Thiazole sulfonamide based ratiometric fluorescent chemosensor with a large spectral shift for zinc sensing

Article abstract of DOI:10.1016/j.tet.2010.10.055

A new thiazole sulfonamide (TTP, 1) based Zn²⁺ selective intrinsic chemosensor has been synthesized and investigated. The chemosensor shows a selective fluorescence enhancement (3.0 fold) with Zn²⁺ over biologically relevant cations (Ca²⁺, Mg²⁺, Na⁺, and K⁺) and biologically non-relevant cations (Cd²⁺) in an aqueous ethanol system. It produces an increase in the quantum yield and a longer emission wavelength shift (64 nm) on Zn²⁺ binding with the potential of a ratiometric assay.

Full text of DOI:10.1016/j.tet.2010.10.055

Tetrahedron 66 (2010) 9925e9932
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Thiazole sulfonamide based ratiometric fluorescent chemosensor with a large
spectral shift for zinc sensing
*
Aasif Helal, Sang Hyun Kim, Hong-Seok Kim
Department of Applied Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
A new thiazole sulfonamide (TTP, 1) based Zn2þ selective intrinsic chemosensor has been synthesized
and investigated. The chemosensor shows a selective fluorescence enhancement (3.0 fold) with Zn2þ
Article history:
Received 25 September 2010
Received in revised form 19 October 2010
Accepted 20 October 2010
Available online 26 October 2010
over biologically relevant cations (Ca2þ, Mg2þ, Naþ, and Kþ) and biologically non-relevant cations (Cd2þ
)
in an aqueous ethanol system. It produces an increase in the quantum yield and a longer emission
wavelength shift (64 nm) on Zn2þ binding with the potential of a ratiometric assay.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Thiazole sulfonamide
Chemosensor
Fluorescence
Ratiometric sensing of Zn2þ
ESIPT
1. Introduction
precision of fluorescence assays several such ratiometric probes
have been developed for intracellular applications at present.4
Fluorescent sensor molecules for the detection of metal ions
have attracted considerable research interest and have led to the
development of highly specific probes with a broad range of
applications in environmental chemistry, biochemistry, and cell
biology.1 Fluorescence-based probes provide high sensitivity and
are particularly well suited for the visualization of in vitro and in
vivo biologically important metal ions.2 An ideal fluorescent che-
mosensor must have a binding-selectivity, i.e., strong affinity for
the target molecule and a good signal-selectivity. However, the
majority of these chemosensors are based on a mechanism in
which the fluorescent output is quenched in the absence of and
restored upon binding of the analyte. Nevertheless, the emission
intensity depends both on the analyte and on the probe concen-
trations, which, in a cellular environment, depends on many fac-
tors, such as the localization of the probe, membrane permeability,
incubation time, and temperature or variations in the cell size. This
problem can be solved by using a fluorophore that displays a shift in
the peak excitation or emission wavelength upon binding of the
analyte. The ratio of the luminescence intensities at two excitation
wavelengths are sufficient to determine the analyte concentration,
independent of sensor concentration or any instrument-related
parameters.3 Due to the simplicity, high sensitivity, and greater
Among a series of biologically important metal ions, zinc has
attracted considerable attention because of its structural signifi-
cance and catalytic functions with metalloproteins.5 Although
these Zn2þ are often tightly bound, chelatable (weakly bound), or
free Zn2þ also exist in several tissues, including brain tissue,6
pancreatic tissue,7 and seminal plasma.8 The estimation of free
zinc has proved to be difficult with classical methods. Moreover,
Zn2þ is invisible to most analytical techniques due to its spectro-
scopically silent nature, so fluorescence techniques stand out as the
method of choice. A variety of Zn2þ selective fluorescent probes
that are based on quinoline,9 fluorescein,10 indole,11 and proteins12
have been developed. But these sensors sometimes show similar
affinities for most of the transition and heavy metals, and also lack
the suitable ratiometric nature, or if ratiometric, then produce
small shifts in the fluorescence wavelength.13,14 Moreover an
emission ratiometric probe is required for imaging using two-
photon excitation fluorescence microscopy, which provides signif-
icant advantages over standard laser confocal approaches.15 Several
chemosensors based on CHEF (chelation-enhanced fluorescence),
ICT (intramolecular charge transfer) or a combination of PET
(photoinduced electron transfer), and ICT mechanisms have been
designed to produce ratiometric changes on Zn2þ binding. These
include di-2-picolylamine (DPA) based receptors, such as Zinbo-
5,16a ZnIC,16b,c tosylamido quinoline-based chemosensors, such as
TSQ,16d Zinquin,16e and Schiff-base based receptors like BPBA,16f
1,10-disubstituted ferrocene.16g Among these, some of the DPA
* Corresponding author. Tel.: þ82 53 950 5588; fax: þ82 53 950 6594; e-mail
address: kimhs@knu.ac.kr (H.-S. Kim).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.10.055

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A. Helal et al. / Tetrahedron 66 (2010) 9925e9932
receptors involve a long synthetic procedure, small emission shift,
lack of selectivity, and low quantum yield. Tosylamido quinoline-
based chemosensors lack selectivity and form complexes with
many metal ions with varying fluorescence intensities, while some
Schiff-base receptors have poor solubility and instability in aqueous
solutions.
