A highly selective fluorescent probe based on Michael addition for fast detection of hydrogen sulfide

Article abstract of DOI:10.1016/j.saa.2016.09.051

A new 4-hydroxy-1,8-naphthalimide-based compound (probe 1) has been designed and synthesized. The colorimetric and fluorescent properties of probe 1 towards hydrogen sulfide (H₂S) were investigated in detail. The results show that the probe 1 c

Full text of DOI:10.1016/j.saa.2016.09.051

A highly selective fluorescent probe based on Michael addition for fast
detection of hydrogen sulfide
Baozhen Gao, Lixia Cui, Yong Pan, Minjie Xue, Boyu Zhu, Guomei
Zhang, Caihong Zhang, Shaomin Shuang, Chuan Dong
PII:
DOI:
Reference:
S1386-1425(16)30576-5
doi:10.1016/j.saa.2016.09.051
SAA 14697
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Accepted date:
5 April 2016
24 August 2016
26 September 2016
Please cite this article as: Baozhen Gao, Lixia Cui, Yong Pan, Minjie Xue, Boyu Zhu,
Guomei Zhang, Caihong Zhang, Shaomin Shuang, Chuan Dong, A highly selective fluo-
rescent probe based on Michael addition for fast detection of hydrogen sulfide, (2016),
doi:10.1016/j.saa.2016.09.051
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ACCEPTED MANUSCRIPT
A highly selective fluorescent probe based on Michael
addition for fast detection of hydrogen sulfide
Baozhen Gaoa, Lixia Cuia, Yong Panb, Minjie Xuea, Boyu Zhua, Guomei Zhanga,
Caihong Zhanga,, Shaomin Shuanga, Chuan Donga,
aSchool of Chemistry and Chemical Engineering, Center of Environmental Science and
Engineering Research, Shanxi University,Taiyuan 030006,China
bState Key Laboratory of NBC Protection for Civilian, Research Institute of Chemical
Defense, Yangfang, Changping District, Beijing, 102205, China;
ABSTRACT: A new 4-hydroxy-1,8-naphthalimide-based compound (probe 1) has
been designed and synthesized. The colorimetric and fluorescent properties of probe 1
towards hydrogen sulfide (H2S) were investigated in detail. The results show that the
probe 1 could selectively and sensitively recognize H2S rather than other reactive
sulfur species. The reaction mechanism of this probe is an intramolecular cyclization
caused by the Michael addition of H2S to give 4-hydroxy-1,8-naphthalimide. The
intramolecular charge transfer of 4-hydroxy-1,8-naphthalimide is significant. Probe 1
quickly responded to H2S and showed a 75-fold fluorescence enhancement in 5
minutes. Moreover, probe 1 could detect H2S quantitatively with a detection limit as
low as 0.23 μM.
Keywords: 1,8-Naphthalimide, Fluorescent probe, Hydrogen sulfide, Michael
addition.
Corresponding authors: E-mail: chzhang@sxu.edu.cn (C. Zhang), dc@sxu.edu.cn (C. Dong); Tel: +86
351 7018613; Fax: +86 351 7018613
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1. Introduction
Hydrogen sulfide (H2S) is a colorless gas with a typical rotten egg odor. It has
long been considered chemical pollutants with toxicity. Exposing in the H2S will
irritate respiratory system[1] and excessive inhalation will make people lose
consciousness, cardiac arrest and even death[2-4]. In addition, the excess H2S in
aerobic environment could have a negative effect on the air and water[5], which could
seriously corrode metal pipes[6] and other serious damages[7]. However, recent
reports show that H2S is the third gaseous compound endogenously produced in vivo
after carbon monoxide (CO) and nitric oxide (NO) [8-10]. It plays important roles in
many biological processes, such as it can influence the proliferation and apoptosis of
human uterine leiomyoma cells[11], protect the lung injury secondary to traumatic
hemorrhagic shock[12], slow down the calcification of human umbilical vein smooth
muscle cells[13,14], regulate the myocardial contractile force[15,16] and promote
liver cancer cells proliferation/anti-apoptosis effects[17]. Therefore, an efficient
method for sensitively and selectively probing H2S is urgently required.
