Fluorescence sensor for sequential detection of zinc and phosphate ions

Article abstract of DOI:10.1016/j.saa.2016.06.026

A new, highly selective turn-on fluorescent chemosensor based on 2-(2′-tosylamidophenyl)thiazole (1) for the detection of zinc and phosphate ions in ethanol was synthesized and characterized. Sensor 1 showed a high selectivity for zinc compared to other cations and sequentially detected hydrogen pyrophosphate and hydrogen phosphate. The fluorescence mechanism can be explained by two different mechanisms: (i) the inhibition of excited-state intramolecular proton transfer (ESIPT) and (ii) chelation-induced enhanced fluorescence by binding with Zn^2?+. The sequential detection of phosphate anions was achieved by the quenching and subsequent revival of ESIPT.

Full text of DOI:10.1016/j.saa.2016.06.026

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 87–94
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Fluorescence sensor for sequential detection of zinc and phosphate ions
a,
Miran An a, Bo-Yeon Kim a, Hansol Seo a, Aasif Helal b, , Hong-Seok Kim
⁎
⁎
a
Department of Applied Chemistry, Kyungpook National University, Daegu 41566, Republic of Korea
b
Center of Research Excellence in Nanotechnology, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
A new, highly selective turn-on fluorescent chemosensor based on 2-(2′-tosylamidophenyl)thiazole (1) for the
detection of zinc and phosphate ions in ethanol was synthesized and characterized. Sensor 1 showed a high se-
lectivity for zinc compared to other cations and sequentially detected hydrogen pyrophosphate and hydrogen
phosphate. The fluorescence mechanism can be explained by two different mechanisms: (i) the inhibition of ex-
cited-state intramolecular proton transfer (ESIPT) and (ii) chelation-induced enhanced fluorescence by binding
with Zn2+. The sequential detection of phosphate anions was achieved by the quenching and subsequent revival
of ESIPT.
Received 1 November 2015
Received in revised form 28 April 2016
Accepted 17 June 2016
Available online 18 June 2016
Keywords:
Fluorogenic chemosensor
Thiazole sulfonamide
Sequential recognition
Zinc
© 2016 Elsevier B.V. All rights reserved.
Phosphate
1. Introduction
because of protonation. One phosphate-recognition strategy is the use
of metal complexes, mostly involving transition metals, in which the
Molecular fluorescent chemosensors for the recognition of cations
and anions have attracted much attention for important and diverse
ecological, biological, and clinical applications [1–4]. Fluorescent
chemosensors have the advantages of real-time monitoring with fast re-
sponse times, intrinsic high sensitivity, and ease of handling compared
to other optical sensors [5,6]. The sensing of Zn2+ ions, the second
most abundant transition metal in the human body, in an aqueous
media is ecologically and biochemically relevant because they play cru-
cial roles in biological systems. Zn2+ ions are vital for many cellular pro-
cesses [7] such as apoptosis [8], DNA synthesis [9], neurotransmission
[10,11], gene expression [12], modulation of diverse ion channels [13],
and signal transduction [14]. Zn2+ ions are also an essential component
of many enzymes, e.g., carbonic anhydrase, transcription factors, and
zinc finger proteins, in which they play catalytic or structural roles
[15]. Clinically, diverse Zn2+-based compounds have been used as
tumor photosensitizers [16], antibacterial/antimicrobial and anticancer
agents [17], radioprotective agents [18], and antidiabetic insulin mi-
metics [19]. Moreover, the hepato and cardio toxicity induced by some
anticancer drugs can be reduced by Zn2+ [20].
metal ion acts as an anchoring point for the phosphate species. Although
different metal ions including transition, lanthanide, and main group
metal ions have been used, one of the most commonly used ions for
this purpose is Zn2+ [28,29].