S
X
S
NH
N
O
NH
N
O S O
N
To develop a zinc-selective ratiometric emission probe, the
photophysics in cation-induced inhibition of excited-state intra-
molecular proton transfer (ESIPT) is often used.17 Recently we have
reported some thiazole-based chemosensors in which the thiazole
ring is substituted with a phenol at position 2 and a pyridine,
phenyl, or another thiazole phenol moiety at position 4 for its use as
a ratiometric fluorescence sensor of zinc,18a anions,18b or the dual
chemosensing of zinc and copper,18c respectively. The thiazole
sulfonamide moiety containing an intramolecular hydrogen bond
undergoes ESIPT and yields a highly Stokes shifted emission from
the proton-transfer tautomer. Coordination of a metal cation will
remove this proton and disrupt the ESIPT process with the emission
of the coordinated species occurring with the normal Stokes shift.19
Moreover introduction of a sulfonamide in the place of a hydroxyl
group induces a rapid rate of ESIPT with a high quantum yield of
tautomer emission within a wide range of solvents.17c,19d
In this paper, the design and development of a novel thiazole
sulfonamide-based ratiometric intrinsic fluorescent chemosensor,
with substitutions at position 2 and 4 of the thiazole ring, regarding
zinc cation in aqueous media is reported. It exhibits a ratiometric
fluorescent response upon the addition of Zn2þ in 10% watere
ethanol, buffered at pH 7.4. The photophysical properties of the
sensor on binding with Zn2þ, the crucial role of the sulfonamide
and the pyridine in the zinc binding, and competitive binding of
Zn2þ with other metal ions are investigated.
1: TTP (X = N)
2: TTB (X = H)
3: BTP
Fig. 1. Structures of chemosensors.
TTB (2) and BTP (3) (Fig.1). Sensors 1, 2, and 3 were prepared in good
yields by the reaction of 2-nitrothiobenzamide with 2-(2-bromoa-
cetyl)pyridine and 2-bromoacetophenone in refluxing ethanol, fol-
lowed by the reduction of the nitro group to amine and then reaction
of the amine with tosyl chloride or benzoyl chloride, as shown in
Scheme 1. The structures of 1, 2, and 3 were confirmed by 1H NMR,
13C NMR, and elemental analysis. The NMR spectrum of 1, 2, and 3
exhibited singlet protons at
assigned to thiazolyl-H, with the NH protons occurring far down-
field. In the case of 1 and 3 doublet protons at 8.65 and 8.52, re-
d 8.18, d 7.34, and d 8.02, respectively,
d
d
spectively, were assigned to the pyridyl-H nearest to the nitrogen.
(Figs. S9e12).