Currently, among the major methods for H2S detection, the fluorescent probe has
attracted more and more attention and become an active field because of its
advantages of high selectivity and sensitivity. A number of fluorescent probes for H2S
have been developed mainly based on specific reactions between H2S and the probes.
These reactions includes H2S reductive reactions[18-20], H2S nucleophilic
reactions[21-23], and copper sulfide precipitation reaction[24-26]. Among these, the
probes based on Michael addition reactions are attractive, because Michael acceptors
are able to react with H2S and promote the intramolecular cyclization to release the
fluorophore, whereas the reactions between Michael acceptors and biological thiols
were reversible and therefore should not consume the probes[27]. Based on this
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strategy, Y. Qian’s group and several other groups presented some probes containing
Michael acceptors for the detection of H2S[28-31], but there are many aspects left to
be improved, such as narrow pH range, long reaction time and complex preparation.
Therefore, the development new probes for H2S detection that are selective, sensitive,
rapid, and applicable to the environmental and biological sensing are still in great
demand.
In this work, we report a new fluorescence probe based on fluorophore, 4-
hydroxy-1,8-naphthalimide, because of its high photostability, large Stokes’ shift and
ratiometric detection capability[32-35]. Michael acceptors, 2-(2-cyano-3-methoxy-3-
oxoprop-1-enyl)-benzoic acid was chosen as H2S accepter. The probe has high
selectivity and good sensitivity to H2S and meets the requirements of practical
applications.
2. Experimental
2.1. Materials
4-Bromo-1,8-naphthalimide,
EDC
(1-(3-dimethylamino-propyl)-3-
ethylcarbodiimide Hydrochloride), DMAP (4-dimethyl-aminopyridine), Methanol,
Sodium metal, Hydrogen bromide and other chemicals were purchased from Sigma-
Aldrich or Aldrich and were used as received. Solvents were dried according to
standard procedures. Na2S·9H2O as the H2S donor was analytical grade and purchased
from Sigma-Aldrich [36]. All other reactive sulfur species, including cysteine (Cys),
homocysteine (Hcy), glutathione (GSH), Na2SO3 and Na2S2O3 were analytical grade.
Probe 1 was synthesized using a modification of a literature method [27,37,38].
2.2 Instruments
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1
A pH meter (Mettler Toledo, Switzerland) was used to determine the pH. H
NMR and 13C NMR spectra were collected on a Bruker Avance DRX 300 MHz
nuclear magnetic resonance spectrometer (Fällanden, Switzerland). Chemical shifts
1
13
were reported relative to CDCl3 (δ7.26) for H NMR and CDCl3 (δ 77.0) for C
NMR. UV absorption spectra were recorded on a Shimadzu UV-265
spectrophotometer (Tokyo, Japan). Fluorescence spectra were taken on a Hitachi
F4500 spectrofluorometer (Tokyo, Japan). A PO-120 quartz cell (10 mm) was
purchased from ShanhaiHuamei Experiment Instrument Plants, China. Fluorescence
measurements were carried out with a slit width of 10 nm (λex=310 nm).
2.3. Preparation and characterization of probe
Synthesis of probe (1) is summarized in Scheme 1. N-Butyl-4-hydroxy-1,8-
naphthalimide (2) and 2-(2-cyano-3-methoxy-3-oxoprop-1-enyl)-benzoic acid (5)
were synthesized following the literature methods.
Dry CH2Cl2 (10 mL) was added to a mixture of N-butyl-4-Hydroxy-1,8-
naphthalimide 2 (54mg, 0.2 mmol), 2-(2-cyano-3-methoxy-3-oxoprop-1-enyl)-
benzoic acid (46 mg, 0.2 mmol) , EDC (38 mg, 0.2 mmol), and DMAP (2.5 mg, 0.02
mmol) in a 25 mL round-bottomed flask at room temperature. The mixture was stirred
for 14h. Solvents were removed under reduced pressure and the crude material was
purified by column chromatography using CH2Cl2 /ethyl acetate (10:0.5, v/v) to
afford the desired probe as a white solid (25 mg, 30%).
