A rhodamine appended tripodal receptor as a ratiometric probe for Hg 2+ ions

Article abstract of DOI:10.1039/c2ob00009a

A new rhodamine appended tripodal receptor 1 has been designed and synthesized. The receptor selectively recognizes Hg²⁺ ions in CH ₃CN-water (4:1, v/v; 10 μM tris HCl buffer, pH 7.0) by displaying a ratiometric change in emission. Additionally, the visual detection is possible by a sharp change in color. The receptor shows in vitro detection of Hg ²⁺ ions in human cervical cancer (HeLa) cells.

Full text of DOI:10.1039/c2ob00009a

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PAPER
A rhodamine appended tripodal receptor as a ratiometric probe for Hg2+
ions†
Kumaresh Ghosh,*a Tanmay Sarkara and Asmita Samadderb
Received 2nd January 2012, Accepted 18th February 2012
DOI: 10.1039/c2ob00009a
A new rhodamine appended tripodal receptor 1 has been designed and synthesized. The receptor
selectively recognizes Hg2+ ions in CH3CN–water (4 : 1, v/v; 10 μM tris HCl buffer, pH 7.0) by
displaying a ratiometric change in emission. Additionally, the visual detection is possible by a sharp
change in color. The receptor shows in vitro detection of Hg2+ ions in human cervical cancer (HeLa) cells.
binder scaffold (see the encircled part in structure 1) is connected
Introduction
to the rhodamine dye. In our earlier publication,7a we reported a
Design and synthesis of fluorescent chemosensors for the selec-
tive sensing and recognition of the toxic heavy metal ions, such
as Hg2+, Pb2+ and Cd2+ ions etc.,1 is of considerable importance
because of the severe immunotoxic, genotoxic, and neurotoxic
effects of those metal ions. Among the different heavy metal
ions, mercury ions (Hg2+) are considered to be dangerous as they
can accumulate in the human body and affect a wide variety of
diseases even in a low concentration, such as prenatal brain
damage, serious cognitive disorders, and Minamata disease.2
Therefore, it is important to develop highly sensitive and selec-
tive assays for Hg2+ ions. In recent years, considerable efforts
have been made in the design of fluorescent chemosensors of
different architectures for the selective sensing of Hg2+ ions.3–6
The receptors belonging to this category are of three types
depending on the nature of their responsive modes (turn-on,
turn-off and ratiometric). Among the three modes, ratiometric
probes (especially, rhodamine-based) for the detection of Hg2+
ions4 are considerably important and they are relatively less in
number than the ‘turn-on’5 based receptors. The ratiometric che-
mosensors offer advantages over the conventional monitoring of
fluorescence intensity at a single wavelength. A dual emission
system can minimize the measurement errors because of the
factors such as phototransformation, receptor concentrations, and
environmental effects.6
new synthesis and the metal binding characteristic features of a
tripodal receptor. To further explore this tripodal centre in wider
aspect, we presently focus our attention with the rhodamine
labelled new structure 1 to detect the metal ion both by fluoro-
metrically and colorimetrically in semi-aqueous system. It is
mentionable that rhodamine B and its derivatives (RhB) are well
known for their good photo stability, high extinction coefficient
(>75 000 cm−1 M−1), and high fluorescence quantum yield.9
The functioning of this moiety in the chemosensor towards
metal ion is related with the switching of the spirocyclic form
(which is colorless and non-fluorescent) to the ring-opened
amide form which is pink and strongly fluorescent.4,5 A number
of rhodamine labeled receptors of this class are known which
show color change and selective “turn-on” response to Hg2+
ions.5 However, in the present case, receptor 1 shows high sensi-
tivity and selectivity towards Hg+2 ions by exhibiting both col-
orimetric and ratiometric fluorescence responses in CH3CN–
water (4 : 1, v/v; 10 μM tris HCl buffer; pH 7.0).
