Pyridinium-based symmetrical diamides as chemosensors in visual sensing of citrate through indicator displacement assay (IDA) and gel formation

Article abstract of DOI:10.1039/c1ob05707c

The design and synthesis of pyridinium-based symmetrical diamides 1 and 2 along with their anion binding studies through 'indicator-diplacement assay' are reported. Both the chemosensors effectively respond in in CH ₃CN-H₂O (4:1 v/v)

Full text of DOI:10.1039/c1ob05707c

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PAPER
Pyridinium-based symmetrical diamides as chemosensors in visual sensing of
citrate through indicator displacement assay (IDA) and gel formation†
Kumaresh Ghosh* and Avik Ranjan Sarkar
Received 4th May 2011, Accepted 23rd June 2011
DOI: 10.1039/c1ob05707c
The design and synthesis of pyridinium-based symmetrical diamides 1 and 2 along with their anion
binding studies through ‘indicator-diplacement assay’ are reported. Both the chemosensors effectively
respond in in CH3CN–H2O (4 : 1 v/v) at pH = 6.3 for the selective naked-eye detection of citrate.
Additionally, chemosensor 2 (c = 6.29 ¥ 10-3 M) forms a stable gel only with citrate in CH3CN, which
validates its visual sensing.
of these are based on positively charged, hydrogen bond groups
or unsaturated metal centers coordinated to 1,3,5-trialkylbenzene
Introduction
The design and synthesis of molecular receptors which respond
scaffolds which adopt a ‘fly-trap’ conformation.
in an “Indicator-Displacement Assay” (IDA) for sensing of
During the course of our work on the recognition of neu-
important analytes is an active area of research in supramolecular
tral, cationic and anionic substrates by our artificial designed
chemistry.1 In this technique, an indicator is first allowed to
bind reversibly to a receptor, and then a competitive analyte
is introduced into the system causing the displacement of the
indicator from the host, which in turn modulates the optical
signal. Anslyn’s group popularized this technique to such an extent
that it is now widely used and recognized as a standard method
for the development of sensors that are able to detect analytes
of biological significance selectively.1a Based on this technique,
the selective sensing of citrate,2 tartarate,3 phosphate,4 heparin,5
nitrate6 etc., is worth mentioning. Given the importance of
carboxylic acids and carboxylates in biology,7 interest in selective
sensing of citrate is appealing. Citric acid is a tricarboxylic acid
that plays an important role in the Krebs cycle to provide the
vast majority of energy used by aerobic cells in human beings.
Citrate is also the source of acetyl CoA, which is required for fatty
acid and cholesterol synthesis which is essential for proliferating
cells, tissue regeneration, embryogenesis and steroid hormone
synthesis. Determination of its levels is important in clinical
conditions and in normal cell metabolism. Diminished citrate
levels in urine have been linked to various aspects of kidney
dysfunction, for example in the pathogenesis of nephrolithiasis
and nephrocalcinosis.8 Several groups have reported different
receptor systems for the recognition of citrate/citric acid.2,9 Many
receptors,10 we herein report simple and easy-to-make receptor
modules 1 and 2 that respond efficiently in the IDA technique for
naked-eye detection of citrate among the other different anions
examined. In addition, receptor 2 formed a stable gel in CH3CN
in the presence of tetrabutylammonium citrate. This is considered
to be worthy in the area of gel chemistry. In recent times, as a
type of typical soft materials, gels have been of particular interest
in materials science due to their intriguing properties between
those of a solid and a liquid.11 Especially, low molecular weight
supramolecular gels which are formed due to interplay of weak
noncovalent forces have received considerable attention due to
their wide range of potential applications as soft materials in drug
delivery, tissue engineering and chemical sensing.12
Department of Chemistry, University of Kalyani, Kalyani, 741235,
India. E-mail: ghosh_k2003@yahoo.co.in; Fax: +913325828282;
Tel: +913325828750
† Electronic supplementary information (ESI) available: Spectral data
of receptor 1 and receptor 2. Figures showing the change in selected
fluorescence and UV-vis titration of receptor 1 and receptor 2 with various
anions, Job plots of receptor 2 with anions from fluorescence and UV-vis.
