Rhodamine-based bis-sulfonamide as a sensing probe for Cu2+ and Hg2+ ions

Article abstract of DOI:10.1039/c2nj40391a

A new rhodamine-based bis-sulfonamide 1 has been designed and synthesized. The receptor selectively recognizes Cu²⁺ and Hg²⁺ ions in CH₃CN-water (4/1, v/v; 10 μM tris HCl buffer, pH 6.8) by showing different emission characteristics and color changes. While Cu²⁺ is sensed through increase in emission, Hg²⁺ is detected by a weak ratiometric change in emission of 1. The receptor shows in vitro detection of both the ions in human cervical cancer (HeLa) cells.

Full text of DOI:10.1039/c2nj40391a

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PAPER
2+
Rhodamine-based bis-sulfonamide as a sensing probe for Cu and
2+
Hg ionsw
a
a
b
Kumaresh Ghosh,* Tanmay Sarkar, Asmita Samadder and
b
Anisur Rahman Khuda-Bukhsh
Received (in Gainesville, FL, USA) 14th May 2012, Accepted 25th July 2012
DOI: 10.1039/c2nj40391a
A new rhodamine-based bis-sulfonamide 1 has been designed and synthesized. The receptor
2
+
2+
selectively recognizes Cu and Hg ions in CH CN–water (4/1, v/v; 10 mM tris HCl buffer,
3
2
pH 6.8) by showing different emission characteristics and color changes. While Cu is sensed
+
2
+
through increase in emission, Hg is detected by a weak ratiometric change in emission of 1.
The receptor shows in vitro detection of both the ions in human cervical cancer (HeLa) cells.
a single wavelength. A dual emission system can minimize the
Introduction
measurement errors because of the factors such as photo transfor-
11
mation, receptor concentrations, and environmental effects.
Design and synthesis of optical chemosensors for the selective
1
recognition of metal ions has attracted a great deal of attention.
6j,9e,12
During the course of our work on sensing of cations
13
and anions of biological significance, we report in this full account
Of particular interest is the development of fluorescent sensors
2+
2+
for heavy transition metal ions such as Hg and Cu , due to
a new rhodamine-based bis sulfonamide which recognizes both
2
their biological and environmental importance. Among the
2+
2+
Cu and Hg ions by exhibiting different emission characteristics
in a semi-aqueous system [CH CN–water (4/1, v/v; 10 mM tris HCl
buffer; pH 6.8)]. In our recent publication, we have shown that
various transition metal ions, the copper ion causes significant
environmental pollution and also serves as a catalytic cofactor
3
for a variety of metalloenzymes. However, exposure to a high
3
9e
2+
2+
Hg ions over a series of ions (even Cu ions) can be selectively
detected by a rhodamine labeled tripodal receptor. Manipulation of
functional groups and their placement under different spacers
sometimes lead to a variety of new structures that show different
recognition features. In relation to this, the present design in
level of copper, even for a short period of time, can cause
gastrointestinal disturbance, while long-term exposure can cause
4
2+
liver or kidney damage. Similarly, the mercury ion (Hg ) is
considered to be dangerous as it can accumulate in the human
body and cause a wide variety of diseases even at a low
concentration, such as prenatal brain damage, serious cognitive
2+
2+
this account represents an example where Hg
and Cu
ions are simultaneously detected in a semi-aqueous system
both colorimetrically and fluorometrically.