As the continuation of our work on the sensing of cations7
and anions8 of biological significance, we report in this full
account a new receptor module 1 in which the tripodal metal
aDepartment of Chemistry, University of Kalyani, Kalyani-741235,
India. E-mail: ghosh_k2003@yahoo.co.in; Fax: +913325828282;
Tel: +913325828750
bDepartment of Zoology, University of Kalyani, Kalyani-741235, India
†Electronic supplementary information (ESI) available: Figures showing
the change in fluorescence and UV-vis titrations of receptor 1 with
various metal ions, Job plot, spectral data (NMR, mass) for 1. See DOI:
10.1039/c2ob00009a
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Scheme 1 (a) (i) Dry MeOH, reflux, 9 h; (ii) NaBH4, dry MeOH,
reflux, 3 h; (iii) Ethyl 2-chloroethanoate, K2CO3, dry acetone, reflux,
7 h; (iv) 5, THF, stirring, 4 h; (b) (v), EtOH, reflux, 9 h.
Fig. 1 Change in fluorescence ratio of 1 (c = 4.41 × 10−5 M) at
580 nm upon addition of 18 equiv. amounts of cations.
Results and discussion
The receptor 1 was synthesised according to Scheme 1. Initially
the intermediate compound 4-benzylmorpholin-2-one 4 was syn-
thesized according to our reported procedure.7c The Schiff base
2, which was obtained from the condensation of benzaldehyde
with 2-aminoethanol, was reduced to the amine 3 using NaBH4
in MeOH. The reaction of the amine 3 with ethyl 2-chloro-
ethanoate under refluxing condition gave 4-benzylmorpholin-2-
one 4 in appreciable yield. Further reaction of the intermediate
compound 4 with the rhodamine labelled amine 510 furnished
the desired compound 1 in good yield. The compound 1 was
1
fully characterized by H NMR, 13C, FTIR, mass and elemental
analyses.
Fig. 2 Fluorescence titration spectra of 1 (c = 4.41 × 10−5 M) in
CH3CN–water (4 : 1, v/v; 10 μM tris HCl buffer, pH 7.0) upon addition
of Hg2+; Inset: Colour change of the receptor solution under illumination
of UV light.
The spectroscopic properties of the receptor 1 towards the
metal ions such as Hg2+, Cu2+, Cd2+, Fe2+, Mg2+, Co2+, Ni2+,
Mn2+, Zn2+, Pb2+ and Ag+ (taken as their perchlorate salts) were
rated in CH3CN–H2O solution (CH3CN : H2O = 4 : 1, v/v,
10 μM tris HCl buffer, pH = 7.0). In this context, a higher
content of water in aqueous measuring solution is seemed to be
desirable. But such an approach is constrained by the limited
solubility of 1 in water. As a reasonable compromise, a 4 : 1
(CH3CN : H2O) aqueous CH3CN solution was used in the sub-
sequent study using 10 μM tris HCl buffer for maintaining pH
7. Initially, the emission of 1 was monitored upon successive
addition of the metal ions to the receptor solution in aq. CH3CN
(CH3CN : H2O = 4 : 1, v/v, 10 μM tris HCl buffer, pH = 7.0). On
excitation at 510 nm, a nonstructured emission at 536 nm under-
went significant quenching upon interaction with all the metal
ions studied. However, the change in fluorescence ratio at
536 nm for all the metal ions was found to be almost the same in
magnitude and there are no distinguishable attributes (Fig. 3S;
ESI†). In contrast, in the case of Hg2+, a new peak at 580 nm
appears with remarkable intensity. Fig. 1, in this regard, shows
the change in fluorescence ratio [(I−I0)/I0] of 1 at 580 nm in the
presence of 18 equiv. amounts of the different metal ions. As
can be seen from Fig. 1, the receptor is highly sensitive to Hg2+
ion. In comparison, other metal ions merely perturbed the emis-
sion of 1 at this wavelength (ESI†).
of the titration of 1 with Hg2+ and exhibits a ratiometric change.
The ratiometric calibration curve is displayed in Fig. 3a.
Importantly, the increase in concentration of buffer in
CH3CN–H2O solution (CH3CN : H2O = 4 : 1, v/v, 35 μM tris
HCl buffer, pH = 7.0) did not alter the ratiometric nature of
emission spectra as well as the extent of change in intensity
while titration was performed with Hg2+ ions (Fig. 5S in ESI†).
This experimental finding (i.e., ratiometric fluorescence change)
distinguishes the Hg2+ ion from the other metal ions examined.
To the best of our knowledge, such rhodamine-based receptor
for ratiometric detection of Hg2+ is a new addition to the existing
reports4 in the literature.