Selected binding constant curves of receptors 1 and 2 from fluorescence and
UV-vis. Indicator displacement experiments, DFT optimized geometry of
1. See DOI: 10.1039/c1ob05707c
Results and discussion
The compounds 1 and 2 were obtained according to the
synthesis shown in Scheme 1. Initially, the symmetrical di-
amide 4 was obtained by coupling of 3-aminopyridine with
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Table 1 Association constants (Ka) for 1 based on UV-vis methods in
CH3CN:H2O (4 : 1 v/v, pH = 6.3,10 mM Tris/HCl buffer)
Guestb
Ka/M-1a
Citrate
Pimelate
Tartarate
Malate
Adipate
Glutarate
Succinate
Malonate
N-Ts glutamate
F-
(2.34 0.01) ¥ 104
(4.05 0.05) ¥ 103
c
c
(1.05 0.01) ¥ 103
(1.17 0.02) ¥ 103
(1.62 0.01) ¥ 103
(1.02 0.01) ¥ 103
(4.99 0.09) ¥ 102
(1.12 0.01) ¥ 103
(9.94 0.07) ¥ 102
(8.98 0.07) ¥ 102
(9.96 0.07) ¥ 102
(1.01 0.03) ¥ 103
(1.61 0.02) ¥ 103
Cl-
Br-
I-
-
H2PO4
AcO-
a Determined by UV-vis method at the wavelength 295 nm. b All guests are
tetrabutylammonium salts. c Association constants were not determined
either due to minimal change or being difficult to determine.
Scheme 1 (i) 3-Aminopyridine, Et3N, dry CH2Cl2; (ii) 5, dry CH3CN and
DMF, reflux, 5 days; (iii) DMF/CH3OH, aq. solution of NH4PF6; (iv) 7,
dry CH3CN and DMF, reflux, 5 days; (v) DMF/CH3OH, aq. solution of
NH4PF6.
can be seen, the receptor 1 shows a greater affinity for citrate over
the other anions.
5-octyloxyisophthaloyl dichloride in dry CH2Cl2. Subsequent
reaction of the diamide 4 with different chloroamides such as
5 and 7 in dry CH3CN under refluxing conditions afforded the
corresponding dichloride salts 6 and 8, respectively. Finally, the
In a similar way, UV-vis and fluorescence titrations of 2 with
the same anionic substrates were conducted to understand its
interaction ability under similar conditions. The chemosensor 2
showed monomer emission at 385 nm upon excitation at 340 nm in
aqueous CH3CN (CH3CN–H2O = 4 : 1 v/v) at pH 6.3 (10 mM Tris
-
chloride anions in 6 and 8 were exchanged with PF6 ions to give
the desired compounds 1 and 2, respectively. All the compounds
were characterised by 1H NMR, 13C NMR, FTIR, mass and
elemental analyses.
-
HCl buffer). Upon complexation of citrate, pimelate and H2PO4
anions, the monomer emission of 2 was significantly perturbed (see
the ESI†). For instance, Fig. 2 represents the change in emission
of 2 upon titration with citrate.
The interaction properties of both 1 and 2 towards a series
of anions of different topologies were studied by fluorescence,
UV-vis and 1H NMR methods in CH3CN–H2O (4 : 1 v/v) at
pH = 6.3 (10 mM Tris HCl buffer). Compound 1, having no
fluorophore, was titrated with a number of anionic guests (taken
as their tetrabutylammonium salts) by the UV method and the
change in absorbance was noted. The change in absorbance of 1
was minor with the gradual addition of anions (see the ESI†) and
the binding stoichiometries of the complexes were evaluated to be
1 : 1 as confirmed by the Job method13 (Fig. 1). Analysis of the
absorbance data gave the binding constant values14 (Table 1). As
Fig. 2 Fluorescence titration spectra of 2 (c = 2.5 ¥ 10-5 M) in
CH3CN–H2O (4 : 1 v/v, pH = 6.3, 10 mM TrisHCl buffer) with the addition
of citrate. Inset: UV titration spectra of 2 (c = 2.5 ¥ 10-5 M) in CH3CN–H2O
(4 : 1 v/v, pH = 6.3, 10 mM TrisHCl buffer) with citrate.