Phosphate is an indispensable constituent of two important
biopolymers—DNA and RNA—and many chemotherapeutic and antivi-
ral drugs [30]. However, the excessive agricultural use of inorganic
phosphates causes excessive algal growth, leading to decomposition
and decreased dissolved oxygen levels [31]. Dihydrogen phosphate
(H2PO−4 ) is the predominant equilibrium species of inorganic phos-
phates at physiological pH. Therefore, H2PO−4 sensing and detection
methods have received much attention [32–35]. Phosphate oxoanions
such as pyrophosphate play crucial roles in many bioenergetic and met-
abolic processes such as ATP hydrolysis and DNA or RNA polymerase re-
actions [36]. The detection of released pyrophosphate has been
investigated as a real-time DNA sequencing method [37]. Studies on tel-
omerase activity (a biomarker for cancer) by evaluating the amount of
pyrophosphate are also important in cancer research [38]. Furthermore,
the pyrophosphate level in synovial fluids has been correlated with the
occurrence of calcium pyrophosphate dihydrate disease, a rheumato-
logic disorder [39]. This species can also be used as a potential biomark-
er for arthritis [40].
There is intense interest in the development of molecular systems
capable of binding inorganic phosphate anions because of their crucial
roles in signal transduction [21,22], energy storage in living organisms,
eutrophication of water bodies [23–25], and catalysis [26,27]. Inorganic
phosphates exhibit diverse shapes and sizes, hydrophobicity, and high
hydration energies, and in some cases, exist only in a limited pH range
The photophysics of the cation-induced inhibition of excited-state
intramolecular proton transfer (ESIPT) has been often used to develop
Zn-selective ratiometric emission probes [41–43]. We have previously
reported some thiazole-based chemosensors in which the thiazole
ring was substituted with a phenol or tosylamide-protected aniline or
naphthol at the position 2 and a pyridine, phenyl, or another thiazole
⁎
Corresponding authors.
E-mail addresses: Aasifh@kfupm.edu.sa (A. Helal), kimhs@knu.ac.kr (H.-S. Kim).
http://dx.doi.org/10.1016/j.saa.2016.06.026
1386-1425/© 2016 Elsevier B.V. All rights reserved.

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M. An et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 87–94
phenol moiety at the position 4 as a ratiometric fluorescence sensor of
Zn or dual chemosensor of Zn and Cu [44–50]. We also reported a thia-
zole-based compound containing a phenol or naphthol at the position 2
and an ester moiety at the position 4 as a very good Al3+ sensor [51,52].
Recently, we reported the synthesis of a phenol/naphthol-containing
thiazole-based sensor for the sequential detection of Ga and HSO4−
[53]. In this study, we prepared compound 1 containing a tosylamide-
protected aniline at the position 2 of a thiazole ring and an ester moiety
at the position 4 (Fig. 1) and investigated its sequential sensing abilities
for different cations and anions based on two different mechanisms of
fluorescence: ESIPT and chelation-induced enhanced fluorescence
(CHEF) [54]. We also synthesized compound 2 to study the effect of
the tosyl group in compound 1 on the sensing of cations and anions.
FAB MS calcd for C12H13N2O2S (M + H)+: 249.0698, found: m/z
249.0699.
2.3. Synthesis of compound 1
Amino compound 2 (100 mg, 0.40 mmol) was added to p-
toluenesulfonyl chloride (92 mg, 0.48 mol) and triethylamine (0.5 mL)
in anhydrous CH2Cl2 (20 mL) and stirred at room temperature for 2 h.
After the reaction was completed, the mixture was treated with
NaHCO3 solution and extracted with CH2Cl2. The organic layer was
dried and concentrated. The residue was purified by column chroma-
tography (SiO2, 10% EtOAc in hexane), affording compound 1
(103 mg, 64% yield). Mp. 133 °C (CH2Cl2/hexane), TLC Rf 0.35 (10%
EtOAc in hexane); 1H NMR (DMSO-d6) δ 1.36 (3H, t, J = 7.1 Hz, CH3),
2.30 (3H, s, CH3), 4.39 (2H, q, J = 7.1 Hz, CH2), 7.24 (1H, d, J = 7.6 Hz,
Ha), 7.27 (2H, d, J = 8.4 Hz, Hg), 7.42 (2H, t, J = 7.4 Hz, Hb,c), 7.57
(2H, d, J = 8.4 Hz, Hf), 7.90 (1H, d, J = 7.6 Hz, Hd), 8.64 (1H, s, He),
11.45 (1H, s, NH); 13C NMR (DMSO-d6) δ 14.1, 20.9, 61.1, 121.8, 122.1,
125.3, 126.7, 129.2, 129.3, 129.7, 131.4, 134.9, 135.9, 143.7, 145.3,
160.2, 166.6; HR-FAB MS calcd for C19H19N2O4S2 (M + H)+: 403.0786,
found: m/z 403.0788.