Furthermore, the plot of ratio of fluorescence response at the
wavelengths 536 and 580 nm is shown in Fig. 3b, which indi-
cates that the response factor is noteworthy for Hg2+ ions. This is
also found to be true when the ratiometric fluorescent response
of 1 was recorded in the presence of 18 equiv. amounts of other
metal ions considered in the study. Fig. 4 signifies this feature. It
is quite comprehensible from Fig. 4 that the ratiometric behav-
iour of 1 is unperturbed in the presence of other metal ions and
thereby indicated its high selectivity and sensitivity towards
Hg2+ ions. As can be seen from Fig. 4, Ag+ and Pb2+ ions which
interfere in Hg2+-ratiometric sensors,11 did not perturb the ratio-
metric behaviour of 1 towards Hg2+ ions.
Fig. 2 describes the emission titration spectra obtained from
the gradual addition of Hg(ClO4)2 solution to the solution of 1
(c = 4.41 × 10−5 M) in CH3CN–H2O (4 : 1, v/v; 10 μM tris HCl
buffer; pH 7.0). While the emission centered at 536 nm
decreases, a new band at 580 nm begins to appear on progression
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Fig. 5 Suggested modes of interaction of 1 with Hg2+ ion in solution.
Fig. 6 Fluorescence Job plot for 1 with Hg+2 in CH3CN–water (4 : 1,
v/v; 10 μM tris HCl buffer; pH 7.0) ([H] = [G] = 4.41 × 10−5 M).
Fig. 3 (a) Ratiometric calibration curve for 1 (c = 4.41 × 10−5 M) with
Hg+2; (b) Ratiometric fluorescent response of 1 (c = 4.41 × 10−5 M)
with the addition of 18 equiv. amounts of each metal ion (c = 8.82 ×
10−4 M) in CH3CN–water (4 : 1, v/v; 10 μM tris HCl buffer; pH = 7.0).
Fig. 7 Nonlinear curve fitting of the fluorescence titration data for 1
(c = 4.41 × 10−5 M) with Hg2+ in CH3CN–water (4 : 1, v/v; 10 μM
tris HCl buffer; pH = 7.0) upon addition of Hg+2
.
Fig. 4 Ratiometric fluorescent response of sensor 1 (c = 4.41 × 10−5
M) to Hg2+ (c = 8.82 × 10−4 M) over the selected metal ions (c = 8.82 ×
10−4 M).
Hg2+ ion strongly (Form 1B in Fig. 5) for which a new peak at
580 nm begins to appear with significant growth on progression
of the titration. The absence of this peak in the cases of all the
metal ions except Hg2+ indicates that the ring opening step is not
occurring during the course of interaction. In the interaction
process, the stoichiometry of the Hg-complex was evaluated to
be 1 : 1, as evident from the Job’s method of continuous vari-
ations (Fig. 6).12 From the titration data, the binding constant
(Ka) for the formation of 1. Hg2+ complex was estimated by a
nonlinear curve fitting procedure and it was found to be (6.31
0.74) × 105 M−1 (Fig. 7). The nonlinear fit was done using
eqn (1)13
In our opinion, the ratiometric response of 1 towards the Hg2+
ion is attributed to the involvement of the binding centre in
different ways. We believe that initially, the aliphatic tertiary
amine nitrogen together with the alcoholic and amide functional-
ities in the tripodal centre interact with the Hg2+ ion (see Form
1A in Fig. 5) in the same way as described in our earlier
report.7c Due to this interaction the emission intensity at 536 nm
decreases. Then the closely spaced spirocyclic form of the rhoda-
mine part is opened and provides an amide ion to chelate the
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Table 1 Binding constant values (Ka in M−1) for 1 with the metal ions
in CH3CN–H2O (4 : 1, v/v; 10 μM tris HCl buffer; pH 7.0) by
fluorescence method
Guests
Ka (M−1) at 536 nm
Hg+2
Cu+2
Fe+2
Mg+2
Co+2
Cd+2
Zn+2
Ni+2
Mn2+
Pb2+
Ag+
(6.31 0.74) × 105
(9.40 0.25) × 104
(3.20 0.37) × 104
(2.43 0.09) × 104
(2.04 0.09) × 104
(1.74 0.13) × 104
(3.50 0.39) × 104
(2.70 0.14) × 104
(4.28 0.82) × 104
(1.50 0.14) × 104
(1.41 0.21) × 104
Fig. 9 Absorption titration spectra of 1 (c = 4.41 × 10−5 M) in
CH3CN–H2O (4 : 1, v/v; 10 μM tris HCl buffer; pH 7.0) upon addition
of Hg+2
.