It is of note that the receptor 2 exhibited a strong excimer
emission at 504 nm along with the monomer emissions at 388
and 408 nm in 5% CH3CN in CHCl3. Upon addition of a small
amount of citrate the excimer emission initially increased and
then decreased on progression of the titration (see the ESI†). The
monomer emission weakly changed in an irregular fashion. Fig. 3
is the combined profile for the change in fluorescence ratio of 2 at
385 nm upon addition of 10 equivalent amounts of each anion in
aqueous CH3CN (CH3CN–H2O = 4 : 1 v/v) at pH 6.3. However,
Fig. 1 UV-vis Job plots for 1 with tetrabutylammonium (a) citrate,
(b) adipate, (c) malonate, (d) pimelate, (e) dihydrogenphosphate and (f)
glutarate at 295 nm in CH3CN–H2O (4 : 1 v/v, pH = 6.3, 10 mM TrisHCl
buffer), where [G] = [H] = 8.5 ¥ 10-5 M at 25 ◦C.
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Fig. 4 Change in fluorescence ratio (DI/I0) of 2 (c = 6.27 ¥ 10-5 M)
upon addition of 3 equiv. amounts of tetrabutylammonium citrate in the
presence of other anions (3 equiv.) in CH3CN–H2O (4 : 1 v/v 10 mM Tris
HCl buffer at pH = 6.3) at 385 nm.
Fig. 3 Fluorescence ratio [(I - I0)/I0] of 2 (c = 6.27 ¥ 10-5 M) at 385 nm
upon addition of 10 equiv. of a particular anion in CH3CN–H2O (4 : 1 v/v,
pH 6.3, 10 mM Tris HCl buffer).
the change in absorbance upon titration was also appreciable (see
the ESI†), particularly with citrate (inset of Fig. 2).
Analysis of both emission and absorbance data gave the binding
constant values14 (Table 2). Like 1, the receptor 2 provided a
greater binding constant with citrate over the other anions studied,
attaining a 1 : 1 binding stoichiometry (Table 2).
Selectivity in the binding process was established by observing
the emission behavior of 2 upon addition of citrate to the solution
of 2 in aqueous CH3CN (CH3CN–H2O = 4 : 1 v/v) at pH 6.3
containing other interfering anions. In this context, Fig. 4 shows
the change in emission behavior when establishing the selectivity
for citrate. It is evident from Fig. 4 that the anions studied
negligibly interfere in the binding of citrate.
Furthermore, we studied the interaction of 2 with the anions
at pH 7.3. Here, the change in emission of 2 was found to be
considerably less than at pH 6.3 (Fig. 5). This suggested the
inefficiency of 2 in slightly alkaline medium.
Fig. 5 Fluorescence ratio [(I - I0)/I0] of 2 (c = 6.27 ¥ 10-5 M) at 385 nm
upon addition of 10 equiv. of a particular anion in CH3CN–H2O (4 : 1 v/v
10 mM Tris HCl buffer) at pHs 6.3 and 7.3.
Once the binding selectivities of 1 and 2 toward citrate were
established, we investigated the naked-eye detection of citrate
following the indicator displacement assay with the dye fluorescein
(uranine) 3. Dye 3, an aromatic di-anion, was found to interact
with both 1 and 2. The absorption and emission properties of
3 were exploited to check its binding affinity for both 1 and 2.
Upon addition of 1 (c = 4.2 ¥ 10-4 M) to a solution of 3 (c = 8.0 ¥
10-5 M) in aqueous CH3CN (CH3CN–H2O = 4 : 1 v/v, 10 mM Tris
HCl buffer, pH = 6.3) both the absorbance and emission of 3 were
significantly reduced (see the ESI†) and the color of the resulting
solution became colorless (Fig. 6).