2. Experimental
2.1. General
Melting points were determined using a Thomas-Hoover capillary
melting point apparatus and uncorrected. The 1H and 13C NMR spectra
were recorded using a Bruker AM-400 spectrometer and Me4Si as the
internal standard. The FAB mass spectra were obtained at the KBSI
Daegu center. The UV–visible absorption spectra were determined
using a Shimadzu UV-1650PC spectrophotometer. The fluorescence
spectra were measured using a Shimadzu RF-5301 fluorescence spec-
trometer equipped with a Xe discharge lamp and 1-cm quartz cells
with a 5-nm slit width. The IR spectra were recorded using a Shimadzu
IR Prestige-21 FTIR spectrometer. All the measurements were carried
out at 298 K. Analytical-grade ethanol was purchased from Merck. All
other materials for the syntheses were purchased from Aldrich Chemi-
cal Co. and used as received without further purification. Compound 3
was obtained following a literature procedure [45], and the quantum
yield (Φ) was calculated as reported [53]. The solutions of metal ions
were prepared from their analytical-grade perchlorate salts, and those
of the anions were prepared from their tetrabutylammonium (TBA)
salts. The working solutions were prepared by further dilution of the
stock solutions.
2.3.1. Synthesis of 1-Zn complex
A mixture of compound 1 (40 mg, 0.10 mmol) and Zn(NO3)2·6H2O
(30 mg, 0.10 mmol) in ethanol/CH2Cl2 (5:5 v/v, 5 mL) was stirred for
1 h. The mixture was stand at room temperature, and the precipitated
complex was filtered off. The filtered cake was washed thoroughly
with ethanol and dried under vacuum, providing the complex (43 mg,
91% yield). HR-FAB mass: calcd for [C19H17N2O4S2Zn]+ 464.9921.
Found: 464.9920.
2.4. UV–visible and fluorescence studies
A solution of host (30 μM) in EtOH was prepared, and the guest
(300 μM) solution was added to the host solution for the UV–visible
study. For fluorescence titration, a solution of host (3 μM) in EtOH was
prepared, and the guest (30 μM) solution was added to the host solu-
tion. In a typical titration experiment, 2 mL of the host solution was
transferred to a fluorescence cell, and the emission spectrum was re-
corded at a fixed wavelength. The guest solution (20 μL) was added
through a microsyringe, and the amount was increased until 10 equiv
of guest. The fluorescence spectrum of each solution was recorded
after each addition. The association constants were determined by
gnuplot using the following equation.
2.2. Synthesis
2.2.1. Synthesis of compound 2
A mixture of compound 3 (200 mg, 0.72 mmol) and 5% Pd/C in eth-
anol (20 mL) was hydrogenated using H2 gas for 10 h. After the removal
of the solvent, CH2Cl2 was added, and the reaction mixture was filtered
through a Celite pad to remove the catalyst. The filtrate was concentrat-
ed, and the residue was crystalized from a mixture of CH2Cl2/hexane,
affording amino compound 2 (159 mg, 89% yield). 1H NMR (DMSO-
d6) δ 1.33 (3H, t, J = 7.1 Hz, CH3), 4.33 (2H, q, J = 7.1 Hz, CH2), 6.62
(1H, ddd, J = 7.2, 6.9, 1.0 Hz, Hb), 6.83 (1H, dd, J = 8.3, 1.0 Hz, Ha),
7.11 (1H, s, NH), 7.18 (1H, ddd, J = 7.1, 7.0, 1.4 Hz, Hc), 7.59 (1H, dd,
J = 7.9, 1.4 Hz, Hd), 8.47 (1H, s, He); 13C NMR (DMSO-d6) δ 14.2, 60.9,
113.1, 115.7, 116.5, 126.7, 128.9, 131.4, 145.7, 146.7, 160.5, 169.3; HR-
plot “gadata.dat” u 1:2 w lp.
f(x) = (a + b ∗ c ∗ x ∗ ∗ 1.00) / (1 + c ∗ x ∗ ∗ 1.00).
fit f(x) “gadata.dat” u 1:2 via a, b, and c.
