Piperazine-based simple structure for selective sensing of Hg2+ and glutathione and construction of a logic circuit mimicking an INHIBIT gate

Article abstract of DOI:10.1039/c3nj00855j

A simple chemosensor (1) has been designed and synthesized. The chemosensor selectively recognizes Hg²⁺ ions in THF-H₂O (3:1, v/v) by showing a significant increase in emission and a bluish color of the solution under exposure to UV light. Change of the fluorophore unit in 1 leads to 2, which also shows selective sensing of Hg²⁺ under similar conditions. Furthermore, while the ensemble of 1 with Hg²⁺ selectively senses reduced glutathione (GSH) over cysteine and homocysteine, the ensemble of 2 with Hg²⁺ has been observed to be inefficient to distinguish glutathione from other biothiols. Thus probe 1 and inputs Hg²⁺ and GSH can be used to develop an INHIBIT logic gate.

Full text of DOI:10.1039/c3nj00855j

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Piperazine-based simple structure for selective sensing
of Hg2+ and glutathione and construction of a logic
circuit mimicking an INHIBIT gate†
Cite this: NewJ.Chem., 2013,
37, 4206
Kumaresh Ghosh,*a Debojyoti Tarafdar,a Asmita Samadderb and
Anisur Rahman Khuda-Bukhshb
A simple chemosensor (1) has been designed and synthesized. The chemosensor selectively recognizes
Hg2+ ions in THF–H2O (3 : 1, v/v) by showing a significant increase in emission and a bluish color of the
solution under exposure to UV light. Change of the fluorophore unit in 1 leads to 2, which also shows
selective sensing of Hg2+ under similar conditions. Furthermore, while the ensemble of 1 with Hg2+
selectively senses reduced glutathione (GSH) over cysteine and homocysteine, the ensemble of 2 with
Hg2+ has been observed to be inefficient to distinguish glutathione from other biothiols. Thus probe 1
and inputs Hg2+ and GSH can be used to develop an INHIBIT logic gate.
Received (in Montpellier, France)
29th July 2013,
Accepted 17th September 2013
DOI: 10.1039/c3nj00855j
www.rsc.org/njc
Introduction
Design and synthesis of simple and easy-to-make optical chemo-
sensors for the selective recognition of toxic metal ions has
drawn much attention.1 Among the different toxic metal ions,
mercury is considered to be dangerous. Its accumulation in a low
concentration in the human body causes a wide variety of
diseases, such as prenatal brain damage, serious cognitive
disorders, and Minamata disease.2 The toxicity of mercury in
living systems is attributed to the measurable affinities of thiol
group containing proteins and enzymes, which result in the
malfunctioning of living cells.3 Therefore, the detection and
sensing of this toxic metal ion is a sensible issue to chemists.
Of the different techniques, fluorescence is of considerable
interest due to its simplicity, high sensitivity and real-time
detection. To date, many fluorescent probes for Hg2+ ions have
been reported.4,5 The majority of the probes for Hg2+ are ‘turn-
off’6 because Hg2+ produces mostly a CHEQ (chelation-enhanced
quenching) effect, presumably because of the large spin–orbit
coupling constant value.7 The design of fluorescence ‘turn-on’
type sensors upon mercury binding is thus a challenging issue
and only a few fluorescence ‘turn-on’ sensors for mercury have
been reported.8
Scheme 1 Structures of 1 and 2.
In this account, we report the simple and easy-to-make PET
(photoinduced electron transfer) systems 1 and 2 (Scheme 1),
which serve dual purposes: (i) the recognition of Hg2+ ions in
THF–H2O (3 : 1, v/v) and (ii) acting as an INHIBIT logic gate in
reporting the selective detection of glutathione over cysteine and
homocysteine. Biothiols such as cysteine, homocysteine and
glutathione play crucial roles in biological systems and therefore,
their recognition and sensing draws attention.9 It is mentionable
that glutathione (GSH) is the most abundant cellular thiol com-
pound that plays a central role in combating oxidative stress in
cells.10 Abnormal levels of GSH are associated with a number of
diseases, including cancer, AIDS, Alzheimer’s and cardiovascular
disease.11 Thus the measurement of GSH in physiological media
has been considered an essential factor in the diagnosis of these
diseases. Several common strategies are known to detect GSH,
such as electrochemical assays,12 high-performance liquid
chromatography (HPLC),13 mass spectrometry,14 fluorescence
spectrometry15 and enzymatic methods.16 Among these reported
detection methods, fluorescent sensing has attracted consider-
able attention due to its simple operation and high sensitivity.
a Department of Chemistry, University of Kalyani, Kalyani-741235, India.
