Synthesis and spectral studies of a novel benzoxazole-derived fluorescent probe for Hg2+

Article abstract of DOI:10.3184/174751916X14599541191262

A novel turn-off fluorescent probe bearing a benzoxazole fluorophore has been synthesised and characterised. Its recognition properties towards different metal ions have been researched by spectrometry. The probe was highly selective and sensitive to Hg

Full text of DOI:10.3184/174751916X14599541191262

280 RESEARCH PAPER
VOL. 40 MAY, 280–283
JOURNAL OF CHEMICAL RESEARCH 2016
Synthesis and spectral studies of a novel benzoxazole-derived fluorescent
probe for Hg2+
Yong Zhanga*, Yi-Chao Wanga, Jia-Geng Meia, Na-Na Yanga, Wei-Na Wangb, Si-Fan Aic and
Zhi-Qiang Fangd
ªCollege of Chemistry and Chemical Engineering, Hubei Polytechnic University, Huangshi 435003, P.R. China
bHigher Vocational Technical School, Hubei Polytechnic University, Huangshi 435003, P.R. China
cCollege of Science, Tianjin University of Science and Technology, Tianjin 300457, P.R. China
dHuangshi Lifuda pharmaceutical chemical co., Ltd., Yangxin, 435229, P.R. China
A novel turn-off fluorescent probe bearing a benzoxazole fluorophore has been synthesised and characterised. Its recognition properties
towards different metal ions have been researched by spectrometry. The probe was highly selective and sensitive to Hg2+ and showed a
–1
complexation ratio towards Hg2+ of 1:1. Its fluorescence intensity varied almost linearly with the concentration of Hg2+ (1.6～8.6 µmol L ),
and the detection limit of Hg2+ was estimated to be 0.68 µmol L–1.
Keywords: benzoxazole, Hg2+, fluorescent probe, selectivity, sensitivity
1
Heavy metals have attracted considerable attention because of their
wide use and subsequent impact on the environment and human
health. One of the very toxic and frequently used heavy metals is
mercury. Mercurial species may be released into the environment
through coal-fired power plants and into water streams from
chemical, gold mining and chlor-alkali industrial plants.1 It’s
reported that exposure to Hg2+ can cause immune, sensory,
neurological, motor and behavioural dysfunctions through the food
chain.2,3 Therefore, it is important to develop new methods for the
detection and quantification of Hg2+. Recently, Hg2+ probes have
beenhighlyvaluablebecauseoftheirhighsensitivityandselectivity,
quick response time and easy signal detection.4–7 Although many
types of Hg2+ ion-sensing ionophores have been introduced to
detect mercuric ions in chemical and biological systems, there is
still need for more probes for the analysis of this ion in varying
types of samples in terms of the sample matrix and concentration
ranges.8,9 Therefore, the development of Hg2+-selective probes is
still a challenge.10–12 Benzoxazole compounds are widely used as
optical probes, fluorescent brighteners and laser dyes due to their
good photophysical and photochemical properties.13–15 Herein,
we report a sensitive and selective fluorescent probe bearing a
benzoxazole fluorophore for the detection of Hg2+.
characterised by H NMR, 13C NMR, IR, MS and elemental
analysis, and its recognition of Hg2+ was studied by spectrometry.
Metal-binding properties showed that 3 is highly selective and
sensitive to Hg2+ in aqueous EtOH solution.
The maximum absorption wavelength of 3 was 320 nm, and its
maximum excitation (λex) and emission (λem) wavelengths were
325 and 412 nm, respectively. Different common metal ions were
used to evaluate the metal ion binding properties and selectivity of
3 (10 µmol L–1) by fluorescence spectra. As shown in Fig. 1, upon
the addition of K+, Ca2+, Na+, Mg2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+,
Zn2+, Cd2+ or Ag+ (5 equiv.), no obvious effect on the fluorescence
emissions were observed, whereas Cu2+ and Pb2+ caused a weak
decrease in the fluorescent intensity. In contrast, the addition of
Hg2+ resulted in a significant quenching in the fluorescent emission
of 3, indicating that 3 displayed a high Hg2+ selectivity. The
selectivity of 3for Hg2+ was further proved by metal ion competition
experiments, which were examined by use of binary mixtures of
Hg2+ and various metal ions (Fig. 2). The fluorescence intensity
of 3 (10 µmol L–1) in the presence of 1 equiv. of Hg2+ was hardly
affected by the addition of 10 equiv. of competing metal ions. It can
be seen that 3 displayed a high selectivity for recognition of Hg2+.
That was attributed to the far higher binding affinity and stability
of 3 to Hg2+ compared to other metal ions. It was also found that
the UV-Vis spectra of 3 showed a strong absorption band at 320
nm, but its absorption intensity decreased upon addition of the
same amount of Hg2+, and the maximum absorption wavelength of
the Hg-complex remained almost unchanged (Fig. 3).
Results and discussion
The benzoxazole-derived fluorescent probe 3 was synthesised by
a nucleophilic substitution reaction of a primary halogenoalkane
and secondary amine, as depicted in Scheme 1. Probe 3 was fully
N
NH2
PPA
+
H2N
COOH
NH2
473K, Ar
OH
O
(1)
S
O
NH
S
N
O
ClCH2COCl, (Et)3N
NH
Cl
THF,
K
KI,
,
3
273K
CO
MeCN, reflux
24h
2
(2)
O
N
NH
N
S
S
O
(3)
Scheme 1 Synthesis of fluorescent probe 3.
* Correspondent. E-mail: zy0340907@163.com

