Chromogenic and fluorogenic sensing of Cu2+ based on coumarin

Article abstract of DOI:10.1016/j.tet.2011.01.093

A new highly selective, reversible, chromogenic, and fluorogenic chemosensor (4) based on thiazole-coumarin moieties for quantification of copper ions in aqueous-DMSO was designed and synthesized. The mechanism of fluorescence was based on ICT, which was modified by the introduction of an electron-donating diethylamino group making it chromogenic and increasing the binding affinity. The selectivity toward copper ions was not affected by the presence of representative alkali metals, alkali earth metals, or other transition metals.

Full text of DOI:10.1016/j.tet.2011.01.093

Tetrahedron 67 (2011) 2794e2802
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Chromogenic and fluorogenic sensing of Cu2þ based on coumarin
,
Aasif Helal a, Mohammad Harun Or Rashid b, Cheol-Ho Choi b, Hong-Seok Kim a
*
a Department of Applied Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea
b Department of 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
Article history:
A new highly selective, reversible, chromogenic, and fluorogenic chemosensor (4) based on thiazolee
coumarin moieties for quantification of copper ions in aqueous-DMSO was designed and synthesized.
The mechanism of fluorescence was based on ICT, which was modified by the introduction of an elec-
tron-donating diethylamino group making it chromogenic and increasing the binding affinity. The se-
lectivity toward copper ions was not affected by the presence of representative alkali metals, alkali earth
metals, or other transition metals.
Received 9 December 2010
Received in revised form 31 January 2011
Accepted 31 January 2011
Available online 4 March 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Fluorescence sensor
Chromogenic sensing
Copper ion
Thiazole
Coumarin
1. Introduction
media, which is a very important factor for potential biological
applications.7
The design and synthesis of chemosensors for highly noxious,
heavy, and transition metal ions are currently a task of prime im-
portance for medical, environmental, and biological applications.1
Presently, one of the most attractive approaches focuses on the
research of novel colorimetric and fluorescent metal ion sensors,
which allow naked-eye detection of color and fluorescent emission
change upon metal ion binding without the use of a spectroscopic
instrument.2 Copper plays an important role in various biological
processes. It is a vital trace element, the third most abundant in
humans, and is present at low level in a variety of cells and tissues,
with the highest concentration in the liver. The average concen-
In chemosensors, a selective binding motif is attached to a flu-
orophore for signal transduction. However, one disadvantage is
that the recognition event is sometimes difficult to detect because
the fluorophore does not directly contact the bound metal ion. In
this aspect, an ideal fluorescent probe would be one whose fluo-
rescent unit is directly involved in the interaction with the metal
ions. We choose coumarin as the fluorophore due to its desirable
photophysical properties, such as a large Stokes shift, visible exci-
tation and emission wavelengths, and a structure that can be easily
modified.8 Moreover, the carbonyl group of coumarin can take part
in the coordination with metal ions.
tration of blood copper in the normal group is 100e150
m
g/dL
It is known that chelating groups such as C]N and C]O exhibit
a high affinity to transition and post-transition metal cations, but
less binding affinity toward alkali metal and alkaline earth metal
cations.9 The carbon in the 3-position bears a partial negative
charge in the ground state for all of the coumarins. Intramolecular
charge transfer (ICT) would be facilitated by attachment of an
electron-withdrawing substituent at the 3-position.10 Introduction
of a thiazole ring at the 3-position of a coumarin moiety enhances
the ICT because of the stronger electron-withdrawing ability of the
thiazole ring. Thus a heteroaromatic ring system, such as a thiazole
with a coumarin as a fluorophore at position 4 and a phenol at
position 2 can chelate with metal ions via the carbonyl oxygen,
thiazole nitrogen, and phenol oxygen atoms that act as a eONOe
donor receptor moiety highly selective for copper.11
(15.7e23.6 m
M).3 Its concentration in the neuronal cytoplasm may
contribute to the etiology of Alzheimer’s or Parkinson’s disease.4,5
The U.S. Environmental Protection Agency (EPA) has set the limit
of copper in drinking water to be 1.3 ppm (w20 mM). As a pollutant
due to its extensive industrial use and an essential trace element in
biological systems, chemosensors for copper(II) based on chromo-
genic probes that are expected to quickly, nondestructively, and
sensitively detect copper ions have drawn a lot of attention.2a,6 So
far, only a few colorimetric and fluorescent sensors based on the
rhodamine chromophore for copper ions have been reported.