the absence and presence of different amounts of Hg2+ions. The
‘h’ proton showed an upfield shift of 0.13 ppm. This upfield
movement is attributed to the increase in electron density arising
from the opening of the spirolactam ring. Indeed, in 13C NMR
the disappearance of the signal at 66.03 ppm for the tertiary
carbon of the spirolactam ring of 1 (labeled as ‘j’; see Fig. 8)
upon addition of 1.2 equiv. amounts of Hg2+ (ESI†) convin-
cingly proved the ring opening14 to give the form 1B in Fig. 5.
Furthermore, the cooperative involvement of the tripodal cavity
in complexation is proved from the downfield shift of the signals
for ‘a’, ‘b’, ‘c’ and ‘d’ type protons by 0.13, 0.22, 0.09 and
0.02 ppm, respectively in the presence of 2 equiv. amounts of
Hg2+ ions. The amide proton (i-type) appeared at 8.34 ppm
underwent a small upfield shift by 0.14 ppm. This suggests that
the amide oxygen instead of nitrogen participates in the metal
coordination.
Fig. 8 Partial 1H NMR of (a) 1 (7.96 × 10−3 M) and with (b) 0.5
equiv. (c) 1 equiv. and (d) 2 equiv. amounts of Hg2+ in CD3Cl contain-
ing 4% d6-DMSO.
Interestingly, during the fluorometric titration of 1 with Hg2+
ions the colourless solution of the receptor became pink. This
pink color is attributed to the opening of the spirolactam ring
and generation of the delocalised xanthene moiety.4,5,15 Under
the illumination of UV light a yellowish brown colour of the
receptor solution containing Hg2+ was noticed (inset of Fig. 2).
This was not observed with other metal ions except Cu2+ and
Fe2+ ions. In case of Cu2+ and Fe2+ ions, the colourless solution
of 1 turned into a very faint pink colour. Therefore, the receptor
1 is also capable of reporting the presence of Hg2+ ion in sol-
ution colorimetrically. But it is interesting to note that the recep-
tor 1 did not show any color change while performing the
titration using 8.7 × 10−6 M concentration of Hg2+. Even the
ratiometric behavior in emission was not observed (Fig. 6S in
ESI†).
I ¼ I0 þ ðIlim ꢀ I0Þ=2CHfCH þ CG þ 1=Ka
ð1Þ
2
1=2
ꢀ ½ðCH þ CG þ 1=KaÞ ꢀ 4CHCGꢁ
g
where I represents the intensity; I0 represents the intensity of
pure host; CH and CG are corresponding concentrations of host
and cationic guest; Ka is the binding constant. The binding con-
stant (Ka) and correlation coefficients (R) were obtained from a
non-linear least-square analysis of I vs. CH and CG.
Using eqn (1), the binding constant values for the other metal
ions were also determined (Table 1) and they were found to be
less than that of Hg2+. This underlines the fact that the strong
interaction of Hg2+ over the rest of the metal ions is due to the
existence of the Form 1B. The formation of 1B through ring
opening was established from both the 1H NMR and FTIR
studies. In FTIR, the amide carbonyl stretching of the spirolac-
tam part appeared at 1683 cm−1 changed to a lower wavenumber
The UV-vis spectrum of 1 (c = 4.41 × 10−5 M) in CH3CN–
H2O (4 : 1, v/v; 10 μM tris HCl buffer; pH 7.0) shows a strong
absorption at 315 nm which decreases followed by an appear-
ance of a new absorption at 555 nm upon gradual addition of
mercury solution and resulted in an isosbestic point at 333 nm
(Fig. 9). The growth of the absorption peak at 555 nm is the con-
sequence of the opening of the spiroring in 1.11c,15 This was not
1
(1678 cm−1) in the presence of Hg2+. Also in H NMR, the ring
protons (e, f and g; see the labeling in Fig. 8) of the rhodamine
part moved to the downfield direction (Δδe = 0.02, Δδf = 0.07
and Δδg = 0.05) in the presence of 2 equiv. amounts of Hg2+
ions. Fig. 8, in this regard, shows the change in 1H NMR of 1 in
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Fig. 10 Change in absorbance of 1-Hg+2 complex in CH3CN–H2O
(4 : 1, v/v; 10 μM tris HCl buffer; pH 7.0) upon addition of KI (c =
1.5 × 10−3 M); Inset: colour change upon addition of KI.