5
disorders, and Minamata disease. Therefore, it is of utmost
interest to develop highly sensitive and selective assays for both
2+
2+
Cu and Hg ions. In recent years, considerable efforts have
been made so far to establish fluorescent chemosensors of different
2
+
2+
architectures for the selective sensing of Cu and Hg ions. In
6,7
2+
this regard, most of the probes for Cu
show fluorescence
quenching and the fluorescent probes which exhibit turn-on
8
response are less in number. On the other hand, the ratiometric
2+
9
probes for the detection of Hg ions are considerably important
and they are relatively less in number than the ‘turn-on’ based
1
0
Rhodamine B and its derivatives (RhB), with their good
photo stability and high fluorescence quantum yield, act as
chemosensors towards metal ions by exhibiting switching
between the spirocyclic form (which is colorless and non-
fluorescent) and the ring-opened amide form, which is pink
receptors. The ratiometric chemosensors give advantages
over the conventional monitoring of fluorescence intensity at
a
Department of Chemistry, University of Kalyani, Kalyani-741235,
India. E-mail: ghosh_k2003@yahoo.co.in; Fax: +91 3325828282;
Tel: +91 3325828750
Department of Zoology, University of Kalyani, Kalyani-741235,
9
,10
and strongly fluorescent.
14a
A closer look into the literature reveals that although a
b
India
w Electronic supplementary information (ESI) available: Figures
showing the change in fluorescence and UV-vis titrations of receptor
1 with various metal ions, Job plot, reversibility test, detection limit,
spectral data for 1 and 3. See DOI: 10.1039/c2nj40391a
number of rhodamine labeled receptors are known for sensing
2+
different metal ions, the simultaneous detection of both Cu
2
and Hg ions by a rhodamine-labeled receptor module is less
+
1
4b–f
in number.
In addition to the few examples on this
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New J. Chem., 2012, 36, 2121–2127 2121

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1
4b–f
aspect,
the present simple structure is a new addendum as
2
a dual probe for sensing both Cu and Hg ions.
+
2+
Results and discussion
The receptor 1 was obtained according to Scheme 1. Coupling of
benzene-1,3-disulfonyl dichloride with the rhodamine labelled
14c
amine 2 afforded the desired compound 1 in good yield.
ꢁ5
Fig. 2 Change in fluorescence ratio of 1 (c = 2.14 ꢀ 10 M) at 580 nm
upon addition of 20 equiv. of cations.
Scheme 1 (i) EtOH, reflux, 9 h; (ii) benzene-1,3-disulfonyl dichloride,
CH Cl , Et N, 10 h.
2
2
3
The metal ion binding properties of 1 towards metal ions
2+
2
+
2+
2+
2+
2+
2+
2+
such as Hg , Cu , Cd , Fe , Mg , Co , Ni , Zn
,
+
2+
Ag and Pb (taken as their perchlorate salts) were investi-
gated in CH CN–H O solution (CH CN : H O = 4 : 1, v/v;
0 mM tris HCl buffer, pH 6.8). Without cations, 1 is almost
3
2
3
2
1
non-fluorescent. However, on excitation at 510 nm, a non-
structured emission at 524 nm increased to a significant extent
2
+
upon interaction with only Cu
ions over the other metal
ions studied. Fig. 1 shows the plot of change in emission of 1 at
the monomer emission (524 nm) in the presence of 20 equiv. of
the different metal ions. It is evident from Fig. 1 that the
ꢁ
5
Fig. 3 Fluorescence titration spectra of 1 (c = 2.14 ꢀ 10 M) in
CH CN–water (4/1, v/v; 10 mM tris HCl buffer, pH 6.8) upon addition
3
2+
ꢁ4
2
+
of Cu (c = 4.18 ꢀ 10 M) (lex = 490 nm); inset: colour change of
receptor is much selective to Cu
ions. Other metal ions
weakly perturbed the emission of 1 at this
wavelength. On progression of titration of 1 with the metal
the receptor solution under illumination of UV light.
2
+
except Hg
2
+
2+
ions, it is observed that only in the presence of Cu and Hg
ions a new peak at 580 nm appears with significant intensity.