1H NMR (300 MHz, CDCl3) δ 9.03 (s, 1H), 8.58-8.68 (m, 3H), 8.26-8.29 (t, 1H,
J = 8.14 Hz), 7.78-7.96 (m, 4H), 7.66-7.69 (t, 1H, J=7.24 Hz), 4.18-4.23 (t, 2H, J =
4.54 Hz), 1.68-1.76 (m, 2H), 1.42-1.47 (m, 2H), 0.97-1.02 (t, 3H, J = 1.57); 13C NMR
(100 MHz, CDCl3) δ 164.4, 163.8, 163.6, 162.1, 156.7, 151.4, 135.4, 134.8, 132.0,
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131.9, 130.5, 127.8, 127.7, 127.6, 125.4, 119.9, 107.6, 53.8, 40.6, 30.4, 20.6, 14.1.
(Fig.S3,S4)
< Insert Scheme 1 >
2.4. General UV–vis and fluorescence spectra measurements
The stock solutions of the probe 1 (2.5 mM) were prepared in acetonitrile. And
phosphate buffer saline (PBS buffer) was prepared in deionized water, while
Na2S·9H2O, cysteine (Cys), glutathione (GSH), homocysteine (Hcy), Na2SO3 and
other solutions were prepared in PBS buffer. Cetrimonium bromide (CTAB) solution
was prepared in deionized water. In this experiment, test solutions were prepared by
placing 16 μL of the probe stock solution into the solution of 4 mL acetonitrile: buffer
(1:1 v/v, PBS buffer, pH 7.4). The requisite volume of Na2S·9H2O were added into
the solutions gradually by using a pipette. 3mL of the solution was transferred into a
1cm quartz cell and fluorescence spectra were measured after addition of analytes for
5 min. UV-Vis and fluorescence spectra were obtained in acetonitrile: buffer (1:1 v/v,
PBS buffer, pH 7.4) solutions.
3. Results and discussion
3.1 Spectroscopic properties of probe 1
The spectroscopic properties of probe 1 were tested in MeCN/buffer (1:1, 50mM
PBS buffer, pH=7.4) solution. As shown in Fig.1, the probe 1 displayed an absorption
band (280-340 nm) and the maximum absorption was about 310nm (ε=4.2×104 M-1
cm-1). The fluorescence spectra were also detected and shown in Fig.1, the probe 1
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showed a weak fluorescence band and the emission maximum at about 450 nm. The
fluorescence quantum yield was 0.032, due to photoinduced electron transfer (PET)
effect caused by carbon-carbon double bond in the a,ß -unsaturated ketone moiety[39].
To examine the response of probe 1 for H2S, probe 1 was treated with 100 μM
H2S, and the signals were recorded after mixing for 5 min. As shown in Fig.1, the
absorption band was decreased and a new band at 450nm (ε=3.7×104 M-1 cm-1)
appeared. Meanwhile, the color of the solution changed from colorless to yellow,
which was clearly recognized by direct visual observation. The fluorescence emission
at 450nm was decreased and a new strong fluorescence emission at 550nm was
appeared. We also noted that the fluorescence of the solution changed from weak blue
to yellow-green under illumination with a 365 nm UV lamp. Clearly, the results
indicated that H2S could be detected through both fluorescence and colorimetric
methods by probe 1.
<insert Fig.1>
3.2 Fluorescence responses of probe 1 for H2S
Preliminary examination has revealed that H2S could turn on the fluorescence of
probe 1 at 550 nm. Thus fluorescence spectra of probe 1 upon titration with H2S were
detected in MeCN/buffer (1:1, 50mM PBS buffer, pH=7.4, 1mM CTAB) solution.
According to the previous work [40, 41], the purpose of CTAB may be attributed to 1)
CTAB can increase the solubility of the probe in aqueous buffers, and 2) CTAB is a
cationic surfactant, which may facilitate the reaction between sulfide anion and the
probe. From Fig.2, we can found that the probe 1(10 μM) displayed an emission at
450 nm in the absence of H2S, with increasing concentration of H2S (0-30 μM) to the
solution of probe 1, a fluorescence emission at 550 nm was observed and enhanced
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gradually, meanwhile, the fluorescence emission at 450nm decreased. The
fluorescence emissions displayed about 75-fold fluorescence enhancement and
minimal changes could be observed when the concentration of H2S went beyond
20μM. The changes in fluorescence intensity should be attributed to the conjugate
addition of H2S to carbon-carbon double bond, which blocked the PET process.