2.5. Theoretical calculations
The geometry of sensor 1 was optimized using gradient-correlated
density functional theory (DFT) according to literature [55–59].
3. Results and discussion
3.1. Synthesis of sensor 1
The required compound 2 was prepared by the reduction of ethyl 2-
(2′-nitrophenyl)thiazoly-4-carboxylate, which was obtained by the re-
action of 2-nitrothiobenzamide with ethyl bromopyruvate in ethanol
[45]. Compound 1 was prepared by the reaction of compound 2 with
p-toluenesulfonyl chloride in the presence of triethylamine in a good
yield (Scheme 1). The 1H NMR spectrum of sensor 1 in DMSO-d6
showed CH3CH2 signals corresponding to the ethyl ester—a triplet at δ
1.36 and a quartet at δ 4.49—as well as one singlet corresponding to
the tosyl CH3 at δ 2.30 and one N–H singlet at δ 11.45. In the 13C NMR
Fig. 1. Structures of chemosensors 1 and 2.

M. An et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 87–94
89
Scheme 1. Synthesis of sensor 1.
spectrum of sensor 1, the signal of ethyl ester appeared at δ 14.1 and
61.1, and the tosyl CH3 signal was observed at δ 20.9. The high-resolu-
tion mass spectrum (HRMS) of sensor 1 showed a molecular ion peak
of (M + H)+ at m/z = 403.0788 (see ESI).
The UV–visible titration of sensor 1 was performed in EtOH. The ab-
sorption bands of compound 1 linearly increased and decreased up to
1 equiv of Zn2+ (Fig. 3a), indicating the formation of a 1:1 complex
with a high binding affinity. This finding is characteristic for the 2-(2′-
tosylaminophenyl)thiazole derivatives when the complexation is ac-
companied by the deprotonation of the N–H proton [45]. Moreover,
three clearly defined isosbestic points (294 nm, 313 nm, and 337 nm)
were observed in the titration spectra of compound 1, indicating the
conversion of free sensor 1 to the corresponding Zn2+ complex [61,
62]. The binding constant of the complex between sensor 1 and Zn2+
was determined by the nonlinear fitting of the corresponding UV–visi-
ble titration data and yielded a value of 1.06 × 104 M−1 with a satisfac-
tory correlation coefficient (R = 0.9983) [63]. The error was estimated
to be ≤7%. The fluorescence titration of compound 1 with Zn2+ was
also performed. The emission band of compound 1 linearly increased
up to 1 equiv of Zn2+ (Fig. 3b). To further quantify the complexation
ratio of sensor 1 with Zn2+ in the complex, Job's method was used, con-
sidering the emission changes at 460 nm as a function of the molar frac-
tion of Zn2+. The maximum emission was observed when the molar
fraction of Zn2+ reached 0.5, indicating a 1:1 stoichiometry in the sensor
1-Zn2+ complex (Fig. S6). The change in the color of the fluorescence
after the introduction of Zn2+ was observed mainly because of the inhi-
bition of ESIPT. Then, Zn2+, which is prone to bind to the sulfonyl oxy-
gen of the tosyl group, oxygen of the ester group, nitrogen of the
thiazole ring, and deprotonated nitrogen of the amide, preferentially
forms a stable chelated complex. Thus, the ESIPT ceases, and CHEF
plays the key role, resulting in an emission at 460 nm. The preferential
binding of Zn2+ with the oxygen of the tosyl and ester group, nitrogen
of the thiazole ring, and amide group was also established by the ener-
gy-minimized structure of sensor 1-Zn2+ complex using B3LYP/6-31G*
set in Fig. 9.