E-mail: ghosh_k2003@yahoo.co.in; Fax: +91 3325828282; Tel: +91 3325828750
b Department of Zoology, University of Kalyani, Kalyani-741235, India
† Electronic supplementary information (ESI) available: Figures showing the
fluorescence and UV-vis titrations of 1 and 2 with various metal ions, Job plot,
binding curves, IR comparison, selectivity test, detection sensitivity, change in
emission of 1 and 2 upon titration with biothiols, MTT assay, 1H NMR, 13C NMR
and mass spectra of 1 and 2. See DOI: 10.1039/c3nj00855j
c
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Results and discussion
Synthesis of chemosensors 1 and 2 was achieved according to
Scheme 2. Piperazine on reaction with chloroacetyl chloride in
the presence of Et3N in dry CH2Cl2 gave dichloroamide 3
(Scheme 2a). On the other hand, the anthracene coupled amine
5, obtained from 9-anthraldehyde via Schiff base formation
followed by reduction with NaBH4 was refluxed with compound
3 in the presence of anhydrous K2CO3 in dry CH3CN to give the
desired compound 1 (Scheme 2b). In a similar way, the pyrene
coupled amine 7 obtained from pyrene aldehyde through a
series of reactions as shown in Scheme 2c was treated with 3 to
afford compound 2. Both compounds 1 and 2 are soluble in
CHCl3, CH2Cl2, DMSO, THF and DMF. They are partially
soluble in CH3CN and CH3OH. All the compounds were char-
acterised spectroscopically.
The metal ion binding properties of 1 towards Hg2+, Cu2+,
Cd2+, Fe2+, Mg2+, Co2+, Ni2+, Zn2+, Ag+ and Pb2+ (taken as their
perchlorate salts; caution: perchlorate salts are sometimes an
explosive hazard17) were initially pursued in THF. First, we
investigated the affinity of chemosensor 1 towards Hg2+ ions by
monitoring the emission upon gradual addition of Hg(ClO4)2 to
the solution of compound 1 (c = 2.5 ꢀ 10ꢁ5 M) in THF. As can be
seen from Fig. 1, upon excitation at 370 nm receptor 1 exhibited
a characteristic weak broad emission centered at 412 nm. While
titrating, the emission at 412 nm increased dramatically upon
the incremental addition of only 1.5 equiv. of Hg2+ ions. During
the titration, the emission intensity at 412 nm progressively
increased with red shift (Dl = 10 nm). Upon interaction with
Hg2+ ions, the solution of 1 turned a bluish color when
observed under exposure to UV irradiation (inset of Fig. 1).
The notable increase in the emission of 1 in the presence of
Hg2+ ions is attributed to the strong coordination of Hg2+ by
tertiary amine nitrogens, which prevents photo-induced elec-
tron transfer (PET) from the binding sites to the excited state of
anthracene. However, among the other cations, only Cu2+ and
Pb2+ weakly perturbed the emission (Fig. 2).
Fig. 1 The change in emission of 1 (c = 2.5 ꢀ 10ꢁ5 M) in THF upon gradual
addition of 1.5 equiv. of Hg(ClO4)2 (c = 1.0 ꢀ 10ꢁ3 M). The inset shows the
fluorescence colour change under UV irradiation.
Fig. 2 Fluorescence ratio (I – I0/I0) of 1 (c = 2.5 ꢀ 10ꢁ5 M) at 420 nm upon
addition of 2 equiv. amounts of particular cations in THF.
fluorescence and UV-vis titrations in THF–H2O (3 : 1, v/v). Upon
excitation at 370 nm, the spectrum of sensor 1 showed a
characteristic emission at 412 nm, attributed to the anthracene
emission. Upon gradual addition of Hg2+ (up to 7 equiv.) to the
solution of compound 1 in THF–H2O (3 : 1, v/v), an enhance-
ment in the emission, accompanied with a 10 nm red-shift was
observed (Fig. 3a). Under the illumination of UV light a bluish
colour of the receptor solution containing Hg2+ was noticed
(inset of Fig. 3a). This was not observed with other metal ions.