However, only few of them exhibit good performance in aqueous
Recently, thiazole based chemosensors 1 and 2, as shown in
Fig. 1, with a phenol at the 2 position and pyridine (1) or another
* 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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.01.093

A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
2795
S
S
S
S
N
N
N
N
Y
N
O
O
OH
X
OH
HO
1
2
3: X = OH, Y = H
4: X = OH, Y = NEt2
5: X = H, Y = NEt2
Fig. 1. Structures of chemosensors.
thiazole phenol moiety (2) at the 4 position of the thiazole ring, for
ratiometric fluorescence sensing of zinc12 and dual sensing of
copper and zinc have been reported.13 In both of these cases, soft
heteroatoms, such as N and S, as an electron donor to metal cations
along with phenolic oxygen, improved the binding selectivity with
zinc and copper.
This paper describes a new coumarin fluorophore at the 4 po-
sition of the thiazole ring. Its reversible, highly selective, chromo-
genic, fluorogenic responses upon the addition of copper ions and
competititive behavior with other cations in aqueous-DMSO (3:1)
containing HEPES buffer (10 mM, pH 7.4) are demonstrated.
Initial studies on the UVevis absorption and fluorescent emis-
sion revealed that 3 showed selectivity toward Cu2þ ions in aque-
ous-DMSO (3:1 v/v). In the absence of Cu2þ ion, 3 showed an
absorption band at 346 nm, that is, attributed to the n/
p tran-
*
sitions of the coumarin moiety. On addition of Cu2þ to the solution
of 3 chelation with the carbonyl of the coumarin enhances the
electron-withdrawing character of the acceptor carbonyl moiety,
resulting in the formation of a new red-shifted absorption band at
375 nm (Table 1) and synchronous decrease in the absorption band
at 346 nm, as shown in Fig. 2b. The absorption bands at 346 and
375 nm linearly decreased and increased, respectively, up to 1 equiv
of Cu2þ ion addition (Fig. 2b inset), indicating the formation of a 1:1
complex. None of the other cations produced such a redshift in
their absorption spectra (Fig. 2a).
2. Results and discussion
Due tothehigh affinity of Cu2þ ions forelectron-donatingnitrogen
and carbonyl oxygen during complexation, two thiazole-coumarin
based analogsd2-(20-hydroxyphenyl)-4-(30-coumarinyl)thiazole (3)
and 2-(20-hydroxyphenyl)-4-[(30-(7-diethylaminocoumarinyl)]thia-
zole (4)dwere synthesized in order to compare the metal ion selec-
tivityand sensitivity. To understand thecrucial roleofboththephenol
and coumarin providing a suitable binding site for Cu2þ, 2-phenyl-4-
[(30-(7-diethylaminocoumarinyl)]-thiazole (5) was also prepared.
Sensors 3 and 4 were obtained by reacting 2-hydroxythiobenzamide
(6) with 3-(2-bromoacetyl)coumarin (7) and 3-(20-bromoacetyl)-7-
diethylaminocoumarin (8), respectively, and 5 was obtained by re-
action of thiobenzamide (9) with 8 in refluxing ethanol as shown in
Scheme 1. The 2-hydroxy-thiobenzamide (6) was prepared by
thionation of 2-hydroxy-benzamide with Lawesson’s reagent. The
3-(20-bromoacetyl)-7-diethylaminocoumarin (8) was prepared by
a condensation reaction of 4-(diethylamino)salicylaldehyde with
ethyl acetoacetate in ethanol followed by bromination.14 The struc-
tures of 3, 4, and 5 were confirmed by 1H NMR, 13C NMR, and ele-
mental analysis.
Table 1
Comparisons of the absorption, emission, and binding properties of sensors 3 and 4
in H2OeDMSO (3:1)
3
3þCu2þ
375
3.9
460
0.3
Dl (nm)
4
4þCu2þ
460
4.2
0
Dl (nm)
lmax (nm)
log
em (nm)
346
3.6
460
29
412
4.3
495
48
e
l
I/I0
Fa
Ka
0.0
0.01
0.35
4.4 ꢀ104
2.2 ꢀ106
a
Quantum yields were obtained using quinine sulfate in 0.5 M H2SO4 as standard.