3.2. UV–visible and fluorescence studies of sensor 1 in the presence of metal
ions
The initial studies on the UV–visible absorption and fluorescence
emission of ethyl 2-(2′-tosylamidophenyl)-4-thiazole carboxylate, sen-
sor 1, were carried out in EtOH. Among the tested solvents, ethanol
showed the best response for sensor 1 in the UV–vis spectrum (Fig
S4). The binding of sensor 1 to Zn2+ in the form of perchlorate salt
was investigated by UV–visible spectroscopy. In the absence of Zn, sen-
sor 1 showed absorption bands with maxima at 286 nm (log ε = 3.98)
and 318 nm (log ε = 3.76), whereas the addition of Zn2+ to compound
1 resulted in a red shift of the absorption band maxima to 296 nm (log
ε = 3.96) and 369 nm (log ε = 3.53) (Fig. 2a). The absorption band at
318 can be attributed to the n–π* transition resulting from the intramo-
lecular hydrogen bonding involving the lone pair electrons on the nitro-
gen of the thiazole ring, whereas the red shift to 369 nm can be
attributed to the favored planar orientation of the complex caused by
metal binding and inhibition of ESIPT [60]. The peak at 288 nm origi-
⁎
nates from the π–π transition of the aromatic rings.
Sensor 1 showed weak fluorescence with a low quantum yield (Φ =
0.0059) at 382 nm and 535 nm, corresponding to the enamine and ke-
tamine peaks, respectively, resulting from ESIPT. The introduction of
Zn2+ leads to the formation of a stable complex with sensor 1 that in-
hibits the ESIPT, and consequently the chelated system produce an
emission band at 460 nm with a larger quantum yield (Φ = 0.0653)
due to CHEF (Fig. 2b) [54]. Similar UV–visible and fluorescence studies
of sensor 2 towards various metal ions did not show any significant se-
lectivity and sensitivity because ESIPT is not possible in compound 2 be-
cause of the low acidity of the free amine compared to that of the
tosylamide-protected compound 1 (Fig. S5).
The selectivity is one of the important criteria for the potential appli-
cation of a cation sensor. To determine the selectivity of sensor 1,
10 equiv of various biologically and non-biologically relevant metal cat-
ions were added to a solution of compound 1 (10 μM), and their binding
was investigated by fluorescence spectroscopy. No cations other than
Fig. 2. (a) UV–visible spectra of compound 1 (30 μM) with 10 equiv of Zn(ClO4)3 and (b) fluorescence spectra of compound 1 (10 μM) with 10 equiv of Zn(ClO4)2 in EtOH. λex = 337 nm.

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M. An et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 87–94
Fig. 3. (a) UV–visible spectra of compound 1 (30 μM) titrated with Zn(ClO4)2 from 0 to 100 equiv in EtOH. Inset plots represent the dependence of the absorbance intensity at 369 nm upon
the addition of Zn(ClO4)2. (b) Fluorescence titration of compound 1 (10 μM) with Zn(ClO4)2 in EtOH, λex = 337 nm. Inset plots represent the dependence of the fluorescence intensity at
460 nm upon the addition of Zn(ClO4)2.
Fig. 4. (a) Fluorescence spectra of compound 1 (10 μM) with 10 equiv of various cations in EtOH. (λex = 337 nm, λem = 460 nm). (b) Fluorescence intensity of compound 1 with various
cations in EtOH at λem = 460 nm.
Zn2+, which produced a prominent fluorescence signal, induced
any distinct emission shift in the spectra of compound 1, except Cd2+
(Fig. 4). The addition of Cd2+, which has coordination properties similar
to those of Zn2+, produced a slight change in the emission spectra. Com-
petition experiments were performed to explore the possibility of using
compound 1 as a practical ion-selective fluorescent chemosensor for
Zn2+. In these experiments, compound 1 (10 μM) was first mixed
with 10 equiv of Zn2+, followed by the addition of 10 equiv of various
competing metal ions. The fluorescence emission spectra showed that
most of the common metal ions exhibited no clear interference with
the detection of Zn2+ (Fig. S7). The plot of the linear relationship be-
tween fluorescence intensity at 460 nm vs. [Zn2+] during the
Fig. 5. (a) Fluorescence spectra of compound 1 (3 μM) upon the addition of Zn2+ (10 equiv) and subsequent addition of anions (10 equiv). (b) Fluorescence responses of 1-Zn2+ in the
presence of various anions (10 equiv) in EtOH. The white bars represent emission upon the addition of 10 equiv of Zn2+ to compound 1 (3 μM). The shaded bars represent the change in
emission that occurs upon the subsequent addition of 10 equiv of various anions to a 3 μM solution of 1-Zn2+. λex = 337 nm. λem = 460 nm.