In the event of other metal ions interacting, the interaction was
weak so the change in emission of 1 was almost negligible. The
corresponding bar plot in Fig. 3b clearly demonstrates this.
To understand the selectivity in the sensing of Hg2+ by 1, we
recorded the emission of 1 upon adding 7 equiv. amounts of
Hg2+ in the presence and absence of 7 equiv. amounts of other
In order to observe the sensing behaviour of 1 towards Hg2+
and other ions in an aqueous environment, we performed
metal ions (Fig. 4a). The pronounced ‘off–on’ type of Hg2+
-
selectivity was noticed even in the presence of other metal ions.
The plot of the fluorescence ratio in Fig. 4b interprets this
aspect.
Simultaneous UV-vis titrations were performed to gain an
insight into the binding interaction of 1 in the ground state.
Scheme 2 a) (i) Piperazine, dry Et3N, dry CH2Cl2, stir, 8 h. b) (ii) n-Butylamine, dry
CH3OH, reflux, 6 h; (iii) NaBH4, CH3OH, reflux, 6 h; (iv) 3, anhydrous K2CO3, dry
CH3CN, reflux, 14 h. c) (v) n-Butylamine, dry C6H6, reflux, 6 h; (vi) NaBH4, CH3OH,
reflux, 6 h; (vii) 3, anhydrous K2CO3, dry CH3CN, reflux, 14 h.
c
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Fig. 5 (a) The change in absorption of 1 (c = 2.5 ꢀ 10ꢁ5 M) in THF–H2O
(3 : 1, v/v) upon gradual addition of 10 equiv. of Hg(ClO4)2 (c = 1.0 ꢀ 10ꢁ3 M).
(b) Fluorescent Job’s plot of receptor 1 (c = 2.5 ꢀ 10ꢁ5 M) with Hg2+ at 412 nm
(lex = 370 nm) in THF–H2O (3 : 1, v/v).
Fig. 3 (a) The change in emission of 1 (c = 2.5 ꢀ 10ꢁ5 M) in THF–H2O (3 : 1, v/v)
upon gradual addition of 7 equiv. of Hg(ClO4)2 (c = 1.0 ꢀ 10ꢁ3 M). The inset
shows the fluorescence colour change under UV irradiation. (b) Fluorescence
ratio (I – I0/I0) of 1 (c = 2.5 ꢀ 10ꢁ5 M) at 420 nm upon addition of 7 equiv.
amounts of particular cations in THF–H2O (3 : 1, v/v).
The intensity of the absorption peaks at 350 nm, 367 nm and
388 nm for anthracene decreased upon complexation of cations
(ESI†). In the presence of Hg2+ the change in intensity of
anthracene absorption was relatively large compared to the
other cations examined. Upon gradual addition of Hg2+ the
absorption peaks for anthracene underwent a red shift (ESI†).
In THF–H2O (3 : 1, v/v) the decrease in the intensity of the
absorption for anthracene upon gradual addition of Hg2+
(Fig. 5a) was noted to be less compared to the case in THF.
Fig. 6 Probable binding structure of 1 with Hg2+
.
In the interaction process, sensor 1 gave a 1 : 1 stoichiometry of the nitrogen centres in complexation according to the
with Hg2+ as confirmed by fluorescence (Fig. 5b) as well as UV mode shown in Fig. 6 was established by downfield chemical
1
Job’s plots (ESI†).18 The association constant19a of the complex shift of the protons of the adjacent –CH2– groups in H NMR
was determined to be (1.33 ꢂ 0.31) ꢀ 104 Mꢁ1 (ESI†). We were (Fig. 7). However, during interaction, the anthracene ring
unable to determine the binding constant values for the other protons underwent a small downfield shift thereby suggesting
cations due to minor changes in emission. Based on these the negligible contribution of cation–p interaction in the bind-
findings, a probable mode of binding of Hg2+ by 1 is repre- ing event.
sented in Fig. 6.