The metal-binding behaviors of TTP (1) were determined by
UVevis and fluorescence spectroscopic studies. In order to achieve
a physiologically acceptable condition the photophysical properties
of TTP (1) were examined in a watereethanol system (1:9). The
UVevis study was carried out in a 10% (v/v) watereethanol solution
buffered by 10 mM HEPES at pH 7.4 with a concentration level of
20
289 nm with a shoulder band at 326 nm (Fig. 2). This can be attrib-
uted to a * transition; this is favored by the planar orientation
mM. Sensor 1 exhibits a maximal absorption band centered at
2. Results and discussion
pep
enforced by intramolecular hydrogen bonding.18,20 When titrated by
Zn2þ (0e10 equiv), the intensity of the maximal absorption band of
TTP (1) decreased, with a concomitant increase of the band at
316 nm and the formation of a shoulder band at 373 nm due to the
deprotonation of the sulfonamide and disruption of the hydrogen
bonding. The absorption bands at 289 and 316 nm linearly decreased
and increased, respectively, up to 1 equiv of zinc (Fig. 2 inset), in-
dicating the formation of a 1:1 complex. Free TTP (1) in a 10% (v/v)
watereethanol solution buffered by 10 mM HEPES at pH 7.4 with
A common fluorophore, such as the amino-benzazoles are often
used to develop ESIPT-based zinc chemosensors. The C]N of the
thiazole ring is analogous to Schiff-base receptors that has a strong
affinity for Zn2þ. The combination of sulfonamide and the thiazole
ring can act as a binding, as well as a fluorophore, resulting in an
intrinsic chemosensor. In order to achieve high fluorescence in-
tensity, selectivity, and affinity for Zn2þ detection, instead of a DPA
unit, we introduced a 2-pyridyl group at position 4 by a simple
synthetic procedure and protected the free amine of the 2-amino-
phenyl group as a tosylamide, as shown in Fig. 1. To gain insight into
the complexation mode of TTP (1) for Zn2þ we also prepared analogs
a concentration level of 2 mM exhibits fluorescence with an emission
band at 524 nm (FF¼0.13, Table 1) with a lex at 339 nm (Fig. 3).
EtOH, reflux
S
NH2
NO2
N
X
NO2
4
S
Br
O
6a: X = N
6b: X = H
5a: X = N
5b: X = H
X
Pd-C/ H2
MeOH, 6 h
S
X
S
Benzoyl chloride
Tosyl chloride
Py, reflux, 8 h
S
NH
N
O
NH
N
Py, reflux, 8 h
NH2
N
O S O
N
X
3: 85%
1: X = N, 80%
2: X = H, 88%
7a: X = N
7b: X = H
Scheme 1. Preparation of chemosensors TTP (1), TTB (2), and BTP (3).

A. Helal et al. / Tetrahedron 66 (2010) 9925e9932
9927
derivatives,17 TTP (1) contains an intramolecular hydrogen bond
that undergoes ESIPT and yields a highly Stokes shifted emission
from the proton-transfer tautomer (Scheme 2).19
This remarkable hypsochromic shift of 64 nm makes TTP (1)
a potential sensor for Zn2þ (Table 1), as compared to DPA, Schiff-
base, and Quinoline-based receptors. The fluorescence enhance-
ment was ten times and I/I0 (fluorescent intensity of receptor
þZn2þ/fluorescent intensity of receptor) was 3 fold more than our
previously reported phenolethiazoleepyridine receptor.18a The
Job’s plot of TTP (1) with Zn2þ also indicates the formation of a 1:1
complex (Fig. 4). The binding constant calculated from the fluo-
rescence titration in a watereethanol system at pH 7.4 was
8.8ꢀ105
M
ꢁ1 (error limit ꢂ10%).21
In order to study the solvatochromic effect on ESIPT, the binding
of Zn2þ with 1 was studied in chloroform, ethanol, THF, acetonitrile,
and DMSO. The fluorescence property of 1 with Zn2þ in all solvents
shows similar behavior, i.e., ratiometric increase in fluorescence
emissions, except in proton-accepting DMSO, in which 1 produces
only normal emission at 454 nm (Table S-1, SI). Since the normal
Fig. 2. Changes in UVevis spectra of
1 (20 mM) upon addition of Zn(ClO4)2 in
state (enamine, N ) possesses a larger dipole moment than the
*
H2OeEtOH (1:9) containing HEPES buffer (10 mM, pH 7.4). Inset: mol ratio plots of
absorbance at 289 and 316 nm.