Fig. 2, in this regard, shows the change in fluorescence ratio
of 1 at 580 nm in the presence of 20 equiv. of the different
metal ions. As can be seen from Fig. 2, the receptor is more
2
+
2+
sensitive to Cu than to Hg ions. Fig. 3 shows the emission
+
2
titration spectra with Cu
ions and also the associated
change in colour under illumination of UV light. Fig. 4, under
identical conditions, describes the emission titration spectra
obtained from the gradual addition of Hg(ClO
4
)
2
solution to
O (4/1, v/v;
0 mM tris HCl buffer; pH 6.8). In this case, while the emission
ꢁ5
the solution of 1 (c = 2.14 ꢀ 10 M) in CH
3 2
CN–H
1
ꢁ5
Fig. 4 Fluorescence titration spectra of 1 (c = 2.14 ꢀ 10 M) in
1
4g
centered at 524 nm decreases to a lesser extent, a new band
CH
3
CN–water (4/1, v/v; 10 mM tris HCl buffer, pH 6.8) upon addition
2+
ꢁ4
at 580 nm begins to appear on progression of the titration of 1
of Hg
(c = 4.18 ꢀ 10 M); inset: colour change of the receptor
solution under illumination of UV light.
2
+
with Hg
and results in a weak ratiometric change. More-
over, in the titration experiment the colour of the solution of 1
2
+
in the presence of Hg
was found to be pinkish yellow on
illumination of UV light (inset of Fig. 4). This is in contrast to the
2
+
2+
case of Cu ions (inset of Fig. 3). Thus, these two ions (Cu and
2+
Hg ) are easily distinguishable from each other. It is further
mentionable that under identical conditions they are distinguishable
from the rest of the other ions examined as the peak at 580 nm was
not observed during the course of titration (ESIw).
2
+
The pronounced OFF–ON type of Cu
selectivity was
ꢁ5
Fig. 1 Change in fluorescence ratio of 1 (c = 2.14 ꢀ 10 M) at 524 nm
further established from fluorescence at 524 nm. Fig. 5, in this
2
+
upon addition of 20 equiv. of cations.
regard, describes the Cu
ion binding induced change in
2
122 New J. Chem., 2012, 36, 2121–2127
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2+
2+
Fig. 7 Suggested modes of interaction of 1 with Cu /Hg ions in
solution.
amide ions to chelate the metal ions strongly (Form 1B in
Fig. 7) for which a new peak at 580 nm begins to appear with
significant growth on progression of the titration. We further
ꢁ
5
2+
2+
2+
Fig. 5 Fluorescent response of sensor 1 (c =2.14 ꢀ 10 M) to Cu
presume that in contrast to Cu
binding, Hg
ions are
ꢁ4
ꢁ4
(
c = 4.18 ꢀ 10 M) over the selected metal ions (c = 4.18 ꢀ 10 M).
initially involved in weak binding with the bis-sulfonamide
moieties due to which change in emission of 1 at 524 nm is less.
The rest of the part related to the spirolactam ring opening is
emission of 1 in the presence and absence of 5 equiv. of other
metal ions. As can be seen from Fig. 5, the interference of the
metal ions considered in the present study is established to be
2
+
similar to the case of Cu and thus the fluorescence intensity
at 580 nm increases although by a lesser extent. To resolve the
issue on the binding of metal ions initially at the disulfonamide
core of 1 followed by the spirolactam ring, we studied the
interaction by considering a model compound 3 where
the rhodamine part is absent (see ESIw). It is evident from
1
5
negligible. The stoichiometries of the complexes of 1 with
2+
2
+
both Cu
and Hg
ions were determined to be 1 : 1 and
1
the binding constant values (K
6
a
) were found to be (9.05 ꢂ
4
ꢁ1
4 ꢁ1 2+
0
.63) ꢀ 10 M and (7.87 ꢂ 0.55) ꢀ 10 M for Cu and
2+
Hg , respectively. The values are close in magnitude and the
2+
Fig. S9a and S10 (ESIw) that upon addition of Cu ions to
the solution of 3 in CH CN–H O (CH CN : H O = 4 : 1, v/v;
2+
small increase in K for Cu is ascribed to its strong chelation
a
3
2
3
2
at the binding core of 1. As representative, Fig. 6a shows the
2
10 mM tris HCl buffer, pH 6.8), both the absorbance and
emission underwent significant changes. This is in contrast to
+
Job plot for the Cu
complex of 1 and Fig. 6b shows the
2
+
binding constant curve obtained from non-linear curve fitting
of the emission titration data. Due to minor changes in
emission we were unable to determine the binding constant
values for other metal ions.