JOURNAL OF CHEMICAL RESEARCH 2016 281
Fig. 1 The effect of metal ions (50 µM) on the fluorescent properties of
probe 3 (10 µM) in 100 mM Tris-HCl buffer (pH 7.4, H2O:EtOH = 4:1) with
an excitation at 325 nm. The samples were incubated for 10 min at room
temperature prior to measurements.
Fig. 2 The effect of Hg2+ on 3 (10 µmol L–1) in the presence of different
metal ions (100 µmol L–1).
Fig. 3 Change in UV-Vis spectra of 3 upon interaction with Hg2+.
Fig. 4 The fluorescence intensity of 3 (10 µmol L–1) and 3 + Hg2+ over a
pH range from 2 to 14 at room temperature.
Fig. 5 The emission intensity of 3 (10 µmol L–1) changes with increasing
Fig. 6 Fluorescence intensity of 3 as a function of concentration of Hg2+
concentrations of Hg2+ (0 30 µmol L–1); the inset shows photograph of
~
(1.6 8.6 µmol L–1).
~
3 (10 µmol L–1) in the absence and presence of 3 equiv. Hg2+ under UV
light (365 nm).
The fluorescence spectrum of 3 was highly stable over a
pH range of 2–11 (Fig. 4). Upon addition of Hg2+, 3 remained
unaffected with increasing pH from 5 to 9, indicated its potential
applications in biological systems. Therefore, 3 can be used for
detection of Hg2+ without the adverse influence of other metal ions
and pH variations close to physiological conditions.
change of the fluorescence spectra of 3 upon addition of Hg2+;
clear “on–off” fluorescence changes of 3 to Hg2+ were observed.
When Hg2+ was added to the solution, a significant decrease of the
fluorescence intensity at 412 nm was observed. It was found that
the fluorescence of 3 was quenched almost completely after the
addition of 3 equiv. of Hg2+. As illustrated in the inset photograph
shown in Fig. 5, an obvious fluorescence change from bright blue
to colourless can be observed under irradiation at 365 nm after the
The sensitivity of the fluorescence response of 3 towards Hg2+
was investigated by fluorescent titration. Fig. 5 shows the gradual