Table 1
Photophysical data and pKa values of 1e3 in H2OeEtOH (1:9)
a
b
Compound
Absorption Max. nm (log
3)
Dl(nm)
Emission Max. nm
Dl (nm)
QuantumYield (
F
)
pKa
Free
Zn2þ
Free
524
Zn2þ
460
Free
0.13
Zn2þ
0.28
1
289 (3.95)
326 (3.64)
331 (3.59)
333 (3.71)
316 (3.78)
373 (3.20)
331 (3.59)
333 (3.71)
27
47
0
64
11.4
2
3
522
540
522
540
0
0
11.8
12.5
0
a
Quantum yields were obtained using quinine sulfate in 0.5 M H2SO4 as standard.
b
Calculated from UVevis absorbance with 20 m
M of 1 in H2OeEtOH (1:9) containing 0.1 M KCl within a pH range 8e13.17c (Fig. S-7, SI).
The addition of the Zn2þ into 1 gave a new 3.0 fold enhanced
tautomer state (ketimine, T ) it is selectively stabilized by polar
*
emission band centered at 460 nm (FF¼0.28, Table 1) with an
isoemissive point at 513 nm. The quantum yield of the Zn2þ bound
species was found to be twice that of some of the DPA (Zinbo-5) and
Schiff-base receptors (BPBA). The peaks at 460 and 524 nm showed
a linear enhancement and diminution, respectively, with an in-
crease of Zn2þ concentration when the ratio of Zn2þ and 1 con-
centration is below or equal to 1:1. When the 1:1 ratio is reached,
however, higher Zn2þ concentration did not lead to any further
emission enhancement (Fig. 3 inset). Like the benzothiazole
solvents like DMSO and as a result the ESIPT equilibrium is shifted
to the normal state, thus increasing its fluorescence intensity.22 On
addition of the Zn2þ in DMSO, this normal emission gradually de-
creases due to chelation-enhanced quenching (CHEQ). The binding
Fig. 3. Changes in fluorescence spectra of 1 (2
mM) in H2OeEtOH (1:9) containing
HEPES buffer (10 mM, pH 7.4), 1.0 mM EGTA and 100 mM KCl with different concen-
tration of free (unbound) Zn2þ (0e20
mM) (lex¼339 nm). Inset: mol ratio plots of
Scheme 2. The possible ESIPT in 1 a 1:1 binding
and binding with Zn2þ in
stoichiometry.
emissions at 460 and 524 nm.

9928
A. Helal et al. / Tetrahedron 66 (2010) 9925e9932
The quenching in the case of Fe2þ, Fe3þ, and Cu2þ is due to the
160
140
120
100
80
very fast and efficient non-radiative decay of the excited states, due
to the electron or energy transfer between the open shell d-orbitals
of the metal ions and TTP (1).23 While Zn2þ, has the closed-shell d-
orbitals, so that energy or electron transfer processes cannot take
place. Coordination of Zn2þ removes the sulfonamide proton and
disrupts ESIPT, thus causing emission with a normal Stokes
shift.17,24
Moreover in a competitive binding experiment the presence of
Naþ, Kþ, Mg2þ, and Ca2þ, which are abundant in cells, did not in-
terfere with the ratiometric response to Zn2þ, even though their
concentrations were 100 times higher than Zn2þ concentration as
illustrated in Fig S-3 (SI). Except in the cases of Fe2þ, Fe3þ, and Cu2þ
,
where the enhancement was smaller when compared to the others.
Also the ratiometric response of 1 for Zn2þ was found to be maxi-
mum as compared to other cations (Fig. 7); thus, TTP selectivity for
Zn2þ detection over other heavy metals and Cd2þ was found to be
better than some Schiff-base and DPA receptors in aqueous ethanol.
0.0
0.2
0.4
2+
0.6
0.8
1.0
2+
[Zn ]/([Zn ]+[1])
Fig. 4. Fluorescence Job’s plot of 1 with Zn(ClO4)2 in H2OeEtOH (1:9) containing
HEPES buffer (10 mM, pH 7.4), 1.0 mM EGTA and 100 mM KCl measured at 460 nm.
constants were calculated to be 4.3ꢀ105 Mꢁ1 in acetonitrile and
7.2ꢀ105
M
ꢁ1 in DMSO (error limit ꢂ10%) (Fig. S-1 and S-2, SI).