the case with Hg ions (Fig. S9b and S11, ESIw) where
complexation induced changes in absorbance and emission
are less. Therefore, the appearance of the more intense emission
2
+
peak at 524 nm for 1 while titration with Cu ions is attributed
2+
2+
The selective fluorescence response of 1 towards Cu ions in
to the greater interaction of Cu
ions at the disulfonamide
the study is explained as due to their best fit in the bis-sulfonamide
core. The intensity of this peak is furthermore sensitive to the
polarity of the solvent. While the solution of Cu-complex of 1 in
CH CN was diluted with CH CN containing 25, 30, 40, 55 and
2+
cleft of 1. The different mode of Cu -induced change in emission
2+
of 1 from that of Hg binding is presumably attributed to the
3
3
involvement of the binding centre in different ways. To our belief,
2+
Cu
65% water, the peak at 524 nm gradually became intensified
(see Fig. S16, ESIw). These findings demonstrate the case of
charge transfer associated with the sulfonamide motif linked to
ions primarily interact with sulfonamide functionalities
involving deprotonation of –NH protons (see Form 1A in
Fig. 7). As a consequence of this interaction the emission
intensity at 524 nm gradually increases. After fixation of the
metal ions by the sulfonamides, the closely spaced spirocyclic
forms of the rhodamine parts are opened which provides
2
+
1f
the Cu
ions. To support the binding mechanism, we
1
recorded the FTIR and H NMR of 1 in the presence and
absence of equiv. amounts of the metal salts. The formation of
1B through the ring opening was established from both the
1
H NMR and FTIR studies. In FTIR, the amide carbonyl
ꢁ
1
stretching of the spirolactam part that appeared at 1673 cm
ꢁ
1
was changed to a lower wavenumber 1671 cm and 1647 cm
ꢁ1
2
+
2+
in the presence of Hg
and Cu
ions, respectively. The
stretching frequency for the –SO – group that appeared at
2
ꢁ
1
468 cm in 1 was found to be unchanged during interaction
1
2
with Hg ions. In the case of interaction with Cu ions a
+
2+
ꢁ1
weak stretching band at 1466 cm was noted and these results
2
thus intimate that the –SO – motifs do not take part in the
metal complexation (ESIw).
In H NMR, the –NH protons that appeared at 7.32 ppm
1
2+
were invisible in the presence of 1 equiv. of Cu and Hg
2+
2
+
3
Fig. 6 (a) Fluorescence Job plot for 1 with Cu in CH CN–water
ꢁ5
ions. The ring protons (e, f and g types; see the labeling of 1 in
(
4/1, v/v; 10 mM tris HCl buffer; pH 6.8) ([H] = [G] = 5 ꢀ 10 M);
Fig. S12, ESIw) of the rhodamine part moved to the downfield
(
b) nonlinear curve fitting of the fluorescence titration data for 1 (c =
5
ꢁ
2+
2+
2
.14 ꢀ 10 M) with Cu in CH
3
CN–water (4/1, v/v; 10 mM tris HCl
e
directions in the presence of 1 equiv. of Cu ions (Dd = 0.04,
buffer; pH = 6.8).