Fig. 12 Change in absorbance from the addition of Hg(ClO4)2 (c = 1.5
× 10−3 M) to the solution of 1-Hg+2 containing KI in CH3CN–H2O
(4 : 1, v/v; 10 μM tris HCl buffer; pH 7.0).
Fig. 13 Change in emission from the addition of Hg(ClO4)2 (c = 1.5 ×
10−3 M) to the solution of 1-Hg+2 containing KI in CH3CN–H2O (4 : 1,
v/v; 10 μM tris HCl buffer; pH 7.0).
Fig. 11 Change in fluorescence of 1-Hg+2 complex (c = 6.81 × 10−5
M) in CH3CN–H2O (4 : 1, v/v; 10 μM tris HCl buffer; pH 7.0) upon
addition of KI (c = 1.5 × 10−3 M); Inset: colour change under exposure
of UV light upon addition of KI.
The potential biological application of the receptor was evalu-
ated for in vitro detection of Hg2+ ions in human cervical cancer
(HeLa) cells. The HeLa cells were incubated with 5 μl of recep-
tor 1 (10 μM in CH3CN–H2O (4 : 1, v/v)) in DMEM (Dulbec-
co’s modified Eagle’s medium) medium (without FBS) for
30 min at 37 °C and washed with phosphate buffered saline
(PBS) buffer (pH = 7.4) to remove excess of receptor 1. DMEM
medium (without FBS) was again added to the cells. The cells
were then treated with 5 μl of mercury perchlorate (30 μM) and
incubated again for 30 min at 37 °C. A control set of cells which
was devoid of Hg2+ ion was kept. The addition of receptor 1 to
the cells did not show any cytotoxicity as evident from the mor-
phology of the cells.
In this regard, Fig. 14a and 14b represent the bright field
images of the cells before and after treatment of the cells with 1,
respectively. Cells incubated with receptor 1 without Hg2+
(Fig. 14c) and cells incubated with Hg2+ without receptor 1
(Fig. 14d) did not show any fluorescence properties. In contrast,
cells incubated with the receptor 1 and then with Hg2+ ions
showed the occurrence of red fluorescence indicating the per-
meability of the receptors inside the cells and binding of Hg2+
with the receptor (Figs. 14e and 14f).
observed when the titrations were conducted with other metal
ions (ESI†). The stoichiometry of the Hg-complex in the ground
state was also found to be 1 : 1 (ESI†).
To check the reversibility in the complexation, a KI-adding
experiment was performed. Addition of KI to the solution of
complex of 1 with Hg2+ brought the reverse change in the
absorption spectra (Fig. 10). A similar finding was observed in
emission (Fig. 11). Interestingly, while KI diminished the absor-
bance and emission significantly, further addition of excess Hg2+
ions could recover both the absorbance and emission signals. In
this regard, Figs. 12 and 13 represent the recovery of both the
absorbance and emission respectively. In the event the pink color
of the solution is also retrieved.
Similar to many reported rhodamine spirolactam-based fluor-
escent probes, the fluorescence enhancement response of 1
toward Hg2+ is most likely the result of the spiro ring-opening
mechanism rather than an ion-catalyzed hydrolysis. The above-
mentioned KI experiment could serve as experimental evidence
to support this reversible spiro ring-opening mechanism. Like
KI, addition of aq. solutions Na2EDTA and cysteine brought
about similar change in both absorbance and emission spectra
(Fig. 7S in ESI†) and further confirmed the reversibility in the
binding process.