4
3
2
1
0
The Zn2þ specific ratiometric response of TTP (1) was further
confirmed by screening with the 10 equiv of biologically relevant
metal ions, such as Naþ, Kþ, Mg2þ, and Ca2þ, and non-biologically
relevant metal ions, such as Cd2þ. As shown in Fig 5 only Zn2þ
among the all tested metal ions induced a distinct emission shift
and enhancement. The paramagnetic transition-metal ions Fe2þ
,
Fe3þ, and Cu2þ coordinate to TTP (1), but partially or completely
quench the fluorescence emission. Successful emission color
change on Zn2þ binding is easily observable by a naked eye ex-
amination of the receptor 1, with different cations. Upon excitation
at 365 nm the green fluorescence of 1 changes to blue fluorescence,
only in the case of Zn2þ as compared to other metal ions (Fig. 6).
a
b
c
d
e
f
g h
i
j k l m n o p q
Fig. 7. Enhancement of fluorescence intensity of 1 (2 mM) in H2OeEtOH (1:9) con-
400
taining HEPES buffer (10 mM, pH 7.4), 1.0 mM EGTA and 100 mM KCl by addition of
10 equiv of respective metal ion. (a) 1 only, (b) 1þZn2þ, (c) 1þAgþ, (d) 1þHg2þ, (e)
1þPb2þ, (f) 1þCa2þ, (g) 1þCu2þ, (h) 1þNi2þ, (i) 1þCo2þ, (j) 1þFe2þ, (k) 1þCd2þ, (l)
1þMg2þ, (m) 1þKþ, (n) 1þNaþ, (o) 1þCsþ, (p) 1þRbþ, (q) 1þFe3þ
.
2+
300
Zn
The detection limit of 1 for Zn2þ was found to be 0.2
mM, which is
sufficient for sensing Zn2þ in the biological system (Fig. S-4, SI).25 All
of these facts indicate that this probe can be employed for a wide
range of biological applications using microscopic techniques.
Due to the spectroscopically and magnetically silent nature of
zinc, it was not possible to examine its ground state behavior in an
ethanolewater system (Fig. S-5, SI).26 However, the ground state
behavior of the zinc complexation and ion recognition was evalu-
ated in DMSO-d6. A partial 1H NMR spectrum of TTP (1), upon ad-
dition of 1 equiv of Zn2þ is shown in Fig 8. Upon addition of 1 equiv
of Zn2þ the NeH proton peak disappeared due to deprotonation
and the signals of Ha and Hb shifted downfield due to the
deshielding effect of the metal ion. However, in the case of protons
Hd, He, and Hf experienced an upfield shift. It possibly resulted from
200
+
2+
2+
Only Host, Ag , Pb , Ca , K ,
+
+
+
+
2+
2+
Na , Cs , Rb , Hg , Mg .
100
2+
2+
2+
Cd
Co , Ni
2+
2+
Cu Fe , Fe
3+
0
400
450
500
550
600
650
Wavelength (nm)
Fig. 5. Fluorescence spectra of 1 (2 mM) in H2OeEtOH (1:9) containing HEPES buffer
(10 mM, pH 7.4), 1.0 mM EGTA and 100 mM KCl upon addition of various metal ions
(10 equiv) with an excitation wavelength of 339 nm.
an aromatic sulfonamideemetal
p-d orbital interaction through
space (Scheme 3).27
Fig. 6. Fluorogenic change in a 20 mM solution of 1 in H2OeEtOH (1:9) in the presence of 10 equiv of every cation upon illumination at 365 nm.

A. Helal et al. / Tetrahedron 66 (2010) 9925e9932
9929
Fig. 8. Partial 1H NMR spectra of 1 with Zn(ClO4)2 in DMSO-d6.
seen in Figs. 9a and b, unlike TTP (1), there are no absorption
spectral changes upon addition of Zn2þ. In the case of the fluores-
cence spectrum there is little enhancement of the emission peak in
TTB (2) on Zn2þ addition and there is no ratiometric change (Figs. 9c
and d). The binding constant of 2 with Zn2þ calculated from the
fluorescence titration (Fig. S-6, SI) in a watereethanol system at pH
7.4 was 1.8ꢀ105 Mꢁ1 (error limit ꢂ10%).21 This strongly suggests
that increasing the Zn2þ coordination number of the TTB (2) by
introducing a pyridine at the position 4, not only increases the zinc
binding ability but also defines the Zn2þ binding mode by making it
ratiometric.