282 JOURNAL OF CHEMICAL RESEARCH 2016
Fig. 7 Changes of fluorescence intensity of 3 (10 µmol L–1) in the
Fig. 8 Job’s plot of 3 and Hg2+ ([3] + [Hg2+] = 10 mmol L–1).
presence of 1.0 equiv. of Hg2+ as a function of time.
412 nm
325 nm
S
+
Hg2
S
S
O
N
S
+
Hg2
O
N
N
N
NH
NH
O
O
+
(3) + Hg2
(3)
fluorescence off
fluorescence on
Scheme 2 The proposed sensing mechanisms of probe 3 for Hg2+.
addition of 3 equiv. of Hg2+ to the solution of 3. Interestingly,
between the chelating unit and the benzoxazole fluorophore,
thus resulting in the fluorescence quenching of the benzoxazole
fluorophore.
In conclusion, we have synthesised a benzoxazole-based
fluorescent probe 3, which is selective and sensitive to Hg2+.
The detection limit of 3 to Hg2+ was determined to be 0.68 µmol
L–1, and 3 forms a 1:1 complex with Hg2+.
the decremental fluorescence emission intensity was found
to be proportional to the concentration of Hg2+ ion in the
range from 1.6 to 8.6 µmol L–1 (Fig. 6), so a linear regression
equation y = –14.5675x + 157.9617 (x is the concentration of
Hg2+, and y is the fluorescence intensity, linear correlation
coefficient R2 = 0.9935) could be applied. From the changes in
Hg2+-dependent fluorescence intensity, the detection limit of 3
to Hg2+ was determined to be 0.68 µmol L–1 according to the
calculation method reported in the literature, thus 3 exhibited
a high sensitivity toward Hg2+.16 The response time of 3 to Hg2+
was also demonstrated to be short (Fig. 7). The intensity of the
fluorescent signal at 412 nm quickly reached the minimum level,
and was unchanged as time went by, therefore, 3 could be used
for real-time tracking of Hg2+. Furthermore, the stoichiometry
of the complex formation of 3 with Hg2+ was determined by a
Job’s plot at room temperature. The emission changes at 412 nm
were plotted against molar fractions of 3 under the conditions
of an invariant total concentration (10 µmol L–1). As a result,
when the molar fraction of [3] / ([3] + [Hg2+]) was about 0.5,
the changes of fluorescence emission approached a maximum,
indicating formation of a 1:1 complex between 3 and Hg2+ (Fig.
8). On the basis of a 1:1 stoichiometry, the association constant
of 3 for Hg2+ was determined to be 1.08 × 106 L·mol–1 according
to the Benesi–Hildebrand equation.17
Experimental
General
Unless otherwise noted, all reagents were purchased from commercial
companies and directly used without further purification. The melting
point was determined with an XT4A micromelting point apparatus and
1
was uncorrected. The H NMR and 13C NMR spectra were recorded
on a Mercury Plus-400 spectrometer in CDCl3. Electrospray ionisation
mass spectra (ESI-MS) were acquired on an Applied Biosystems API
2000 LC/MS/MS system. IR spectra were performed on a Perkin-
Elmer Spectrum BX FT-IR instrument in tablets with potassium
bromide. Elemental analyses were carried out on a Perkin-Elmer 2400
instrument. Fluorescence spectra were performed on a FluoroMax-P
spectrofluorimeter.
Synthesis
The intermediates 4-(benzo[d]oxazol-2-yl)aniline (1) and bis[2-
(ethylthio)ethyl]amine were prepared according to the reported
procedure.21,22
N-[4-(benzo[d]oxazol-2-yl)phenyl]-2-chloroacetamide (2): Chloroacetyl
chloride (1.12 g, 10 mmol) was added dropwise to a solution of the
intermediate 1 (1.05 g, 5 mmol) and triethylamine (1.01 g, 10 mmol) in
anhydrous tetrahydrofuran (40 mL), cooled in an ice bath. The resulting
mixture was stirred rapidly for 4 h, then the solvent was evaporated to give
the crude product, which was further purified via column chromatography
Based on above experiments and some recent reports, we
propose a possible complexation mechanism between 3 and
Hg2+, as shown in Scheme 2.18–20 In the chelation by 3, two
sulfur atoms, one amino–nitrogen atom and one oxygen atom
of amide might bind with Hg2+, the sulfur, oxygen and nitrogen
atoms donating their lone pair of electrons to the empty orbitals
of Hg2+. The capture of Hg2+ caused electron or energy transfer

JOURNAL OF CHEMICAL RESEARCH 2016 283
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This work was supported by the National Natural Science
Foundation of China (Grant No. 51173060), College Students’
Science and Technology Innovation Project of Hubei
Polytechnic University (No. 14cx16) and Young Teachers
Training in Enterprise Program of the Higher Education
Institutions of Hubei Province, China (No. XD2014677).
Received 5 February 2016; accepted 26 February 2016
Paper 1603904 doi: 10.3184/174751916X14599541191262
Published online: 20 April 2016
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