The KI-adding experiments which could serve as experimental
evidence to support the reversibility in structural change, were
also applied to human cervical cancer (HeLa) cells. Red
coloured cells obtained from the incubation of the receptor
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through a change in color as well as emission in ratiometric
fashion. This ratiometric chemosensor for Hg2+ ions is a new
example among the few reports in the literature.4 The coopera-
tive action of the binding groups of the tripodal cavity and the
amide ion generated from the spirolactam ring opening favors
the strong chelation of Hg2+ over a series of other studied
cations. The selectivity towards Hg2+ over the heavy transition
metal ion Cu2+ (usually interfere with the binding of Hg2+) is
noteworthy with respect to ratiometric emission characteristic of
1. Additionally, the chemosensor is also noted to be efficient in
reporting the presence of Hg2+ inside the cell. Thus, the exper-
imental findings in this full account together with our previous
observations underlines the truth that the 4-substituted morpho-
lin-2-one can be opened up in various ways7a,c to meet the mol-
ecular diversity very easily for the sensing of different cations.
Experimental
4-Benzylmorpholin-2-one (4)
Ethanolamine (0.51 g, 8.29 mmol) was added to a solution of
benzaldehyde (0.8 g, 7.54 mmol) in dry methanol (30 mL) and
the reaction mixture was refluxed for 9 h. The solution was
cooled and stirred at 0 °C for 2 h to give yellow precipitate. The
precipitate was filtered off and washed with methanol several
times and finally dried under vacuum to give foam like yellow
solid 2 (0.97 g, 87%), mp: 110–112 °C. In the next step, NaBH4
(0.304 g, 8.04 mmol) was added to a stirred solution of Schiff-
base 2 (0.8 g, 5.36 mmol) in dry methanol (30 mL) portion wise
at 0 °C under nitrogen atmosphere. The reaction mixture was
refluxed for 3 h. The solvent was removed under vacuum; water
was added and extracted with CH2Cl2 (30 mL × 3). The organic
layer was separated and dried over Na2SO4 and the solvent was
removed in a rotary evaporator. Finally, the crude product was
purified by column chromatography using 2% methanol in
CHCl3 as eluent to give yellowish solid 3 (0.64 g, 79%), mp:
92 °C.
Fig. 14 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 μM) for 1 h at 37 °C, (c) Fluorescence image of cells
treated with 1 (10 μM) for 1 h at 37 °C, (d) Fluorescence image of cells
treated with Hg(ClO4)2 (30 μM) for 1 h at 37 °C, (e) Red fluorescence
images of cells upon treatment with receptor 1 (10 μM) and then Hg
(ClO4)2 (30 μM) for 1 h at 37 °C, (f) Red fluorescence images of cells
upon treatment with 1 (10 μM) and then Hg(ClO4)2 (30.0 μM) for 2 h at
37 °C; λex = 510 nm.
K2CO3 (0.5 g, 3.97 mmol) was added to a solution of 3
(0.5 g, 3.31 mmol) in dry acetone (40 mL), followed by addition
of ethyl 2-chloroethanoate (0.48 g, 3.97 mmol). The reaction
mixture was refluxed for 7 h under nitrogen atmosphere. After
completion of the reaction, K2CO3 was filtered off and the
filtrate was evaporated on a rotary evaporator. Water was added
to the residue and the aqueous layer was extracted with CH2Cl2
(30 mL × 3). The combined organic layer was dried over anhy-
drous Na2SO4 and concentrated in vacuo. Purification of this
crude product by column chromatography gave the lactone 4
(0.49 g, 78%) as light yellow solid, mp 122 °C; 1H NMR
(400 MHz, CDCl3): δ 7.36–7.27 (m, 5H), 4.40 (t, 2H, J = 4 Hz),
3.61 (s, 2H), 3.33 (s, 2H), 2.67 (t, 2H, J = 4 Hz) ppm; FT IR
(ν in cm−1, KBr) 3474, 3080, 2959, 2810, 1747, 1455.
Fig. 15 Fluorescence images of HeLa cells: (a) Red fluorescence
images of cells upon treatment with receptor 1 (10 μM) and then Hg
(ClO4)2 (30 μM) after 1 h, (b) Red fluorescence images of cells upon
addition of KI (30 μM) to the ensemble of 1 (10 μM) and Hg(ClO4)2
(30 μM) after 1 h.
followed by treatment with Hg2+ became invisible in fluor-
escence upon addition of KI (30 μM) (Fig. 15).