M. An et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 87–94
91
Fig. 6. (a) UV–visible titrations of 1-Zn2+ complex (30 μM) with HP2O37− (10 equiv) in EtOH. Inset plots represent the relationship between the absorbance intensity at 369 nm and the
HP2O37− concentration. (b) Fluorescence titrations of 1-Zn2+ complex (10 μM) with HP2O37− (10 equiv) in EtOH. Inset plots represent the relationship between the fluorescence intensity
at 460 nm and the HP2O37− concentration.
and 535 nm by displacing Zn2+ from sensor 1 and restoring ESIPT,
Table 1
which had been inhibited by Zn2+. In contrast, the addition of F−
,
Binding constants Ka (M−1) of 1-Zn2+ and with anionsa.
OAc−, and CN− enhanced the fluorescence by displacing the ligand
and removing the acidic N–H proton, thereby inhibiting ESIPT. Compe-
tition experiments were performed to investigate whether this property
of 1-Zn2+ was influenced by CN− or any other anions at concentrations
exceeding the concentrations of HP2O37− and H2PO4− by as much as 10
times. These experiments showed that 1-Zn2+ complex had a high se-
lectivity and sensitivity for HP2O37− and H2PO4− ions.
UV
Fluorescence
1-Zn2+
1.06 × 104
1.46 × 105
5.20 × 105
0.98 × 104
1.40 × 105
5.66 × 105
1-Zn2+-HP2O37−
1-Zn2+-H2PO−4
a
Estimated errors are 7–16%.
Upon the slow addition of HP2O37−, the peak at 460 nm, resulting
from the addition of Zn2+ to compound 1, was gradually quenched be-
cause of the disappearance of CHEF and restoration of ESIPT until a total
fluorescence titrations of sensor 1 (10 μM) with Zn(ClO4)2 (100 equiv)
showed that the limit of detection of Zn2+ by sensor 1 was 20 μM in
EtOH (R = 0.981) (Fig. S8) [63].
of 1 equiv of HP2O37− was added. A further increase in the HP2O37−
-
concentration did not lead to any additional quenching. The binding
constants of 1-Zn2+ to HP2O73− as determined by UV–visible and fluo-
rescence titrations were 1.46 × 105 M−1 and 1.40 × 105 M−1, respec-
tively. (Fig. 6 and Table 1). The emission intensity of 1-Zn2+ at
460 nm steadily decreased until a total of 1 equiv of HP2O37− was
added (inset plot in Fig. 6b). The Job's plot showed a 1:1 stoichiometry
between 1-Zn2+ and HP2O37− (Fig. S9). The same results were obtained
from the UV–visible and fluorescence titrations of 1-Zn2+ with H2PO4−
(Fig. S10). However, the fluorescence titration of 2-Zn2+ (10 μM) with
10 equiv of anions did not show any significant activity (Fig. S11).
A linear decrease in the fluorescence intensity of sensor 1-Zn2+ at
460 nm upon the gradual addition of a solution of HP2O37− and H2PO4−
indicated the limits of detection of HP2O37− and H2PO4− as 1 μM and
3.3. Fluorescence based selectivity of 1-Zn2+ ensemble towards anions
The binding of sensor 1 with Zn2+ was weak compared to the other
thiazole-based sensors reported previously (Table S1) [44,45,48].
Therefore, the anion selectivity of 1-Zn2+ ensemble was investigated.