Sulfonamides tightly bind only to the zinc holoenzyme and not
to the metal-free apoenzyme. This property clearly facilitates the
use of sulfonamide fluorophores to measure zinc binding to a car-
bonic anhydrase II-based biosensor.29 To gain an insight into the
complexation mode of Zn2þ and the role of sulfonamide, we pre-
pared BTP (3).
S
S
2+
Zn
N
N
NH
O2S
N
O2S
Zn
N
N
π-d orbital interaction of
sulfonamide and metal
coordinate bond with Zn2+
Scheme 3. Chelation of
1
with Zn2þ showing the aromatic-metal
p-d orbital
interaction.
To understand the crucial role of the pyridine unit in position 4
of the thiazole ring, which acts as an additional binding site for the
Zn2þ with the aid of the sulfonamide, we have synthesized the
corresponding phenyl derivative TTB (2) bearing the phenyl unit in
position 4 of the thiazole ring.28 Subsequently, it was tested for
absorption and fluorescence changes upon addition of the Zn2þ. As
In contrast to 1, BTP (3,
), which has benzamide in the place of
sulfonamide did not reveal any significant changes in absorption
and fluorescence emission upon addition up to 10 equiv of Zn2þ as
0.5
(a)
0.5
(b)
0.4
0.4
289 nm
0.3
0.3
316 nm
333 nm
333 nm
0.2
0.2
326 nm
331 nm
331 nm
0.1
0.1
373 nm
0.0
0.0
300
350
400
450
300
350
400
450
400
300
200
100
0
400
300
200
100
0
(c)
(d)
460 nm
522 nm
522 nm
524 nm
540 nm
540 nm
400
450
500
550
600
650
400
450
500
550
600
650
Wavelength (nm)
Fig. 9. UVevis spectra of 1 (
Fluorescence spectra of 1 (
), 2 (
), 2 (
) and 3 (
) and 3 (
): (a) in the absence, (b) in the presence of 10 equiv of Zn(ClO4)2 in H2OeEtOH (1:9) containing HEPES buffer (10 mM, pH 7.4).
): (c) in the absence and (d) in the presence of 10 equiv of Zn(ClO4)2 in H2OeEtOH (1:9) containing HEPES buffer (10 mM, pH 7.4).

9930
A. Helal et al. / Tetrahedron 66 (2010) 9925e9932
shown in Fig. 9. The pKa value of 3 (pKa¼12.5) was found to be
greater than 1 (pKa¼11.4), which makes the NeH proton less acidic
and resulting in a very low tautomer emission in 3 than 1 (Table 1,
and Fig. 9). Thus, it is notable that the sulfonamide group of 1 plays
two important roles in Zn2þ complexation: (a) it increases the
acidity of the NeH proton (Table 1) leading to a stronger intra-
molecular hydrogen bonding that results in a rapid rate of ESIPT
the ratiometric fluorescence signal and strong binding affinity with
Zn2þ in aqueous ethanol. Thus, the TTP receptor involves simple
synthetic procedures, overcomes the poor solubility and instability
of the Schiff-base receptors in an aqueous system, lack of selectivity
of the tosylamido quinoline receptors, and is suitable for ratio-
metric imaging of Zn2þ in biological system.
and quantum yield of the tautomer emission (
F
¼0.13), and (b) the
4. Experimental section
4.1. General methods
sulfonamide Nꢁ anion is a stronger donor30 than the parent neutral
amino, or the benzamide group, so it enhances the fluorescence
and forms a stronger stable binding with Zn2þ
(
F
¼0.28).
We prepared the complex of TTP (1) with Zn2þ in H2OeEtOH
(1:9) and characterized by HR-FAB mass (Fig. S-8, SI). The HR mass
spectra of 1-Zn2þ shows a 1:1 stoichiometry with the molecular ion
peak at m/z 469.9975, which can be assigned as the signal for
[MþZnꢁH]þ.
Melting points were determined using a ThomaseHoover cap-
illary melting point apparatus and are uncorrected. 1H and 13C NMR
spectra were recorded on a Bruker AM-400 FT NMR spectrometer
using Me4Si as the internal standard. Mass-spectral data were
obtained fromthe KoreaBasic Science Institute (Daegu) on a Jeol JMS
700 High resolution mass spectrometer. UVevis absorption spectra
were determined on a Shimadzu UV-1650PC spectrophotometer.