Dd = 0.01 and Dd = 0.06). Similar observation was found in
f
g
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New J. Chem., 2012, 36, 2121–2127 2123

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2+
the presence of 1 equiv. of Hg ions i.e. ring protons shifted
downfield (Dd = 0.02, Dd = 0.07 and Dd = 0.03) with
e
f
g
significant broadening. Fig. S12 (ESIw), in this regard, shows
the change in H NMR of 1. The downfield shifting of the ‘e’,
1
‘
f’ and ‘g’ type protons indicated ring opening of the spiro-
lactam part in 1. During interaction while the ‘h’ type proton
8.23 ppm) showed a slight upfield shift of 0.05 ppm in the
(
2
+
presence of Hg , it remained positionally unaltered in the
2
presence of Cu
+
ions. Such negligible or no change in
chemical shift of the ‘h’ type proton indicates that the metal
ion is more closely spaced to the spirolactam moiety of
rhodamine rather than the disulfonamide core owing to
charge–charge interaction.
ꢁ
5
Fig. 9 Absorption titration spectra of 1 (c = 2.14 ꢀ 10 M) in
The ring opening in 1 to form the metal chelated species of
CH
3
CN–H
2+
2
O (4/1, v/v; 10 mM tris HCl buffer; pH 6.8) upon addition
1
type 1B was further confirmed by C NMR. Fig. 8 represents
3
ꢁ4
of Cu (c = 4.18 ꢀ 10 M).
1
the change in C NMR of 1 in the presence of 1 equiv. of each
3
2
+
2+
of Cu
5.6 ppm for the tertiary carbon of the spirolactam ring of 1
labeled ‘i’) is in favor of the opening of the spirolactam ring to
introduce the form 1B.
and Hg
ions. The disappearance of the signal at
6
(
2
+
As shown in Fig. 9, without Cu ions, 1 scarcely shows an
absorption at 555 nm, indicating that 1 exists as spirolactam
2+
2+
form. Addition of Cu (Fig. 9) and Hg (Fig. 10) separately to
ꢁ
5
the solution of 1 (c = 2.14 ꢀ 10 M) in CH
3 2
CN–H O (4/1, v/v;
1
0 mM tris HCl buffer; pH 6.8) brought about strong absorption
at 555 nm along with clear colour change from colourless to
pink, as is normally noticed for rhodamine-based probes. The
appearance of pink color is attributed to the opening of the
spirolactam rings and generation of the delocalized xanthene
ꢁ
5
1
4a
Fig. 10 Absorption titration spectra of 1 (c = 2.14 ꢀ 10 M) in
moieties.
This was not observed when the titrations were
CH
3
CN–H
2
O (4/1, v/v; 10 mM tris HCl buffer; pH 6.8) upon addition
conducted with other metal ions (ESIw). The stoichiometries of
both Cu- and Hg-complexes in the ground state were established
to be 1 : 1 (ESIw).
2+
ꢁ4
of Hg (c = 4.18 ꢀ 10 M).
Careful scrutiny shows that the intensity of the pink colors
the interference of the nitrate ion at the bis-sulfonamide
binding core of 1.
2
+
2+
arising from the addition of Cu
different. It is mentionable that Cu(NO ) instead of Cu(ClO )
4 2
and Hg
ions is little
In order to be confirmed with the reversible nature of the
complexation, fluorescence and absorption spectra of the
copper and mercury complexes of 1 in CH CN–H O (4/1, v/v;
3
2
opened the spirolactam ring weakly as confirmed from the
appearance of the faint pink colour of the solution as well as
a weakly intense emission peak at 580 nm (ESIw). A similar
3
2
10 mM tris HCl buffer; pH 6.8) were observed upon addition
of KI. Addition of KI did not bring the reverse change in
both emission and absorption spectra. The pink color of
the solution did not vanish. Similar experiments were also
performed with addition of EDTA solution in large excess to
situation was observed with the addition of Hg(NO
3 2
) to
the solution of 1 (ESIw). This is assumed to be due to
2
+
2+
the solutions of the complexes of 1 with Cu and Hg ions
but we failed to prove the reversibility. Only in the presence of
ethylene diamine the pink colour of the mercury complex of 1
was discharged and both the absorption and emission spectra
of the free receptor were retrieved. In the case of the copper
complex, the pink colour turned into faint blue color upon
addition of ethylene diamine. Interestingly, while the emission
spectrum for the free receptor was retrieved in this case, the
absorption spectra appeared to be different. This occurred
due to the simultaneous formation of the blue colored
copper–ethylene diamine complex in solution (ESIw).