Because H2PO−4 and HP2O73− can form strong complexes with Zn2+
,
the use of 1-Zn2+ complex as a chemosensor for the detection of
HP2O73− and H2PO4− was investigated. Complex 1-Zn2+ (3 μM) was
treated with 10 equiv of various anions including F−, Cl−, Br−, I−
,
NO3−, ClO4−, H2PO4−, HP2O73−, OAc−, and CN−. As shown in Fig. 5, the ad-
dition of HP2O37− and H2PO−4 caused remarkable complete fluorescence
quenching, whereas the addition of F−, OAc−, and CN− enhanced the
fluorescence. Because of the high Zn-phosphate affinity, the addition
of HP2O37− and H2PO−4 caused the disappearance of the peak at
460 nm, and the enamine and ketamine peaks appeared at 382 nm
1 μM, respectively for 1-Zn2+ (R1 = 0.986 for HP2O73−, and R2
=
0.971 for H2PO−4 ) in EtOH (Fig. S12). [63].
The complexation properties of 1-Zn2+ complex were further stud-
ied by fluorescence. The addition of up to 10 equiv of CN− to 1-Zn2+
Fig. 7. Fluorescence changes in 1-Zn2+ (1:10, 3 μM) upon the addition of (a) 0–10 equiv and (b) 10–100 equiv of CN− in EtOH. (c) Plots represent the relationship between the emission
intensity at 460 nm and the concentration of CN−
.

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M. An et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 87–94
Scheme 2. Plausible complexation mechanism between sensor 1, Zn2+, and anions.
complex in solution (3 μM) enhanced the fluorescence, due to the en-
hancement of the CHEF on anion binding with the metal of the 1-
Zn2+ complex, and with further addition of up to 60 equiv of CN−, the
emission intensity gradually decreased, the fluorescence was quenched,
and peaks appeared at 382 nm and 535 nm, resulting from the displace-
ment of Zn2+ from 1-Zn2+ complex that inhibits the CHEF mechanism
and restores the ESIPT (Fig. 7, Scheme 2). Similar trends were observed
in the fluorescence spectra of 1-Zn2+ upon the addition of 10–
100 equiv of F− (Fig. S13).
sulfonamide NH proton, which was exchanged with deuterio-ethanol,
whereas all other protons, i.e., the signals of aminophenyl protons Ha,
Hb, Hc, and Hd, and p-tosylamide protons Hf, Hg, and Hh shifted down-
field except the proton of thiazole He that shifted upfield compared to
its 1H NMR spectra in DMSO-d6 (Fig S1). The addition of 2 equiv of
Zn(ClO4)2 to a solution of compound 1 showed 7% complex formation,
as obtained from the integration of the complexed and free proton He.
The addition of 5 equiv of Zn(ClO4)2 to a solution of compound 1 trig-
gered ~11% of complex formation and showed significant upfield shifts
of the aminophenyl protons Ha, Hb, and Hc from δ 7.199 to 6.930 ppm
(Ha), δ 7.735 to 7.533 ppm (Hb), and δ 7.433 to 7.257 ppm (Hc), respec-
tively, and the other proton Hd shifted downfield from δ 7.790 to
7.911 ppm. Thiazole proton He, and p-tosyl protons Hf, Hg, and Hh
shifted downfield from δ 8.396, 7.571, 7.131, and 2.285 ppm to 8.726,
7.742, 7.324, and 2.381 ppm, respectively (Fig. 8). Moreover, the ethoxy
methylene proton also shifted from δ 4.464 ppm to δ 4.566 ppm (Fig.
S14). These NMR chemical shifts explain the chelation effect of Zn2+
with sensor 1. Further addition of 100 equiv of Zn2+ to a solution of
3.4. NMR binding study
To further investigate the nature of Zn binding to sensor 1, a 1H NMR
study of sensor 1 in the presence of Zn2+ was performed in CD3CN. The
addition of 1–2 equiv of Zn(ClO4)2 to a solution of sensor 1 in CD3CN did
not produce any significant shifts in any of the proton signals.