Fluorescence spectra were measured on a Shimadzu RF-5301 fluo-
rescence spectrometer equipped with a xenon discharge lamp and
1 cm quartz cells. All of the measurements were carried out at 298 K.
Absolute ethanol of analytical grade was purchased from Merck.
Deionized water (double distilled) was used throughout the ex-
periment as an aqueous layer. All other materials used for synthesis
HartreeeFock 3-21G calculations using Spartan 04 of 1 and its
Zn2þ complex support our hypothesis that the C]N of the thiazole,
nitrogen of the pyridine ring, and the sulfur of the sulfonamide are
responsible for Zn2þ coordination having a bond length of 1.92 A,
ꢀ
ꢀ
ꢀ
2.01 A, and 2.82 A, respectively. The energy-minimized calculations
also revealed that the benzene ring, along with the thiazole, and
pyridine rings, are on the same plane as the Zn2þ while the tosyl
group makes an angle of 95.54ꢃ (Fig. 10).
Fig. 10. Energy minimized structures of (a) 1 and (b) 1ꢁZn2þ complex as predicted by HartreeeFock 3-21G calculations.
Thus we have prepared an intrinsic fluorescent probe whose
fluorescent unit is directly involved in the interactionwith metal ions
were purchased from Aldrich Chemical Co. and used without fur-
ther purification. 2-Nitrobenzthioamide (4)31 and 2-(bromoacetyl)
pyridine (5a),32 compounds 6b and 7b33 were prepared in accor-
dance with the procedure in the literature. The solutions of metal
ions were prepared from their perchlorate salts of analytical grade
and then subsequently diluted to prepare working solutions. HEPES
buffer solutions of different pH were prepared using proper
amount of HEPES and KOH (all of analytical grade) under adjust-
ment by a pH meter. All fluorescence experiments were carried out
at pH 7.4 in buffered Zn2þ solution comprising of 10 mM HEPES
(buffer), 1.0 mM EGTA as Zn2þ chelator, and 100 mM KCl in accor-
dance with the procedure reported in the literature.5b
helping in quick, sensitive, and selective detection of Zn2þ
.
3. Conclusion
In conclusion, we have developed a new sulfonamideethiazo-
leepyridine based ratiometric intrinsic fluorescent probe TTP (1),
highly selective for Zn2þ over other biologically relevant, non-rel-
evant, and transitional metal cations on the basis of the ESIPT
mechanism. Upon complexation, the Zn2þ-bound species exhibits
a significantly large blue shift (64 nm), enhancement (3.0 fold) in
the emission spectrum, and an increase in the quantum yield,
which amplifies the recognition event to a greater extent than
other commonly used DPA and Schiff-base receptors, as well as our
previously reported phenolethiazoleepyridine compound. The TTP
utilizes the C]N of the thiazole ring, similar to the Schiff-base re-
ceptor and the pyridine ring like the DPA receptor, for production of
4.2. Determination of quantum yields
Quantum yields were determined by the relative comparison
procedure.34 The quinine sulfate with the quantum yield of 0.54
in sulfuric acid (0.5 M) was used as the standard. The general

A. Helal et al. / Tetrahedron 66 (2010) 9925e9932
9931
equation used in the determination of relative quantum yields is
shown as follows:
diluted with water and extracted with EtOAc. The organic layer was
dried over anhydrous sodium sulfate and concentrated. The residue
was purified by column chromatography (SiO2, EtOAcehexane-1:4)
to give 3 (143 mg, 85% yield). Mp: 104e105ꢃC (CH2Cl2 ehexane); 1H
ꢀ
ꢁ
Qx=Qst ¼ ðFx=FstÞ ꢀ ðAst=AxÞ ꢀ n2x=ns2t
NMR (400 MHz, CDCl3)
d
7.06 (t, J¼7.3 Hz, 1H), 7.16 (dt, J¼5.3, 1.8 Hz,
Where Q is the quantum yields; F is the integrated area under the
corrected emission spectrum; A is the absorbance at the excitation
wavelength; n is the refractive index of the solution; and the sub-
1H), 7.29 (t, J¼7.8 Hz, 2H), 7.38 (t, J¼7.3 Hz, 1H), 7.46 (q, J¼7.3 Hz,
2H), 7.59 (d, J¼7.8 Hz, 1H), 7.71 (d, J¼6.8 Hz, 1H), 7.90 (d, J¼7.4 Hz,
2H), 8.02 (s, 1H), 8.52 (d, J¼4.3 Hz, 1H), 8.84 (d, J¼8.6 Hz, 1H), 12.54
(s, 1H, NH); 13C NMR (100 MHz, CDCl3)
d 117.1, 119.8, 121.2, 121.4,
scripts
x and st refer to the unknown, and the standard,
respectively.