To determine the detection limit of the receptor 1 towards
2
+
2+
and Hg
both Cu
ions, emission titrations of 1 were
conducted upon addition of the said metal ions in different
concentrations. In regard to this, Fig. 11 represents the change
in emission spectra of 1 as well as the associated colour change
13
2+
2+
Fig. 8
C NMR (CDCl
3
, 100 MHz) of (a) 1, (b) 1ꢃCu and (c) 1ꢃHg
.
2
124 New J. Chem., 2012, 36, 2121–2127
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Fig. 12 Fluorescence and bright field images of HELA cell lines: (a)
bright field image of normal cells, (b) bright field image of cells treated
with probe 1 (10.0 mM) for 1 h at 37 1C, (c) fluorescence image of cells
treated with probe 1 (10 mM) for 1 h at 37 1C, (d) fluorescence image of
ꢁ
5
Fig. 11 Change in fluorescence spectra of 1 (c = 2.14 ꢀ 10 M) in
CH CN–H O (4/1, v/v; 10 mM tris HCl buffer; pH 6.8) upon addition
of (a) Cu (c = 4.18 ꢀ 10 M), (b) Cu (c = 4.18 ꢀ 10 M);
3
2
+
2
ꢁ4
2+
ꢁ5
cells treated with Hg(ClO
cells treated with Cu(ClO
images of cells upon treatment with probe 1 (10.0 mM) and then with
Hg(ClO (30.0 mM) for 1 h, (g) red fluorescence images of cells upon
treatment with probe 1 (10.0 mM) and then with Cu(ClO ) (30.0 mM)
4
)
2
(30 mM) for 1 h, (e) fluorescence image of
2+
ꢁ6
4 2
) (30 mM) for 1 h, (f) red fluorescence
(
c) Cu (c = 4.18 ꢀ 10 M). Associated colour changes are shown
in the inset of the figures.
4 2
)
2
+
4
2
of the receptor solution with the variation of Cu
ion
for 1 h (for red fluorescence image: lex = 510 nm), (h) green
fluorescence images of cells upon treatment with probe 1 (10.0 mM)
and then with Hg(ClO ) (30.0 mM) for 1 h, (i) green fluorescence
4 2
concentrations in CH
3 2
CN–H O (4/1, v/v; 10 mM tris HCl
buffer; pH 6.8). The spectral and colour changes of 1 upon the
2
+
addition of Hg
ions of varying concentrations in
images of cells upon treatment with probe 1 (10.0 mM) and then with
Cu(ClO ) (30.0 mM) for 1 h (for green fluorescence image l range:
CH CN–H O (4/1, v/v; 10 mM tris HCl buffer; pH 6.8) can
3
2
4
2
ex
be found in the ESI.w However, in both cases it is observed
that the receptor 1 convincingly reports the detection of both
511 nm to 534 nm and for red fluorescence image lex range: 567 nm
to 617 nm).
2
+
2+
Cu
and Hg
ions up to their concentration ranges of
ꢁ5
B10 M through colour change. Thus the detection limit
of receptor 1 in the present case is observed to be B21 mM
which is comparable with the reported values 10–25 mM.
The potential biological application of the receptor was
1
4b–f
2
+
2+
evaluated for in vitro detection of Cu
and Hg
ions in
human cervical cancer (HeLa) cells. The HeLa cells were
incubated with 5 mL of receptor 1 (10 mM in CH CN–H O
3
2
(
4/1, v/v)) in DMEM (Dulbecco’s modified Eagle’s medium)
(without FBS) for 30 min at 37 1C and washed with phosphate
buffered saline (PBS) buffer (pH = 7.4) to remove the excess of
receptor 1. DMEM (without FBS) was again added to the cells.