The 1H NMR study of sensor 1 was performed in the presence of
Zn2+ in CD3CD2OD. The 1H NMR spectra of compound 1 showed no
Fig. 8. A partial 1H NMR spectra of compound 1 (4.5 μM) in CD3CD2OD at 25 °C: (i) Free 1, (ii) 1 + Zn2+ (5 equiv), and (iii) 1 + Zn2+ (5 equiv) + H2PO−4 (10 equiv).

M. An et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 87–94
93
Fig. 9. DFT based energy-minimized structure of 1-Zn2+ complex using B3LYP/6-31 G* as the basis set in EtOH as an implicit medium; a) vertical view, b) horizontal view, and c) complex
electrostatic potential map (the blue to red trend shows the positive to negative potential property range in kJ/mol). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
compound 1 formed 21% of complex with band broadening. These re-
sults clearly indicate that the formation of a complex between com-
pound 1 and Zn2+ was very weak in CD3CD2OD. The addition of
H2PO−4 (10 equiv) to the resulting mixture of sensor 1 and Zn2+
(5 equiv) regenerated sensor 1 by removing Zn2+ from 1-Zn2+ com-
plex as Zn(H2PO4) is shown in Fig. 8.
(sulfonyl group), N (thiazole), and O (ester) as well as Zn2+ ions were
located on the same plane (Fig. 9b), indicating that sensor 1 formed a
stable chelated complex. Thus, CHEF plays the key role in the emission
at 460 nm.
The applications of sensor 1 to detect metal ions including Zn2+ and
anions in real analytes will be carried out in due course.
3.5. IR and HRMS study
4. Conclusions
A complex of sensor 1 with Zn(NO3)2 in a mixture of ethanol/CH2Cl2
(5:5 v/v) was prepared and characterized by IR (KBr) and HR-FAB mass.
The IR spectrum of compound 1 showed a characteristic N–H stretching
at 3142 cm−1, ester C_O stretching at 1732 cm−1, and thiazole C_N
stretching at 1602 cm−1. Complex 1-Zn showed the disappearance of
N–H stretching at 3142 cm−1 due to N–Zn formation (Figs. S15–S16).
The HRMS spectra of complex 1-Zn showed a 1:1 stoichiometry with
the molecular ion peak at m/z 464.9920, corresponding to [1 + Zn-
1]+ (Fig. S17). These data confirmed the 1:1 formation of 1-Zn complex.
In
tosylamidophenyl)thiazole-based fluorescent chemosensor 1 was syn-
thesized. Sensor showed highly selective “on–off”-type
conclusion,
a
highly
Zn-selective
2-(2′-
1
a
fluoroionophoric switching behavior towards Zn2+, and partial en-
hancement with Cd+2 even in the presence of diverse common interfer-
ing metals ions such as Co+2, Ni2+, and Cu+2. The fluorescence was
based on ESIPT followed by CHEF mechanism. Moreover, 1-Zn2+ com-
plex showed a high selectivity and sensitivity towards HP2O37− and
H2PO−4 that completely quenched the fluorescence of the complex by
displacing the Zn2+
.
3.6. DFT study
Acknowledgment
To understand the complexation of Zn2+ with sensor 1, B3LYP/6-
31G* level energy minimization calculation was performed using SPAR-
TAN 10 software in ethanol (SM8 solvent model) as the implicit medi-
um [55–59]. The calculated energy-minimized structure of 1-Zn2+
complex indicated the interaction distances between Zn2+ and the
deprotonated nitrogen of the sulfonamide (because of a higher reso-
nance stabilization through complexation) (b), nitrogen of the thiazole
ring (a), sulfonyl oxygen of the tosyl group (c), and oxygen of the ester
ethoxy group (d) in Fig. 9. A distance of 1.90 Å and 1.96 Å were calculat-
ed for the Zn\\O bond (c) between the tosyl S–O and Zn–N bonds (b)
between the deprotonated N and Zn2+. Other two interactions between
Zn2+ and thiazole C_N and the oxygen of ethoxy ester group were cal-
culated to be 1.97 Å and 1.98 Å, respectively. The band-gap energies
This research was supported by the Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by the
Ministry of Science, ICT, and Future Planning (2013R1A1A2006777).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.saa.2016.06.026.
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