Fluorescence spectra of 3 in aqueous-DMSO (3:1 v/v) showed
the emission peak at 460 nm corresponding to the charge transfer
(CT) from the phenol and thiazole to the coumarin moiety (Fig. S9).
On the addition of Cu2þ up to 1 equiv the emission peak is partially
quenched, but no further quenching occurred upon the addition of
the analyte up to 10 equiv (Fig. 3b). From fluorescence titration the
binding constant of 3 with Cu2þ was calculated to be 4.4 ꢀ 10 4 Mꢁ1
O
S
Br
+
NH2
N
EtOH
90%
O
OH
S
6
O
OH
O
O
7
3
O
O
S
Br
EtOH
68%
N
N
N
O
N
N
O
O
O
O
OH
S
8
4
O
O
Br
NH2
EtOH
70%
+
N
S
9
O
8
5
Scheme 1. Synthesis of 3, 4, and 5.

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A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
Fig. 2. UVevis spectra of 3 (20 mM) (a) with different cations (10 equiv), (b) upon the addition of Cu(ClO4)2 (200 mM) in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4).
Inset: mol ratio plot of absorbance at 375 nm.
Fig. 3. Fluorescence spectra of 3 (20 mM) in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4) (a) upon the addition of 10 equiv of various metal cations, (b) as a function of
added Cu(ClO4)2. Inset: mol ratio plot of emissions at 460 nm (lex¼361 nm).
(error limits ꢂ10%) (Fig. 7a).15 Moreover, iron and cobalt ions also
partially quench the fluorescence of 3 (Fig. 3a).
of the sensor in the free and complex forms, and in the binding
constant (Table 1).17
To enhance the fluorescence intensity, quantum yield, binding
ability and selectivity toward Cu2þ, an electron-donating dieth-
ylamino group was introduced into coumarin at the 7-position in
conjugation with the electron-withdrawing carbonyl group. This
increases the CTcharacter by producing a large change in the dipole
moment upon excitation from the ground to the excited state
resulting in very large Stokes’ shifts between their absorption and
fluorescence maxima,16 increasing the molar extinction coefficient
UVevis properties of 4 were investigated in aqueous-DMSO
(3:1) at a concentration level of 20 mM. Compound 4 displayed an
obvious absorption band in the visible region peaked at 412 nm,
which could be assigned to the CT absorbance, as observed in other
compounds with ICT character (Fig. 4). However, upon the addition
of Cu2þ to the solution of 4 a new red-shifted absorption band at
460 nm is enhanced gradually while the absorption band at 412 nm
decreased synchronously, with an isobestic point at 435 nm as
Fig. 4. UVevis spectra of 4 (20
mM) (a) with different cations (10 equiv), (b) upon the addition of Cu(ClO4)2 (200 mM) in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4).
Inset: mol ratio plot of absorbance at 460 nm.

A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
2797
shown in Fig. 4b. The absorption bands at 412 and 460 nm linearly
decreased and increased, respectively, up to 1 equiv of Cu2þ (Fig. 4b
inset), indicating the formation of a 1:1 complex with a strong
binding affinity. The Job’s plot of 4 with Cu2þ also confirmed the
observed by the naked-eye (Fig. 5a). This response was selective for
Cu2þ under these conditions. The addition of 10 equiv of the other
cations as their perchlorate salts resulted in no appreciable changes
(Fig. 4a).
Fig. 5. (a) Chromogenic changes of a 20
mM solution of 4 in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4) in the presence of 10 equiv of each cation. (b) Fluorogenic
changes of a 20 M solution of 4 in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4) in the presence of 10 equiv of each cation upon illumination at 365 nm.