Table 2 Association constants (Ka) for 2 based on UV-vis methods in
CH3CN–H2O (4 : 1 v/v, pH = 6.3,10 mM Tris/HCl buffer)
Guesta
Ka/M-1b
Ka/M-1c
Citrate
Tartarate
Malate
Pimelate
Adipate
Glutarate
Succinate
Malonate
N-Ts glutamate
F-
(9.05 0.06) ¥ 104
(7.02 0.7) ¥ 104
d
d
d
d
(3.97 0.05) ¥ 103
(1.62 0.04) ¥ 103
(1.50 0.02) ¥ 103
(1.49 0.02) ¥ 103
(1.54 0.01) ¥ 103
(6.24 0.08) ¥ 102
(6.88 0.1) ¥ 103
(1.18 0.01) ¥ 103
d
(1.8 0.02) ¥ 103
d
d
d
d
d
d
d
Fig. 6 (1) Dye 3, (2) receptor 1 + dye 3 (1 : 1) = A. A with 1 equiv. amount
of (3) Cl-, (4) pimelate, (5) glutarate, (6) H2PO4-, (7) tartarate, (8) malate,
(9) adipate, (10) N-Ts glutamate, (11) malonate, (12) acetate, (13) glutarate,
(14) citrate; [3] = 8.0 ¥ 10-5 M, [1] = 4.2 ¥ 10-4 M, [G] = 4.62 ¥ 10-3 M.
Cl-
(1.07 0.02) ¥ 103
Br-
d
I-
d
-
H2PO4
(3.47 0.01) ¥ 103
(1.81 0.03) ¥ 103
(8.21 0.02) ¥ 103
AcO-
d
Gradual addition of 2 (c = 4.2 ¥ 10-5 M) to a solution of 3
(c = 8.5 ¥ 10-5 M) in CH3CN–H2O (4 : 1 v/v, 10 mM Tris HCl
buffer, pH = 6.3) also resulted in similar changes in absorption and
emission characteristics of 3 to that of 1 with 3. Upon interaction
with 2, the solution of 3 became colorless (Fig. 7).
a All guests are tetrabutylammonium salts. b Determined by fluorescence
method at the wavelength 385 nm. c Determined by UV-vis method at the
wavelength 340 nm. d Association constants were not determined either
due to minimal change or being difficult to determine.
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Fig. 7 (1) Dye 3, (2) receptor 2 + dye 3 (1 : 1) = B. B with 1 equiv. amount
of (3) Cl-, (4) pimelate, (5) succinate, (6) H2PO4-, (7) tartarate, (8) malate,
(9) adipate, (10) N-Ts glutamate, (11) malonate, (12) acetate, (13) glutarate,
(14) citrate; [3] = 8.5 ¥ 10-5 M, [2] = 4.2 ¥ 10-4 M, [G] = 2.0 ¥ 10-3 M in
CH3CN–H2O (4 : 1 v/v, 10 mM Tris HCl buffer, pH = 6.3).
The 1 : 1 stoichiometries for 1 and 2 with the dye 3
were established using Job’s method of continuous variation13
(Fig. 8). Analysis of absorption titration data in both cases gave
the binding constant values14 (1.08 0.001) ¥ 104 M-1 and (3.89
0.1) ¥ 104 M-1 for 1 and 2 with the dye 3, respectively (see the ESI†).
Moreover, fluorometric titration resulted in the binding constant
value (7.59 0.3) ¥ 104 M-1 for 2 with 3. However, these values are
close in magnitude to the binding constant values for 1 and 2 with
citrate, and thus the combinations of 1/3 and 2/3 will be ideal
ensembles for the sensing of citrate. The selectivity of an indicator
displacement assay is meaningful when the receptor has the same
or slightly lower affinity than the analyte of choice, but a larger
affinity than other competing analytes.15
Fig. 9 (a) Change in emission of dye 3 (c = 8.0 ¥ 10-5 M) upon increasing
addition of 1 (c = 4.2 ¥ 10-4 M) and (b) retrieval of emission upon increasing
addition of citrate (c = 4.62 ¥ 10-3 M) to the ensemble 1/3 in CH3CN–H2O
(4 : 1, v/v, pH 6.3, 10 mM Tris/HCl buffer).
The strong binding of citrate to 1 and 2 was further understood
from the 1H NMR. During titration of 1 (c = 6.29 ¥ 10-3 M) with
citrate, the amide protons Ha and Hb moved in the downfield di-
rection significantly. While the pyridinium protons Ho¢ underwent
downfield shift, the Ho protons showed upfield chemical shift, and
thereby we suggested a binding structure where the pyridinium
groups are involved in binding in a tilted fashion (Fig. 10). The
non planar structure of the binding site is also evident from the
DFT optimized structure of 116 (see the ESI†).