During interaction, the signals for the protons of types ‘a’,
The amide group, being a chelator, may coordinate with the ‘b’ and ‘c’ of the anthracene ring moved downfield by 0.12, 0.03
metal ion either through the amide nitrogen or the carbonyl and 0.03 ppm, respectively. The protons of the methylene
oxygen. The involvement of the amide nitrogen in coordination groups of types ‘g’, ‘h’ and ‘f’ also underwent downfield shifts
in the present case was confirmed by FTIR. The signal for by 0.03, 0.23 and 0.20 ppm, respectively. This change in
amide carbonyl stretching appeared at 1643 cmꢁ1 and moved to chemical shift of the methylene protons indicated the involve-
1650 cmꢁ1 upon complexation (ESI†). In addition, the participation ment of the nitrogen centers in complexation. A small downfield
Fig. 4 (a) The change in emission of 1 (c = 2.5 ꢀ 10ꢁ5 M) in THF–H2O (3 : 1, v/v) upon addition of 7 equiv. of Hg(ClO4)2 (c = 1.0 ꢀ 10ꢁ3 M) in the presence and absence
of 7 equiv. of other metal ions. (b) Competitive selectivity of 1 (c = 2.5 ꢀ 10ꢁ5 M) towards Hg2+ in the presence of different cations in THF–H2O (3 : 1, v/v).
c
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Fig. 7 Partial 1H NMR (400 MHz, CDCl3) of (i) 1 (c = 1 ꢀ 10ꢁ3 M) and (ii) 1 in the
presence of 1 equiv. amount of Hg(ClO4)2 (see the labeling of the protons of 1 in
Fig. 6).
chemical shift (0.01 ppm) of protons of type ‘i’ of the piperazine
unit in the presence of Hg2+ ions cannot be ignored from its
participation in complexation. However, the absence of excimer
emission of 1 in the presence of Hg2+ signifies that the appended
anthracenes are apart from the distance required for p-stacking
either intra or intermolecularly. This information overall sup-
ports our proposition in Fig. 6.
Fig. 8 Emission spectra for the 1ꢃHg2+ complex upon addition of 30 equiv.
amounts of glutathione in THF–H2O (3 : 1, v/v; 10 mmol HEPES, pH 7.4).
Thus, the dimension of the open cavity as well as the
coordination abilities of the two pendant arms makes 1 a good
host for Hg2+. The fluorometric behaviour of 1 with Hg2+ ions
was also checked in THF–H2O (3 : 1, v/v) using HEPES buffer
(10 mmol, pH 7.4). Here we noted a similar change and
selectivity of 1 towards Hg2+ ions (ESI†). It is mentionable that
compound 1 was able to detect Hg2+ ions at concentrations as
low as 3 ꢀ 10ꢁ4 M (ESI†).
We also synthesized compound 2, which consists of an
identical binding site with pyrene as the signalling probe
instead of anthracene. As expected, with the gradual addition
of Hg2+ ions, receptor 2 also showed an increase in emission at
386 nm and exhibited a 1 : 1 stoichiometry of the complex
(ESI†). The increase in emission in the presence of Hg2+ ions
is attributed to the formation of a complex like the mode shown
in Fig. 6 that results in the inhibition of the PET process (ESI†)
Fig. 9 The change in the fluorescence ratio at 420 nm for the 1ꢃHg2+ ensemble
ꢁ3
upon addition of 30 equiv. of various thiols (c = 1 ꢀ 10
(3 : 1, v/v; 10 mmol HEPES buffer, pH 7.4; lex = 370 nm).