A good linear relationship (R=0.98) was found between the fluorescence
intensity at 550 nm and the concentration of H2S from 0 to 20 μM (insert Fig.2). The
detection limit for H2S, based on the definition by IUPAC (CDL=3Sb/m) was
calculated to be 0.23 μM. Probe 1 shows a high sensitivity towards H2S which is
comparable to other reported H2S probes [27,29,42,43,44] and can be applied to
quantitative detection of H2S.
<insert Fig.2>
3.3 Selectivity over biothiols
To examine the selectivity of probe 1 toward H2S, probe 1(10 μM) was treated
2
2
with different reactive sulfur species, including Cys, GSH, Hcy, SO3 − , S2O3 − (all at
100 μM) in MeCN/buffer (1:1, 50mM PBS buffer, pH=7.4, 1mM CTAB), and
fluorescence signals were recorded after mixing for 5 min. The fluorescence intensity
at 550nm of probe 1 showed 75-fold fluorescence enhancement upon reaction with
H2S, whereas little fluorescence intensity changes were observed in the presence of
2
2
−
−
other reactive sulfur species (Cys, GSH, Hcy, SO3 and S2O3 ), an error of ±10.0%
in the relative fluorescence intensity was considered to be tolerable. Moreover, when
2−
2−
reactive sulfur species, including SO3 , S2O3 , GSH, Cys and Hcy, co-existed with
H2S, we observed almost the same fluorescence enhancement as that only treated by
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100 mM H2S. These results clearly demonstrated the probe 1 has the excellent
selectivity even when H2S co-existed with reactive sulfur species.
<insert Fig.3>
3.4 pH effects and time-dependence in the detection process of H2S
The pH effects on fluorescence signals of probe 1 have been investigated. As
shown in Fig. 4, the fluorescence of probe 1 appeared to be stable. In the presence of
H2S, a large emission enhancement at 550nm is observed over a wide pH range of 4-8.
It is known that H2S is a weak diprotic acid, in aqueous state under different pH, there
is an equilibrium distribution of three species including H2S, HS- , and S2-. The Ka2 of
H2S is 1.00 x 10-14, so there shouldn't be much S2- at most pHs employed, the major
form of H2S in pH=4~8 exists as HS- and H2S, the ratio of [HS-]:[H2S] was calculated
based on Ka1= 9.55 x 10-8 of H2S and list in Table 1. From the Table 1, we can find
that H2S is over 1000 times greater in concentration than HS- at pH 4.00, as the pH
decreases the equilibrium shifts toward fully protonated form, [HS-]:[H2S] =2.39:1.00
at pH 7.4. At pH 8, [HS-]:[H2S] =9.55:1.00 and the fluorescence intensity at 550nm
plummeted. Such findings showed that H2S is in fact the donor analyte and probe 1
could be used in biological applications and environmental analysis.
<insert Fig.4 and Table 1>
We also carried out time-dependent fluorescence changes of probe 1 to H2S at
room temperature. As depicted in Fig. 5, the fluorescence intensity at 550 nm
increased dramatically when H2S was added in the solution and the fluorescence
intensity were able to reach a maximum in less than 5 min. It showed that the reaction
between probe 1 and H2S was completed within 5 min in PBS buffer at room
temperature.
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<insert Fig.5>
3.5 Proposed mechanism
Some references reported that simple thiols reacted readily with some Michael
acceptors at physiological pH[27,45], so Michael acceptor, 2-(2-cyano-3-methoxy-3-
oxoprop-1-enyl)-benzoic acid, was be used as H2S receptors in probe 1. As shown in
scheme 2, probe 1 can react with H2S to form a free SH containing intermediate A2,
the SH group should undergo a spontaneous cyclization to form by-product A3 and
release the fluorophore A4, 4-hydroxy-1,8-naphthalimide. It shows long-wavelength
fluorescence emission due to the stronger electron donating ability of hydroxyl group.
The hypothesis was confirmed by the reaction between probe 1(0.1 mmol) and
Na2S·9H2O (1.0 mmol) in CH3CN: PBS buffer (1:9) solution (20mL). The cyclization
1
13
product A3 was isolated and confirmed by HNMR and CNMR (Fig.S5, Fig.S6).