m
formation of a 1:1 complex (Fig. S1). The changes in the absorption
spectra upon the complexation are due to the direct interaction
between the bound cation and the carbonyl group of the coumarin
moiety. The Cu2þ ion reinforces the electron-withdrawing charac-
ter of the carbonyl group in coumarin, which leads to a bath-
ochromic shift of 48 nm in the absorption spectrum (Table 1). In
terms of electronic interaction, the dipole moment of 7-dieth-
ylaminocoumarin in the excited state is higher than that of the
ground state because of the photoinduced CT occurring from the
diethylamino, a nonbonding electron donor group, to the carbonyl
group. Therefore, when Cu2þ ions coordinate with the carbonyl
group, the excited state is more stable than the ground state so that
the absorption spectrum is shifted toward the red. In this intrinsic
chromogenic sensor, the ligandeCu2þ binding was accompanied by
the solution color change from light green to yellow, which is easily
Similarly, fluorescence titration of 4 with Cu2þ was carried out in
aqueous-DMSO (3:1) at a concentration level of 0.2 M. The addition
m
of Cu2þ to the solution of 4 causes a complete quenching of the
fluorescent emission and the peak at 495 nm is switched off when
excited at 435 nm (Figs. 5b and 6b). The long wavelength of excita-
tion (435 nm) and emission (495 nm) can prevent damage to living
biological samples, interference by autofluorescence from native
cellular species, and the influence of background fluorescence.18
Thus introduction of 7-diethylaminocoumarin, a typical dye with
enhanced ICT character, as a fluorophore, increases the absorbance
and emission efficiency and the quantum yield of sensor 4 (
F
¼0.35)
as compared to 3 (
F
¼0.01) (Table 1). Cu2þ, being a paramagnetic
cation with open shell d-orbitals, quenches the fluorescence of the
fluorophore upon binding with it. Presumably this is due to the
electron or charge transfer between the metal cation and thiazole,
(b)
(a)
Fig. 6. Fluorescence spectra of 4 (0.2
mM) (a) with different cations (10 equiv) (b) as a function of added Cu(ClO4)2 in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4).
(
lex¼435 nm). Inset: mol ratio plot of emission at 495 nm.

2798
A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
phenol, and coumarin fluorophore providing avery fast and efficient
nonradiative decay of the excited state.2a,19 Compound 4 undergoes
a complete quenching of fluorescence (99% quenching) as compared
to 3. The binding mode of 4 with Cu2þ from the results of fluores-
cence titration spectra (Fig. 6b inset) was 1:1 with a binding constant
of 2.2 ꢀ106 Mꢁ1 (error limits ꢂ10%) (Fig. 7b).15
moiety inhibit the n/
p transitions in 3 and the CT in 4. Further
*
increase of the pH from 5.0 to 6.0 the absorbance reaches its
maximum and displays no obvious change in its characteristic
within the range of 6.0e9.0. Increasing the pH (10.0e12.0) results
in blue shift of the absorbance in both the probes due to the
deprotonation of the phenol (Figs. 8a, Figs. S-2a and S-3a).19c
(a)
(b)
Fig. 7. Linear regression curve of (a) 3 and (b) 4 obtained by plotting I0/(IꢁI0) as a function of 1/[Cu2þ] in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4). (lex¼361 nm
for 3 and 435 nm for 4).
The selectivity and tolerance of 4 for Cu2þ overother metal cations
were investigated by adding 10 equiv of metal cations to 0.2 M so-
The difference in the absorbance of the probes and its copper
complexes is negligible at low (2.0e5.0) and high (10.0e12.0) pH.
But at pH 6.0 there is a redshift to 375 nm in 3 and 460 nm in 4 on
addition of Cu2þ this redshift and the characteristic absorbance
were unchanged within the pH range from 6.0 to 9.0 suggesting
that the Cu2þ complex of both the sensors are stable over this range
(Figs. S2b and S3b).
Next the effect of pH on the emission spectrum was investigated
(Fig. 8b). In both 3 and 4 decreasing pH protonates the nitrogen of
the thiazole ring and the tertiary nitrogen of 4 thus inhibiting the
ICT to the coumarin resulting in decrease of fluorescence. At pH 6.0
both 3 and 4 show a maximum intensity and it remain unchanged
till pH 9.0. At higher pH (>9.0) the fluorescence intensity is de-
creased due to the enhancement of negative charge density and
formation of phenolate (Figs. S4a, S5a).19f
m
lution of 4. In the case of Cu2þ the molecular fluorescence is quenched
to a maximum level and therefore a high molecular sensitivity is
attained. Nevertheless, there was no quenching with any other metal
ions,asshowninFig. 6a.Thisselectivityisduetothefactthatalthough
transition metals do not differ too much in size, but they can establish
coordinative interactions at very different energies, which can be
used for discriminative purposes, especially for fluorescent sensing.20
Thisphenomenonisconsistentwithcopperthatoccurshighestonthe
IrvingeWilliams series.21 Copper(II) has a particularly high thermo-
dynamic affinity for the typical eONOe donor, i.e., the imino nitrogen
of the thiazole ring, carbonyl group of the coumarin moiety, and
a strong tendency to promote deprotonation of phenolic proton
during complex formation,9 with fast metal-to-ligand binding ki-
netics that are not possible with the other transition metal ions.