2-(Benzyl(2-hydroxyethyl)amino)-N-(2-(3′,6′-bis(diethylamino)-
3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)acetamide (1)
Amine 5 (0.211 g, 0.436 mmol), which was obtained according
to the reported procedure,10 was added dropwise to a stirred sol-
ution of compound 4 (0.1 g, 0.522 mmol) in THF (20 mL). Stir-
ring was continued for 4 h. After completion of reaction,
Conclusions
In conclusion, we have thus synthesized a rhodamine labeled
flexible tripodal cavity 1 which selectively recognizes Hg2+ ions
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monitored by TLC, THF was evaporated off and water was
added to the residue. The aqueous layer was extracted with
CHCl3 (25 mL × 3) and dried over anhydrous Na2SO4. Purifi-
cation of the crude mass by silica gel column chromatography
using 3% CH3OH in CHCl3 as eluent yielded the product 1
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1
(0.268 g, 90%), mp 142 °C; H NMR (400 MHz, CDCl3): δ
8.44 (bt, 1H), 8.01 (d, 1H, J = 8 Hz), 7.50 (dd, 2H, J1 = 8 Hz, J2
= 4 Hz), 7.43 (d, 2H, J = 8 Hz), 7.31 (t, 1H, J = 8 Hz),
7.16–7.09 (m, 4H), 6.36–6.34 (m, 4H), 6.16 (dd, 2H, J1 = 8 Hz,
J2 = 4 Hz), 3.77 (t, 2H, J = 4 Hz), 3.67 (s, 2H), 3.38–3.28 (m,
10H), 3.05 (s, 2H), 2.84 (m, 2H), 2.73–2.71 (m, 2H), 1.15 (t,
12H, J = 7.20 Hz), 13C NMR (100 MHz, CDCl3): δ 171.4,
170.1, 153.9, 153.2, 148.9, 137.9, 133.0,130.2, 129.3, 128.9,
128.4, 128.3, 127.2, 123.9, 123.0, 108.2, 104.2, 97.6, 66.0,
59.7, 59.4, 58.8, 58.5, 44.3, 40.5, 39.2, 12.5; Mass (LCMS)
676.2 (M + 1)+, Anal. Calcd for C41H49N5O4: C, 72.86; H, 7.31;
N, 10.36. Found: C, 72.84; H, 7.29; N, 10.39.
General procedure for fluorescence and UV-vis titrations
Stock solutions of the receptor were prepared in 4 : 1(v/v)
CH3CN : H2O containing 10 mM Tris/HCl buffer (pH = 7.0) in
the concentration range ∼10−5 M. 2.5 ml of the receptor solution
was taken in the cuvette. Stock solutions of guests in the concen-
tration range ∼10−4 M, were prepared in the same solvents and
were individually added in different amounts to the receptor sol-
ution. 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.
The metal solution was successively added in different amounts
to the receptor solution (2.5 mL) in the cuvette and the absorp-
tion spectra were recorded. Both fluorescence and UV-vis titra-
tion experiments were carried out at 25 °C.
Job plots
The stoichiometry was determined by the continuous variation
method (Job plot).12 In this method, solutions of host and guests
of equal concentrations were prepared in the solvents used in the
experiment. Then host and guest solutions were mixed in differ-
ent proportions maintaining a total volume of 3 mL of the
mixture. All the prepared solutions were kept for 1 h with
occasional shaking at room temperature. Then emission and
absorbance of the solutions of different compositions were
recorded. The concentration of the complex, i.e. [HG], was cal-
culated using the equation [HG] = ΔI/I0 × [H] or [HG] = ΔA/A0
× [H] where ΔI/I0 and ΔA/A0 indicate the relative emission and
absorbance intensities respectively. [H] corresponds the concen-
tration of pure host. The mole fraction of the host (XH) was
plotted against concentration of the complex [HG]. In the plot,
the mole fraction of the host at which the concentration of the
host–guest complex concentration [HG] is at a maximum, gives
the stoichiometry of the complex.
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Acknowledgements
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We thank DST and UGC, New Delhi, India for providing facili-
ties in the department. TS thanks CSIR, New Delhi, India for a
fellowship.
3242 | Org. Biomol. Chem., 2012, 10, 3236–3243
This journal is © The Royal Society of Chemistry 2012

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