M) in THF–H2O
from the binding site to the excited state of the fluorophore. reports the ability of the 1ꢃHg2+ ensemble to selectively sense
The binding constant of 2 with Hg2+ was calculated19b to be GSH over cysteine and homocysteine under physiological
1.32 ꢀ 104
M
ꢁ1. Compound 2 also showed strong selectivity conditions.
towards Hg2+ over other metal ions with a detection limit of 3 ꢀ
During interaction of glutathione with the mercury complex
10ꢁ4 M. This observation shows that the change of the aromatic of 1 the bluish color of the solution of 1ꢃHg2+ observed under
surface in the design has little effect on the binding and UV exposure was discharged. Further addition of Hg2+ retrieved
selection; rather the chelating centres contribute more to the the color (Fig. 10). The spectral changes, in this aspect, can be
binding of metal ions.
found in the ESI†. Thus the overall observations indicate that
However, to test the reversibility of binding in such sensors while compound 1 with simple architecture is excellent to act as
we titrated the Hg2+ complex of 1 with biothiols such as a reversible ‘off–on’ molecular switch for Hg2+ ions, the 1ꢃHg2+
cysteine, homocysteine and glutathione, which play important ensemble behaves as an ‘on–off’ switch to GSH.
roles in maintaining the appropriate redox status of biological
A similar finding was not fully achieved with sensor 2 (ESI†).
systems.8 The gradual addition of cysteine and homocysteine In contrast, the 2ꢃHg2+ complex showed less selectivity towards
up to 30 equiv. to a solution of the mercury complex of 1 in GSH as both cysteine and homocysteine resulted in significant
THF–H2O (3 : 1, v/v; using 10 mmol HEPES buffer, pH 7.4) quenching of the emission of 2ꢃHg2+. Therefore, the ‘on–off’
induced partial quenching of the emission of 1ꢃHg2+ (ESI†). switching in the presence of Hg2+ and all the biothiols was
In contrast, under identical conditions, increasing the addition observed without any distinguishing features (ESI†).
of reduced glutathione (up to 30 equiv.) to the solution of the
In an effort to construct the molecular logic gate, we
1ꢃHg2+ complex (Fig. 8) resulted in complete quenching of the investigated the ‘‘off–on’’ switching process in 1 by a sequence
emission indicating that Hg2+ ions are completely removed dependent emission output between these chemical inputs.
from the binding centres. The corresponding plot in Fig. 9 Herein, the simple molecular system 1 correlates very well with
c
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Fig. 12 Fluorescence and bright field images of HeLa cells: (a) bright field image
of normal cells, (b) bright field image of cells treated with receptor 1 (10 mM) for
1 h at 37 1C, (c) fluorescence image of cells treated with 1 (10 mM) for 1 h at 37 1C,
(d) fluorescence image of cells treated with Hg(ClO4)2 (30 mM) for 1 h at 37 1C, (e)
blue fluorescence images of the cells upon treatment with receptor 1 (10 mM)
and then Hg(ClO4)2 (30 mM) for 30 min at 37 1C, (f) blue fluorescence images of
the cells upon treatment with 1 (10 mM) and then Hg(ClO4)2 (30 mM) for 1 h at
37 1C; lex = 370 nm.
Fig. 10 Fluorescence experiment showing the reversible off–on–off switching
for Hg2+ and GSH; above are the vials showing the visual fluorescent colour
change at every cycle, where a = 1; b = 1 + Hg2+; c = b + GSH; d = c + Hg2+; e = d +
GSH (Hg2+ = 14 equiv., GSH = 30 equiv.).
the cells with 1, respectively. Cells incubated with sensor 1
without Hg2+ (Fig. 12c) and cells incubated with Hg2+ without
sensor 1 (Fig. 12d) did not show any fluorescence properties. In
contrast, cells incubated with sensor 1 and then with Hg2+ ions
showed the occurrence of blue fluorescence indicating the
permeability of sensor 1 inside the cells as well as the binding
of Hg2+ with the sensor (Fig. 12e and f).
The glutathione induced reversible ‘‘off–on’’ experiment
that could serve as experimental evidence to support the
reversible as well as selective sensing of glutathione over
cysteine and homocysteine was also applied to human cervical
cancer (HeLa) cells. The blue coloured cells obtained from
incubation with the receptor followed by treatment with Hg2+
showed no fluorescence upon addition of GSH (80 mM) (Fig. 13).
The addition of sensor 1 to the cells did not show any
cytotoxicity as is evident from the morphology of the cells
(Fig. 12) as well as the MTT assay depicted in the ESI.†20 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. The cell permeability and cytotoxicity
of 2 were also documented in the same way as was done for 1
and the results were noticed to be nearly identical (ESI†).