For the biological application of chemosensors, the sensing
should operate in a wide range of pH. So the effects of pH on the
absorbance and emission intensities of 3 and 4 in the absence and
presence of Cu2þ were investigated in the pH range from 2.0 to 12.0
(Fig. 8). Decreasing the pH decreases the absorbance, of 3 at 346 nm
and 4 at 412 nm, as the protonation of the thiazole and coumarin
The effect of pH on the Cu2þ complex of 3 and 4 shows that
increase in pH increases the amount of quenching, reaching its
maximum at pH 6.0 after which it displayed no pH-sensitivity till
pH 9.0. With further increase of pH, the fluorescence intensity of 3,
4 and their Cu2þ complexes become closer, probably because the
formation of hydroxo-complex of Cu2þ is favored in this condition
(Figs. S4b, S5b).19f Thus chemosensors 3 and 4 displayed virtually
(a)
(b)
Fig. 8. Effect of pH on the (a) absorbance and (b) emission intensities of 3, 4 and their Cu2þ complexes in H2OeDMSO (3:1) containing HEPES buffer (10 mM). (lex¼361 nm for 3 and
435 nm for 4).

A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
2799
no physiological pH-sensitivity and fluorescence oneoff can be
controlled by Cu2þ ion binding within the pH range of 6.0e9.0.
In contrast to 4, compound 5, which has no hydroxyl group in
the phenyl ring, did not reveal any significant changes in absorption
and fluorescence emission upon addition up to 10 equiv of Cu2þ
ions, as shown in Fig. 9, which indicates the importance of a phe-
nolic group as a binding site at the 2 position of the thiazole ring.
electrons are distributed over the phenol, thiazole, and coumarin
ring. On the other hand the LUMO of 4 shows the density of electrons
over the coumarin and thiazole ring, which indicates charge or
electron transfer from the phenolic group to a large extent accom-
panied by charge transfer from the diethylamino group of the cou-
marin resulting in a peak at 495 nm. On complexation with copper,
the SOMO of 4eCu2þ shows the electron density transferredover the
Fig. 9. UVevis spectra of 4 ( ) and 5 ( ) (a) in the absence, (b) in presence of 10 equiv of Cu(ClO )2 in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4). Fluorescence
––
––
4
spectra of 4 ( ) and 5 ( ) (c) in the absence and (d) in the presence of 10 equiv of Cu(ClO ) in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4).
––
––
4
2
Competitive binding experiments with different metal ions
(10.0 equiv) and Cu2þ ion (1.0 equiv) as shown in Fig. 10 showed
that they did not interfere with the quenching of Cu2þ by 4. It was
Cu2þ ion (Fig. 11). Thus, localization of SOMO over the Cu2þ ion
causes the fluorescence quenching of 4 upon addition of Cu
2þ 6b The
.
contributions of each electronic oscillator to the lowest energy
transition (from the ground state to the first excited state) are listed
in Table 2. The electronic oscillators of 4eCu2þ corresponding to the
HOMO/SOMO transitions were calculated to be 54%, which is
significantly higher and at such a density change may cause the
complex to quench the fluorescence. Thus, the fluorescence
also found that 4 shows 0.04 mM of detection limit able to suffi-
ciently sense the Cu2þ concentration in the blood system and in
drinking water (Fig. S6).22
An NMR study for complexation was not available due to the
paramagnetic property of Cu2þ. Instead, the complex of 4 was
prepared with Cu2þ in ethanoleDMSO (9:1) and characterized by
HR-FAB mass (Fig. S7). The mass spectra of 4eCu2þ shows a 1:1
stoichiometry (between 4 and Cu2þ) with the molecular ion peak at
m/z 532.0557, which corresponds to (4þCu2þþDMSOeH)þ. EPR
spectroscopic study of the 4eCu2þ complex showed that the copper
retains its oxidation state of 2þ during complexation (Fig. S8).