A similar titration experiment was applied to 2 (c = 6.29 ¥
10-3 M) with citrate, but we failed to complete the titration (Fig. 11)
due to the formation of gel in the NMR tube. In this case, the
amide protons as well as the pyridinium Ho protons moved in the
downfield direction with significant broadening.
Fig. 8 Job plots of dye 3 (c = 8.0 ¥ 10-5 M) with (a) 1 (c = 8.0 ¥ 10-5 M)
and (b) 2 (c = 8.0 ¥ 10-5 M) at 502 nm from UV-vis in CH3CN–H2O (4 : 1
v/v, pH = 6.3, 10 mM Tris/HCl buffer) at 25 ◦C.
Upon addition of citrate to the ensemble of 1/3, dye 3 is
displaced from the binding cavity and both its absorbance and
fluorescence are restored. In this regard, for example, while Fig. 9a
shows the gradual reduction in emission of 3 (lex = 480 nm and
lem = 530 nm) upon successive addition of 1, Fig. 9b demonstrates
the change in emission of 3, which is retrieved upon gradual
addition of citrate to the ensemble of 1/3.
The color of the solution turned into the original color of the
dye (Fig. 6). The case with the ensemble of 2/3 was similar (see
the ESI†). Anions that are less efficiently bound by both 1 and 2
than 3 were not capable of displacing dye 3. Even substrates such
as malate, tartrate etc., which are closely related to citrate, did not
restore the original greenish yellow color of the dye solution. They
were also unable to restore absorbance as well as fluorescence.
Figs. 6 and 7 corroborate the naked eye detection of citrate via the
IDA approach by both 1 and 2, respectively. Importantly, both 1
and 2 did not respond in naked eye detection of citrate via the IDA
approach at pH 7.3 (see the ESI†).
Due to strong interaction, chemosensor 2 formed a gel in the
presence of citrate in CH3CN in the concentration range ~10-3 M.
Thus, citrate in the present example acts as a chemical stimulus
in gel formation. However, under identical conditions, 1 did not
produce a gel with citrate, thereby suggesting a role of pyrene in
maintaining a hydrophobic/hydrophilic balance in 2. A survey
of the literature reveals that citrate-selective gel formation by a
suitable abiotic receptor which can be useful in visual sensing
of citrate by the naked eye is unknown in the literature. To our
belief, the present example is the first report on visual recognition
of citrate through gelation of an organic molecule. Receptor 2
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1
Fig. 11 Change in H NMR of 2 (c = 6.29 ¥ 10-3 M) upon addition of
citrate (c = 2.17 ¥ 10-2 M).
Fig. 12 Photographs showing phase changes for a gel of 2 (c = 6.29 ¥
10-3 M) with citrate in CH3CN.
Fig. 10 (A) Change in 1H NMR of 1 (c = 6.29 ¥ 10-3 M) upon addition
of citrate (c = 2.17 ¥ 10-2 M). (B) Suggested hydrogen bonding structure of
1 with citrate.
(c = 6.29 ¥ 10-3 M) on mixing with tetrabutylammonium citrate
in CH3CN produced a gel (Fig. 12) which underwent a gel-to-
sol phase transition at 92 ◦C. On cooling the sol below 92 ◦C,
gel formation again took place. Under similar conditions no such
anion binding-induced gel formation took place in presence of the
other anions.
Importantly, the gel formation took place only in CH3CN
and not in other solvent combinations such as CH3CN/CHCl3,
CH3CN/H2O. In this respect, Table 3 describes the gelation study
of 2 in various solvents at 25 ◦C. The gel was characterized by
recording SEM images (Fig. 13). The SEM images showed the
presence of densely coiled fibers and also globular particles. The
different pictures based on resolution are represented in Fig. 13.
Fig. 13 SEM image of dried gel; scale bar: (A) 1 mm, (B) 10 mm.