Fig. 11 (i) The logic circuit of receptor 1 with chemical inputs of Hg2+ and GSH,
mimicking an INHIBIT gate; (ii) truth table for the INHIBIT gate of 1 with chemical
inputs of Hg2+ and GSH.
an INHIBIT logic gate. In considering the INHIBIT logic gate
properties of 1, Hg2+ and GSH act as inputs while the emission
intensity at 420 nm (I420) is the output (Fig. 11i and ii). The
output is zero and the gate is OFF when (a) both the Hg2+ and
GSH are absent, (b) GSH alone is present, or (c) both Hg2+ and
GSH are present. The output is one and the gate is ON only
when Hg2+ alone is present. Thus 1 can act as an INHIBIT
logic gate.
Finally, the potentiality of 1 in biological systems was
evaluated for in vitro detection of Hg2+ ions in human cervical
cancer (HeLa) cells. The HeLa cells were incubated with 5 ml of
sensor 1 (10 mM) in THF–H2O (3 : 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, pH = 7.4) to remove
excess 1. DMEM (without FBS) was again added to the cells. The
cells were then treated with 5 ml of Hg(ClO4)2 (30 mM) and
incubated again for 30 min at 37 1C. A control set of cells
which was devoid of Hg2+ ions was kept. The addition of 1 to
the cells did not show any cytotoxicity as is evident from the
morphology of the cells. Herein, Fig. 12a and b represent the
bright field images of the cells before and after treatment of
Fig. 13 Fluorescence images of HeLa cells: (a) blue fluorescence images of cells
upon treatment with sensor 1 (10 mM) and then Hg(ClO4)2 (30 mM) after 1 h, (b)
fluorescence images of cells upon addition of GSH (80 mM) to the ensemble of 1
(10 mM) and Hg(ClO4)2 (30 mM) after 1 h.
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Conclusion
reduced by refluxing the mixture with NaBH4 (0.19885 g,
5.26 mmol) for 6 h. The reaction was quenched by evaporation
of the solvent under reduced pressure. CHCl3–water (2: 1) was
added to the residue and the compound was extracted with
CHCl3. The organic phase was dried over anhydrous Na2SO4 and
evaporated in vacuo. The crude product was purified by column-
chromatography using petroleum ether–EtOAc, 80: 20 (v/v) as
eluent to afford amine 7 as a pale yellow solid (0.910 g, yield:
In conclusion, we have developed the simple and easy-to-make
piperazine-based PET sensory systems 1 and 2, which show
excellent responses towards Hg2+ ion in semi-aqueous systems
by exhibiting increased emission and fluorescence. Further-
more, between 1 and 2, sensor 1 in its complexed form with
Hg2+ has been established as a good ensemble to detect
glutathione (GSH) over cysteine and homocysteine. Sensor 1
operates as an INHIBIT logic gate when Hg2+ and GSH are
considered as the possible inputs. In addition, compounds 1
and 2 are both cell permeable and able to detect intracellular
Hg2+ ions through fluorescence imaging.
1
90.4%), m.p. 95 1C; H NMR (400 MHz, CDCl3): d 8.36 (d, 1H,
J = 8 Hz), 8.18–8.11 (m, 4H), 8.03–7.97 (m, 4H), 4.87 (br s, 1H,
NH), 4.48 (s, 2H, ArCH2), 2.79 (t, 2H, J = 8 Hz, NHCH2), 1.60–1.53
(m, 2H, NHCH2CH2), 1.42–1.33 (m, 2H, NHCH2CH2CH2), 0.92
(t, 3H, J = 8 Hz, NHCH2CH2CH2CH3). FTIR (KBr, cmꢁ1): 3400,
2950, 2926, 2858, 2813, 1442.
Receptor 1. To a stirred solution of amine 5 (0.347 g,
1.32 mmol) in 20 ml dry CH3CN, anhydrous K2CO3 (0.3 g,
2.17 mmol) was added and the mixture was stirred for 30 min.