To examine the reversibility of 4 toward Cu2þ, an aqueous-DMSO
quenching behavior is mainly due to the HOMO/SOMO of 4eCu2þ
,
which evidently show the photoinduced electron transfer from the
thiazole, phenolic, and coumarin ring to the Cu2þ ion.
Similar charge transfer from the phenol and thiazole to the
coumarin moiety causes fluorescence in 3 and HOMO/SOMO
transition in 3eCu2þ also caused the partial fluorescence quenching
(Fig. S9). For this purpose, the TD-B3LYP/6-31G(d) calculated os-
cillator strength f1 to the lowest energy transition, are listed in Table
3 that shows that the oscillator strength is at maximum in the case
of 4 (f¼0.345), which becomes almost zero (f¼0.0001) in the lowest
energy transition of 4 upon complexation with Cuþ2 that implies
the complete quenching in 4eCuþ2 (Table 3). While in the case of 3,
the fluorescence intensity being less than 4, the oscillator strength
f1 is sevenfold less (f¼0.050) as compared to 4 and in the 3eCu2þ
complex the oscillator strength is fourfold excess (f¼0.0004) than 4
resulting in partial quenching.
(3:1) solution of EDTA (2.0
m
M) was added to the complexed solution
of 4 (0.2
m
M) and Cu2þ (2.0
mM). As expected, an absorption peak at
412 nm and a fluorescence signal with a maximum at 495 nm were
completely recovered, demonstrating the binding is chemically re-
versible and not a cation-catalyzed reaction (Scheme 2).
To understand the fluorescence quenching behavior of 3 and 4
upon addition of Cu2þ the frontier molecularorbitals [HOMO, LUMO,
and SOMO (singly occupied molecular orbital)] of 3, 4, 3eCu2þ, and
4eCu2þ were calculated. For compound 4, the HOMO shows that the

2800
A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
400
350
300
250
200
150
100
50
DMSO of analytical grade was purchased from Merck. Deionized
water (double distilled) was used throughout the experiment as an
aqueous layer. All other materials for synthesis were purchased
from Aldrich Chemical Co. and used without further purification.
Compounds 7 and 9 were obtained from Aldrich Chemicals, 2-
hydroxythiobenzamide (6),12 and 3-(2-bromoacetyl)-7-dieth-
ylaminocoumarin (8)14 were prepared by the procedure in the lit-
erature. 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, KOH (1 N), and
HCl (1 N) (all of analytical grade) under adjustment by a pH meter.
Quantum yield (
F) were calculated according to the literature
procedure.23
Density functional optimizations were carried out using Gamess.24
The geometries of each of the compounds were optimized using
density functional theory (DFT) with B3-LYP functional25 at the 6-31G
(d,p) basis set. The excited state electronic transition calculations were
done using time-dependent density functional theory (TD-DFT)26
with the same functional and basis set using a suite of Gaussian 03
programs.
0
a
b c d e
f
g
h
i
j
k
l m n o p
Fig. 10. Metal ion selectivity of (4): bars indicate the fluorescence intensity (435 nm
excitation, 495 nm emission). Perchlorate salts of various metal ions (10.0 equiv) were
added to 4 (0.2
m
M) and Cu2þ (1.0 equiv) (a) 4 only, (b) AgþþCu2þ, (c) Pb2þþCu2þ, (d)
Ca2þþCu2þ, (e) Ni2þþCu2þ, (f) Co2þþCu2þ, (g) KþþCu2þ, (h) Zn2þþCu2þ, (i) Al3þþCu2þ
,
(j) Fe2þþCu2þ, (k) NaþþCu2þ, (l) Cd2þþCu2þ, (m) Mg2þþCu2þ, (n) Hg2þþCu2þ, (o)
CsþþCu2þ, (p) RbþþCu2þ in H2OeDMSO (3:1) containing HEPES buffer (10 mM, pH 7.4).
4.2. Calculation of binding constant
The binding constant (Ka) value of 3 and 4 with Cu2þ was de-
termined from the emission intensity data following the steady-
Scheme 2.
