulfide in PBS
buffer (10 mM, pH7.4,containin1 mM CTAB). λex = 350
nm, λem = 466 nm. (1) probe, (2) S2−, (3) SO32−+S2−, (4)
SO24−+S2−, (5) SCN−+S2−, (6) F−+S2−, (7) Cl−+S2−, (8)
Br−+S2−, (9) I−+S2−, (10) PO34−+S2−, (11) HPO24−+S2−
,
(12) AcO−+S2−, (13) HCO3−+S2−, (14) N−3 +S2−, (15)
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