Experimental
Syntheses
1,10-(Piperazine-1,4-diyl(2-chloroethanone)) (3). To a stirred Compound 3 (0.15 g, 0.63 mmol) was then added and the
solution of piperazine (1 g, 11.61 mmol) in 40 ml dry CH2Cl2, reaction mixture was refluxed for 14 h and the progress of
chloroacetyl chloride (2.77 ml, 34.83 mmol) and dry Et3N the reaction was monitored by TLC. After completion of the
(4.850 ml, 34.82 mmol) were added dropwise. The reaction reaction CH3CN was evaporated in a vacuum. CHCl3–water
mixture was stirred at room temperature for 8 h. The solvent (3 : 1, v/v) was added to the residue and the compound was
was evaporated under vacuum. The residue was taken in water extracted with CHCl3. The combined organic layer was dried
(40 mL), and the product was extracted with CHCl3 (3 ꢀ 40 mL). over anhydrous Na2SO4 and the solvent was evaporated
The combined organic layer was dried over anhydrous sodium in vacuo. The crude product was purified through column
sulphate and evaporated in vacuo. The crude product was chromatography using 25% EtOAc in petroleum ether as eluent
purified by column chromatography using ethyl acetate– to give the desired receptor 1 as a yellow solid (0.34 g, yield:
petroleum ether (1 : 1, v/v) as eluent to give the desired product 78.21%), m.p. 180 1C; 1H NMR (400 MHz, CDCl3): d 8.41 (s, 2H),
3 as a pale brown solid (2 g, yield: 72.1%); m.p. 133 1C; 1H NMR 8.36 (d, 4H, J = 8 Hz), 7.99 (d, 4H, J = 8 Hz), 7.49–7.31 (m, 8H),
(400 MHz, CDCl3): d 4.10 (s, 4H), 3.72–3.56 (m, 8H); FTIR 4.53 (s, 4H), 3.03 (s, 4H, J = 16 Hz), 2.81–2.75 (m, 8H), 2.54–2.51
(KBr, cmꢁ1): 2991, 2908, 2879, 1338.
(m, 4H), 1.74–1.70 (m, 4H), 1.38–1.34 (m, 4H), 0.92 (t, 6H, J =
Anthracen-9-ylmethyl-butyl-amine (5). To a solution of 8 Hz); 13C NMR (100 MHz, CDCl3): d 169.6, 131.3, 131.2, 129.1,
9-anthranaldehyde (0.4 g, 1.94 mmol) in dry methanol, n-butyl 128.8, 127.9, 125.9, 125.0, 124.4, 56.9, 55.6, 50.3, 44.4, 41.4,
amine (0.193 ml, 1.94 mmol) was added and the reaction 29.1, 20.8, 14.0; FTIR (KBr, cmꢁ1): 2895, 2836, 2862, 2836, 1643;
mixture was refluxed for 6 h. The Schiff base 4 thus formed HRMS (TOF MS ES+): (M + H)+ required 693.4169, found
in situ was reduced by refluxing the mixture with NaBH4 693.4207.
(0.150 g, 3.96 mmol) for 6 h. The progress of the reduction
Receptor 2. To a stirred solution of amine 7 (0.700 g,
was monitored by thin layer chromatography (TLC) (petroleum 2.44 mmol) in 25 ml dry CH3CN, anhydrous K2CO3 (0.460 g,
ether–EtOAc, 80 : 20, v/v). After completion of the reaction, the 3.32 mmol) was added and the mixture was stirred for 30 min.