3. Conclusion
state fluorometric method15 in which I0 refer to the intensities of
fluorescence of the solutions containing free ligand. From the fluo-
rescence titration when I0/(IꢁI0) is plotted against the reciprocal of
the cation concentration (1/[M]) the stability constant is obtained
from the ratio intercept/slope with a good linear correlation co-
efficient (R¼0.98647 for 3 and 0.99027 for 4) as shown in Fig. 7.
In summary, we have developed a new thiazole-coumarin based
highly selective, sensitive, reversible, chromogenic, and fluorogenic
chemosensor 4 for copper ions in aqueous-DMSO (3:1) containing
HEPES buffer (10 mM, pH7.4). Itutilizes strongcoordination ofcopper
cations on the phenolic oxygen, thiazole nitrogen, and carbonyl ox-
ygen atom in coumarin. Introduction of an electron-donating dieth-
ylamino grouptothe coumarinringenhanced theICT. Chemosensor 4
also exhibits fluorescence quenching upon binding of Cu2þ ion with
an ‘‘oneoff’ type fluoroionophoric switching property. Its fluorescent
signal can be revived by the addition of an EDTA solution. Finally, we
have also elaborated on the mechanism of fluorescence quenching in
terms of the occupancy of the frontier orbitals.
4.3. Calculation of detection limit
The fluorescence spectra of the chemosensor 4 in various con-
centrations of free Cu2þ were normalized between the maximum
and the minimum intensities as shown in Fig S6. A linear regression
curve was fitted to the eight intermediate values. The point at
which this line crossed the ordinate axis was taken as the detection
limit and equaled approximately 0.04 m .
M of Cu2þ 22
4. Experimental section
4.4. Synthesis of chemosensor
4.1. General methods
4.4.1. Chemosensor 3. A solution of 2-hydroxythiobenzamide (6,
200 mg, 1.48 mmol) and 3-(20-bromoacetyl)coumarin (7, 470 mg,
1.78 mmol) in ethanol (15 mL) was refluxed for 2 h. The reaction
progress was monitored by TLC analysis. The solvent was removed
in vacuo and the residue was washed with water, neutralized with
1 N NaOH and extracted with EtOAc. The organic layer was dried
over anhydrous Na2SO4 and concentrated. The residue was purified
by column chromatography (10% EtOAc in hexane) to give 3 in 90%
Melting points were determined using a Thomas-Hoover cap-
illary melting point apparatus and are uncorrected. The 1H and 13
C
NMR spectra were recorded on a Bruker AM-400 spectrometer
using Me4Si as the internal standard. FAB mass and EPR were taken
at KBSI Daegu branch. The UVevis absorption spectra were de-
termined on a Shimadzu UV-1650PC spectrophotometer. Fluores-
cence spectra were measured on a Shimadzu RF-5301 fluorescence
spectrometer equipped with a xenon discharge lamp and 1 cm
quartz cells. All of the measurements were operated at 298 K.
yield. Mp 142e143 ꢃC. 1H NMR (DMSO-d6)
d
6.98 (t, J¼7.6 Hz, 1H),
7.04 (d, J¼8.4 Hz, 1H), 7.42e7.30 (m, 3H), 7.59 (t, J¼8.4 Hz, 1H), 7.90
(d, J¼8.0 Hz, 1H), 8.32 (dd, J¼8.0, 1.5 Hz, 1H), 8.40 (s, 1H), 8.90

A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
2801
Fig. 11. B3LYP/6-31G .
calculated molecular orbitals of 4 and 4eCu2þ
*
96.0, 108.0, 109.4, 112.8, 116.6, 116.9, 119.0, 119.5, 127.7, 130.1, 131.1,
140.2, 147.4, 150.7, 155.2, 155.6, 159.9, 162.4. Anal. Calcd for
C22H20N2O3S: C, 67.33; H, 5.14; N, 7.14; S, 8.17. Found C, 67.04; H,
5.19; N, 7.15; S, 7.90.