123.2, 123.5, 127.9, 128.7, 128.9, 131.4, 131.7, 135.9, 137.1, 137.2, 149.4,
151.3, 155.0, 166.7, 169.2. Anal. Calcd for C21H15N3OS$CH2Cl2: C,
59.73; H, 3.87; N, 9.50; S, 7.25. Found: C, 60.10; H, 3.94; N, 9.53; S,
6.99.
4.3. Synthesis
4.3.1. 2(20-Nitrophenyl)-4-pyridylthiazole (6a). A mixture of
4
(100 mg, 0.55 mmol) and 5a (185 mg, 0.66 mmol) in ethanol
(15 mL) was refluxed for 4 h. The solvent was removed under vacuo
and the residue was washed with water, neutralized with 1 N
NaOH, and extracted with EtOAc. The organic layer was dried over
anhydrous sodium sulfate and concentrated. The residue was pu-
rified using column chromatography (SiO2, EtOAcehexane-1:2) to
give 6a (124 mg, 80% yield). Mp: 88ꢃC (CH2Cl2 ehexane); 1H NMR
4.3.5. Zn2þe1 complex. A mixture of 1 (100 mg, 0.24 mmol) and Zn
(ClO4)2 $ 6H2O (110 mg, 0.29 mmol) in ethanolewater (v/v 9:1,
5 mL) was refluxed for 3 h. The mixture was cooled to room tem-
perature and the precipitated complex was filtered off. The filtered
cake was washed thoroughly with water, ethanol, and diethyl ether,
and dried under vacuum to provide the complex (85 mg, 74% yield).
HR-FAB mass: calcd for (C21H16O2N3S2Zn) 469.9975. Found:
469.9972.
(400 MHz, CDCl3)
d
7.51 (dt, J¼7.8, 1.5 Hz, 1H), 7.58 (dt, J¼7.8, 1.5 Hz,
1H), 7.74e7.70 (m, 3H), 8.01 (d, J¼7.8 Hz,1H), 8.18 (s, 1H), 8.25e8.22
(m, 1H), 8.53 (d, J¼4.4 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d 119.0,
Acknowledgements
121.7, 123.3, 125.6, 126.9, 130.6, 131.1, 132.1, 134.4, 137.5, 149.1, 151.8,
156.1, 161.9. Anal. Calcd for C14H9N3O2S: C, 59.35; H, 3.20; N, 14.83;
S, 11.32. Found: C, 59.14; H, 3.11; N, 14.79; S, 11.40.
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea (NRF),
and funded by the Ministry of Education, Science and Technology
(20100010070). A.H. was also supported by the Research Institute
of Industrial Technology at Kyungpook National University.
4.3.2. Chemosensor 1. A solution of 6a (200 mg, 0.70 mmol) in
methanol (10 mL) was hydrogenated with 10% Pd/C under 1 atm of
hydrogen for 6 h. After the catalyst was removed by filtration the
filtrate was concentrated and dried to give the crude amine 7a. To
the solution of the crude amine (7a, 120 mg, 0.47 mmol) in pyridine
(20 mL) p-toluenesulfonyl chloride (112 mg, 0.59 mmol) was added
and refluxed for 8 h. The mixture was diluted with water and
extracted with EtOAc. The organic layer was dried over anhydrous
sodium sulfate and concentrated. The residue was purified by col-
umn chromatography (SiO2, EtOAcehexane-1:2) to give 1 (154 mg,
80% yield). Mp: 165ꢃC (CH2Cl2ehexane); 1H NMR (400 MHz, CDCl3)
Supplementary data
Supplementary data related to this article can be found online
version at doi:10.1016/j.tet.2010.10.055.
References and notes
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(d, J¼8.3 Hz, 2H), 7.64 (d, J¼8.3 Hz, 1H), 7.72 (d, J¼8.3 Hz, 1H), 7.91
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