solvent was evaporated under reduced pressure. Then, CHCl3– Compound 3 (0.264 g, 1.11 mmol) was then added and the
water (2 : 1, v/v) was added to the residue and the compound reaction mixture was refluxed for 14 h. After completion of the
was extracted with CHCl3. The organic phase was dried over reaction, CH3CN was evaporated in a vacuum. CHCl3–water
anhydrous Na2SO4 and evaporated in vacuo. The crude product (3 : 1, v/v) was added to the residue and the compound was
was purified by column-chromatography using petroleum extracted with CHCl3. The combined organic layer was dried
ether–EtOAc, 80 : 20 (v/v) as eluent to afford amine 5 as a yellow over anhydrous Na2SO4 and the solvent was evaporated
gummy product (0.42 g, yield: 82.3%), 1H NMR (400 MHz, in vacuo. The crude product was purified through column
CDCl3): d 8.53 (s, 1H), 8.34 (d, 2H, J = 8 Hz), 8.01 (d, 2H, J = chromatography using 25% EtOAc in petroleum ether as eluent
8 Hz), 7.55 (t, 2H, J = 8 Hz), 7.46 (t, 2H, J = 8 Hz), 4.75 (s, 2H, to give the desired receptor 2 as a white solid (0.68 g, 0.917 mmol)
ArCH2), 2.87 (t, 2H, J = 8 Hz, NHCH2), 1.88 (br s, 1H, NH), 1.62– yield 78.2%, m.p. 135 1C; 1H NMR (400 MHz, CDCl3): d 8.39
1.55 (m, 2H, NHCH2CH2), 1.41–1.31 (m, 2H, NHCH2CH2CH2), (d, 1H, J = 8 Hz), 8.29 (d, 1H, J = 8 Hz), 8.17 (d, 2H, J = 8 Hz),
0.91 (t, 3H, J = 8 Hz, NHCH2CH2CH2CH3); FTIR (KBr, cmꢁ1): 8.09–8.03 (m, 4H), 8.02–7.99 (m, 3H), 7.97–7.92 (m, 3H), 7.88–
3412, 3046, 2953, 2911, 1444.
7.86 (m, 2H), 7.83 (d, 1H, J = 8 Hz), 7.77 (d, 1H, J = 8 Hz), 4.20
Butyl-pyren-1-ylmethylene-amine (7). To a solution of pyrene- (d, 4H, J = 8 Hz), 3.13 (d, 4H, J = 8 Hz), 2.82–2.78 (m, 4H), 2.67–
1-carboxaldehyde (1 g, 4.34 mmol) in dry benzene, n-butyl amine 2.56 (m, 8H), 1.59–1.53 (m, 4H), 1.42–1.21 (m, 4H), 0.89 (t, 6H,
(0.474 ml, 0.477 mmol) was added and the reaction mixture was J = 8 Hz); 13C NMR (100 MHz, CDCl3): d 169.5, 131.4, 131.2,
allowed to reflux for 6 h. The Schiff base 6 formed in situ was 129.8, 128.7, 127.4, 127.3, 127.0, 126.0 (2C unresolved), 125.4,
c
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125.3 (2C unresolved), 124.9, 124.6, 124.3, 123.5, 57.3, 55.2, The cells were incubated in 96-well microplates for 24 hours
44.7, 41.6, 29.7, 29.0, 20.7, 14.0; FTIR (KBr, cmꢁ1): 2952, 2898, along with the receptors at different concentrations. A set of
2822, 2800, 1644; HRMS (TOF MS ES+): (M + H)+ required HeLa cells not exposed to any of the receptors were kept as an
741.4169, found 741.7592.
untreated control. Other sets of cells were incubated with
receptor 1 through 5 ml of stock solution. The cells were allowed
to grow for the next 24 hours. MTT was then added to each well
General procedure for fluorescence and UV-vis titrations
Stock solutions of the receptors were prepared in THF and and incubated for the next 4 h. The intracellular formazan
3 : 1 (v/v) THF–H2O in the concentration range of B10ꢁ5 M. crystals formed were solubilized with dimethyl sulfoxide
2 ml of the receptor solution was taken in the cuvette. Stock (DMSO) and the absorbance of the solution was measured at
solutions of guests in the concentration range of B10ꢁ4 M, 595 nm by using a microplate reader (Thermo scientific,
were prepared in the same solvents and were individually Multiskan ELISA, USA). The percentage of cell survival was
added in different amounts to the receptor solution. Upon calculated as: (mean experimental absorbance/mean control
addition of metal ions, the change in emission of the receptor absorbance) ꢀ100%.
was noted. The same stock solutions of receptors and guests
were used to perform the UV-vis titration experiment. The
solutions of the metal salts were successively added in different
Acknowledgements
We thank DST and UGC, New Delhi, India for providing
facilities in the department. DT thanks CSIR, New Delhi, India
for a fellowship.
amounts to the receptor solution (2 mL) taken in the cuvette
and the absorption spectra were recorded. Both fluorescence
and UV-vis titration experiments were carried out at 25 1C.
Job plots
References
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the plot, the mole fraction of the host at which the concen-
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stoichiometry of the complex.
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