4 2
The cells were next treated with 5 mL of Cu(ClO ) (30 mM) and
incubated again for 30 min at 37 1C. A control set of cells which
2
+
was devoid of Cu ions was kept. A similar experiment was
done for Hg(ClO . Fig. 12a and b represent the bright field
images of the cells before and after treatment of the cells with 1,
)
4 2
Fig. 13 MTT assay for the receptor 1.
2
+
respectively. Cells incubated with receptor 1 without Hg and
The addition of receptor 1 to the cells did not show any
cytotoxicity as evident from the morphology of the cells
(Fig. 12) as well as from the MTT assay (Fig. 13). The viability
was more than 90%, 90% and 91% for normal, acetonitrile-
and receptor-treated cells respectively. Since the percentage of
viable cells of all the series was above 90% this would suggest
that the receptor was not cytotoxic when exposed to cultured
cells in vitro.
2
Cu (Fig. 12c) and cells incubated with Hg without receptor
+
2+
1
(Fig. 12d) did not show any fluorescence property. This was
2
+
also true when cells were incubated with Cu without receptor
(Fig. 12e). In contrast, cells incubated with the receptor 1 and
1
2
then with Hg ions showed the occurrence of red fluorescence
+
(
Fig. 12f). Again cells incubated with the receptor 1 and then
2
+
with Cu
ions showed the occurrence of red fluorescence
(
Fig. 12g). These facts indicate the permeability of the receptor
2+
2
+
inside the cells and the binding of Hg , Cu ions with the
receptor. Fig. 12h and i show the occurrence of green fluores-
Conclusions
2+
cence upon incubation of receptor 1 and then Hg (Fig. 12h)
2+
and Cu (Fig. 12i) with the probe, respectively.
In conclusion, we have shown that the rhodamine labeled
2+
2+
receptor 1 is capable of detecting Cu
and Hg
ions
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simultaneously in aq. CH
characteristics as well as color. Among few examples
rhodamine labeled receptors for simultaneous detection of
3
CN by exhibiting different emission
volume of 3 mL of the mixture. All the prepared solutions
1
4b–f
on
were kept for 1 h with occasional shaking at room tempera-
ture. Then emission and absorbance of the solutions of
different compositions were recorded. The concentration of
the complex i.e., [HG] was calculated using the equation
[HG] = DI/I ꢀ [H] or [HG]=DA/A ꢀ [H] where DI/I and
2
+
2+
Cu and Hg ions, the present example in this full account
is a new addendum. The dimension of the isophthaloyl cleft
and the cooperative action of the sulfonamides along with the
amide parts of the rhodamines favor the strong chelation of
0
0
0
0
DA/A indicate the relative emission and absorbance intensi-
2
+
2+
Cu and Hg ions over the other cations examined. Further-
ties respectively. [H] corresponds the concentration of the pure
more, the chemosensor 1 is found to be efficient in reporting the
+
H
host. Mole fraction of the host (X ) was plotted against
2
2+
presence of both Cu and Hg ions inside the cell without
showing any cytotoxicity.
concentration of the complex [HG]. In the plot, the mole
fraction of the host at which the concentration of the
host–guest complex [HG] is maximum gives the stoichiometry
of the complex.
Experimental
Method for MTT assay
0
0
2
3
-(Benzyl(2-hydroxyethyl)amino)-N-(2-(3 ,6 bis(diethylamino)-
0
-oxospiro[isoindoline-1,9 -xanthene]-2-yl)ethyl)acetamide (1)
Reagents. MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,S-diphenyl-
tetrazolium bromide] and all other reagents were purchased
from Sigma-Aldrich Inc. (St-Louis, MO, USA); Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum,
penicillin, streptomycin, neomycin (PSN) antibiotics were
purchased from Gibco BRL (Grand Island, NY, USA).