Table 2
The contributions of each electronic oscillator (orbital transitions) to the lowest
energy transition
Electronic oscillators
4 (%)
Electronic oscillators
4eCu2þ (%)
H-1/L
H/L
22.70
62.41
4.96
H-16/S
H-8/S
H-1/S
H/S
7.41
19.75
19.14
53.70
4.4.3. Chemosensor 5. This compound was obtained by the same
procedure as above using thiobenzamide (9, 200 mg, 1.4 mmol) and
8 (530 mg, 1.6 mmol) in ethanol (15 mL). The product was purified
by column chromatography (10% EtOAc in hexane) to give 5 in 68%
H-1/Lþ1
H/Lþ1
9.93
yield. Mp 155e156 ꢃC. 1H NMR (CDCl3)
d
1.22 (t, J¼7.0 Hz, 6H), 3.41
Table 3
(q, J¼7.1 Hz, 4H), 6.53 (s, 1H), 6.63 (d, J¼8.0 Hz, 1H), 7.42 (d,
J¼8.8 Hz, 1H), 7.45 (t, J¼7.3 Hz, 3H), 8.04 (d, J¼8.0 Hz, 2H), 8.29 (s,
The TD-B3LYP/6-31G(d) calculated oscillator strength f1 to the lowest energy
transition
1H), 8.73 (s, 1H); 13C NMR (CDCl3)
d 12.5, 44.9, 97.1, 109.0, 109.4,
3
3eCu2þ
4
4eCu2þ
114.4, 116.8, 126.6, 128.9, 129.6, 130.0, 133.7, 140.6, 149.9, 150.6,
155.9, 160.9, 166.7. Anal. Calcd for C22H20N2O2S: C, 70.19; H, 5.35; N,
7.44; S, 8.52. Found C, 69.70; H, 5.53; N, 7.39; S, 8.31.
f1
0.050
0.0004
0.345
0.0001
4.4.4. Cu2þe4 complex. A mixture of 4 (100 mg, 0.25 mmol) and Cu
(ClO4)2$6H2O (110 mg, 0.29 mmol) in ethanoleDMSO (v/v 9:1,
5 mL) was refluxed for 6 h. The mixture was cooled to room tem-
perature and the precipitated complex was filtered. The filtered
cake was washed thoroughly with water, ethanol, and dried under
vacuum to provide the complex (100 mg, 74% yield). HR-FAB mass:
calcd for (C22H19O3N2S$Cu$DMSO) 532.0552; found: 532.0557.
(s, 1H), 11.17 (s, 1H, OH); 13C NMR (DMSO-d6)
119.2, 119.4, 120.1, 120.2, 124.7, 127.8, 128.9, 131.2, 131.8, 139.3, 146.1,
152.5, 155.2, 158.9, 162.6. Anal. Calcd for C18H11NO3S: C, 67.28; H,
3.45; N, 4.36; S, 9.98. Found C, 67.24; H, 3.33; N, 4.35; S, 9.58.
d
115.8, 116.5, 118.9,
4.4.2. Chemosensor 4. This compound was obtained by the same
procedure as above using 2-hydroxythiobenzamide (6, 200 mg,
1.3 mmol) and 3-(20-bromoacetyl)-7-diethylaminocoumarin (8,
530 mg, 1.6 mmol) in ethanol (15 mL). The product was purified by
column chromatography (25% EtOAc in hexane) to give 4 in 68%
Acknowledgements
yield. Mp 164e165 ꢃC. 1H NMR (DMSO-d6)
d
1.12 (t, J¼7.0 Hz, 6H),
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). The Research Institute of Industrial Technology at
Kyungpook National University also supported A.H.
3.43 (q, J¼7.1 Hz, 4H), 6.56 (d, J¼2.0 Hz, 1H), 6.73 (dd, J¼8.8, 2.2 Hz,
1H), 6.99 (t, J¼7.8 Hz, 1H), 7.05 (d, J¼7.8 Hz, 1H), 7.33 (dt, J¼8.3,
1.8 Hz, 1H), 7.64 (d, J¼9.1 Hz, 1H), 8.23 (s, 1H), 8.29 (dd, J¼7.8, 1.7 Hz,
1H), 8.75 (s, 1H), 11.22 (s, 1H, OH); 13C NMR (DMSO-d6)
d 12.4, 44.1,

2802
A. Helal et al. / Tetrahedron 67 (2011) 2794e2802
8. (a) Leray, I.; Asfari, Z.; Vicens, J.; Valeur, B J. Chem. Soc., Perkin Trans. 2 2002,
Supplementary data
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H. S.; Yoon, J.; Lee, C.-H.; Kim, J. S J. Org. Chem. 2007, 72, 4242.
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2011.01.093. These data include
MOL files and InChiKeys of the most important compounds
described in this article.
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