Biothiols (RSH) such as Cys, Hcy and GSH could react with probe1 in an analogous
fashion to yield B1, which does not contain sulfhydryl group. Therefore, B1 cannot
undergo an intramolecular nucleophilic attack. Clearly, probe 1 can be used to
selectively trap H2S and they are hardly consumed by thiols.
<insert Scheme 2>
4. Conclusions
In summary, we reported the development of a new 4-hydroxy-1,8-
naphthalimide-based fluorescence off–on probe for H2S detection. The probe
exhibited good selectivity and sensitivity toward H2S even in the presence of biothiols.
The fluorescence emission displayed 75-fold enhancement for H2S in MeCN/buffer
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(1:1, 50mM PBS buffer, pH=7.4) solution within 5min. Moreover, the detection
mechanism of probe to H2S was proposed, after H2S added, the new emission
enhanced was caused by H2S-mediated Michael addition followed by intramolecular
cyclization reaction.
Acknowledgments
The work described in this paper was supported by the National Nature Science
Foundation of China (No.21475080), State Key Laboratory of NBC protection for
civilian, the Nature Science Foundation of Shanxi Province (2014011016-2) and
Postdoctoral Science Foundation of China (2013M530120).
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Captions
Scheme 1. The synthesis route of probe 1.
Scheme 2. Proposed detection mechanism of probe 1 against H2S.
Fig.1 The UV-Vis and fluorescence spectra of probe 1 (10 μM) in MeCN/buffer (1:1,
50 mM PBS buffer, pH=7.4, 1 mM CTAB) solution without and with addition of
Na2S (100 uM) for 5 min at room temperature, λex=310 nm. Inset: the color changes
of probe without and with addition of Na2S. left: under ambient lighting, right: under
UV irradiation.
Fig.2 The emission spectra of probe 1 (10 μM) in MeCN/buffer (1:1, 50 mM PBS
buffer, pH=7.4, 1 mM CTAB) solution with different concentrations of Na2S (0, 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 30 μM) for 5 min at room temperature, λex=310 nm.
Insert is the concentration of Na2S dependence of the intensity of fluorescence
emission (F550).
Fig.3 The fluorescence intensity of probe 1 (10 μM) in MeCN/buffer (1:1, 50 mM
PBS buffer, pH=7.4, 1 mM CTAB) solution in the presence of various reactive sulfur
species: (1) control; (2) 100μM Na2S2O3; (3) 100μM Na2SO3; (4) 100μM GSH; (5)
100μM Hcy; (6) 100μM Cys; (7) 100μM Na2S; (8) 100μM Na2S+100μM Na2S2O3; (9)
100μM Na2S+100μM Na2SO3; (10) 100μM Na2S+100μM GSH; (11) 100μM
Na2S+100μM Hcy; (12) 100μM Na2S+100μM Cys. λex=310 nm. Each spectrum was
acquired 5 min after various analytes addition at 25 oC.
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Fig.4 pH-dependent fluorescence intensity (550nm) changes of probe 1 (10 μM) in
the presence of Na2S (100 μM).
Fig.5 Time-dependent fluorescence response of the probe 1 (10 uM) to Na2S (100 uM)
in MeCN/buffer (1:1, 50 mM PBS buffer, pH=7.4, 1 mM CTAB) solution.
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Scheme 1
17

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Scheme 2
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Fig.1
19

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Fig.2
20

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Fig.3
21

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Fig.4
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Fig.5
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Table 1 The ratio of [HS-]:[H2S] at different pH
pH=4
pH=5
pH=6
pH=7
pH=7.4
pH=8
9.55×10-4:1 9.55×10-3:1 9.55×10-2:1
0.96:1.00
2.39:1.00
9.55:1.00
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Graphical abstract
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Highlights
 A new colorimetric and ratiometric fluorescence probe for hydrogen sulfide is
developed.
 The use of Michael acceptor as the sensing group of H2S shows high selectivity
and sensitivity.
 Probe 1 for hydrogen sulfide detection is based on intramolecular charge transfer
(ICT) mechanism.
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