To a stirred solution of benzene-1,3-disulfonyl dichloride
(
0.055 g, 0.197 mmol) in dry CH
2 2
Cl (20 mL), the amine 2
(
0.2 g, 0.412 mmol), which was obtained according to the
14c
reported procedure,
was added dropwise followed by the
3
addition of Et N. Stirring was continued for 12 h. After comple-
tion of reaction, monitored by TLC, solvent was evaporated and
water was added to the residue. The aqueous layer was extracted
with CHCl (25 mL ꢀ 3) and dried over anhydrous Na SO .
Cell culture. Human cervical cancer cell line HeLa was
procured from NCCS Pune, India. The cells were cultured at
5
3
2
4
5 ꢀ 10 cells per mL in DMEM supplemented with 10% fetal
Purification of the crude mass by silica gel column chromato-
graphy using 3% CH OH in CHCl as eluent yielded the desired
bovine serum and 1% PSN antibiotic at 37 1C, 5% CO for
2
3
3
experimental purpose.
compound 1 (0.170 g, 74%), mp 142 1C.
1
Assessment of percentage of viable cells. The percentage
viability of HeLa cells, after being exposed to the receptors,
was evaluated by MTT assay (Mossman, 1983). The cells were
incubated in 96-well microplates for 24 hours along with the
receptors at different concentration. A series of normal cells
H NMR (400 MHz, CDCl
m, 4H), 7.48–7.42 (m, 5H), 7.31 (t, 2H, J = 4 Hz), 7.01 (m, 2H),
.33–6.30 (br s, 6H), 6.19–6.13 (s, 6H, unresolved), 3.33–3.28
m, 16H), 3.13–3.05 (m, 4H), 2.75–2.74 (m, 4H), 1.14 (s, 24H);
3
): d 8.23 (s, 1H), 7.91–7.87
(
6
(
1
3
6 3
C NMR (100 MHz, d -CDCl ): d 169.6, 153.5, 153.1, 148.9,
(
without any exposure) and a series of positive control cells
1
1
41.6, 132.9, 130.3, 130.2, 129.7, 128.3, 128.2, 125.6, 123.8, 122.9,
ꢁ
1
,
(exposed to similar concentrations of acetonitrile, the ‘‘vehicle
solvent’’ of the receptors, as that of the exposure of the
receptors) were taken. The intracellular formazan crystals
formed were solubilized with dimethyl sulfoxide (DMSO) and
the absorbance of the solution was measured at 595 nm by using
a microplate reader (Thermo scientific, Multiskan ELISA, USA).
The calculated cell survival rate = percentage of MTT inhibition
as follows: percentage of survival = (mean experimental absor-
bance/mean control absorbance) ꢀ 100%.
08.3, 104.3, 97.7, 65.6, 44.3, 44.3, 39.9,12.5; FT IR (n in cm
KBr): 3171, 2969, 2928, 1677, 1633, 1546, 1468; MS (FAB) m/z
+ +
171.5 (M + H) , 1170.4 (M ).
1
General procedure for fluorescence and UV-vis titrations
Stock solutions of the receptor were prepared in 4 : 1 (v/v)
CH CN : H O containing 10 mM Tris–HCl buffer (pH = 6.8)
3
2
ꢁ
5
in the concentration range B10 M. 2.5 mL of the receptor
solution was taken in a cuvette. Stock solutions of guests in the
ꢁ
4
concentration range B10 M were prepared in the same
solvents and were individually added in different amounts to
the receptor solution. Upon addition of metal ions, the change
in emission of the receptor was noted. The same stock solutions for
receptor and guests were used to perform the UV-vis titration
experiment. Solution of the metal salts was successively added in
different amounts to the receptor solution (2.5 mL) taken in the
cuvette and the absorption spectra were recorded. Both fluorescence
and UV-vis titration experiments were carried out at 25 1C.
Acknowledgements
We thank DST (for FIST and PURSE programs) and UGC (for
SAP), New Delhi, India, for providing facilities in the department.
TS thanks CSIR, New Delhi, India, for a fellowship.
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New J. Chem., 2012, 36, 2121–2127 2127