Furthermore, it is established for the first time that sensor 2 is
also capable of sensing citrate visually through gel formation in
CH3CN solvent. The conventional N–H ◊ ◊ ◊ O, and unconventional
C–H ◊ ◊ ◊ O hydrogen bondings and charge–charge interaction are
the weak forces that interplay cooperatively into the clefts of 1 and
Table 3 Gelatination study of 2 with tetrabutylammonium citrate in
various solvents at 25 ◦
C
Solvent
mgc/mg ml-1
Gel status
CH3CN
7.9
7.9
7.9
7.9
7.9
formed (opaque gel)
not formed (ppt)
not formed (s)
not formed (ppt)
not formed (ppt)
5% CH3CN in CHCl3
1% DMSO in CH3CN
10% CH3CN in CHCl3
Conclusion
CH3CN–H2O 4 : 1 (v/v)
In conclusion, we have presented here two new sensors, 1 and
2, for the naked eye detection of citrate over a series of other
anions in a semi-aqueous system through the IDA technique.
mgc = minimum gelatination concentration, s = soluble, ppt = precipitate.
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2 to make them selective towards citrate ion. Further progress in
this direction is underway in our laboratory.
2-Chloro-N-(4, 6-dihydropyren-1-yl)acetamide (7). To a stirred
solution of 1-aminopyrene (0.57 g, 2.31 mmol) in CHCl3 (30 mL),
chloroacetyl chloride (0.29 g, 2.54 mmol) was added dropwise
◦
at 0 C. Then water (10 mL) was added to the reaction mixture
Experimental
followed by addition of K2CO3 (0.48 g, 3.46 mmol). A catalytic
amount of tetrabutylammonium hydrogensulphate was added to
the reaction mixture and the mixture was stirred for 4 h. After
completion of the reaction, as monitored by TLC, the organic
layer was separated and dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated under reduced pressure. The residual
mass was purified by silica gel column chromatography using 1%
CH3OH in CHCl3 as eluent to afford pure compound 7 (0.630 g,
General methods
Materials were obtained from commercial suppliers and were used
without further purification. Thin layer chromatography (TLC)
was carried out using Merck 60 F254 plates with a thickness of
0.25 mm. Melting points were determined on a hot-plate melting
point apparatus in an open-mouth capillary and are uncorrected.
1H and 13C NMR spectra were recorded on a Bruker 400 MHz
instrument. For NMR spectra, CDCl3, CD3CN and d6-DMSO
were used as solvents, using TMS as an internal standard. IR
spectra were recorded on a Perkin Elmer Spectrum One, using
KBr discs. Fluorescence spectra were recorded on a Perkin Elmer
Model LS 55 spectrophotometer and UV-vis spectra were recorded
on a Perkin Elmer Model Lambda 25.
◦
1
92%), mp. 204 C. H NMR (CDCl3, 400 MHz): d 9.05 (s, 1H,
amide NH), 8.44 (d, 1H, J = 8 Hz), 8.22–8.14 (m, 4H), 8.07–8.01
(m, 4H), 4.44 (s, 2H); 13C NMR [d6-DMSO (for homogeneity of
the solution), 100 MHz]: d 165.8, 130.9, 130.7, 130.4, 128.6, 127.3,
127.1, 126.8, 126.4, 125.3, 125.0, 124.9, 124.3, 124.0, 123.7, 123.3,
122.0, 57.4; FTIR (KBr, n in cm-1): 3234, 3114, 3021, 2957, 1663,
1598, 1525, 840; Mass (LCMS): 295.2 (M + 2)+, 294.2 (M + 1)+.
5-(Octyloxy)-N1, N3-di(pyridin-3-yl)isophthalamide (4). The
bisamide 4 was obtained by reaction of 3-aminopyridine (0.59 g,
6.34 mmol) with 5-(octyloxy)isophthaloyl dichloride (0.7 g,
2.11 mmol) in 60 mL dry CH2Cl2 in the presence of Et3N
(1 mL) under nitrogen atmosphere. The reaction mixture was
stirred overnight. After completion of the reaction, the solvent
was removed under vacuum. The residual mass was extracted
with a CHCl3/CH3OH mixture solvent (3 ¥ 30 mL). The organic
layer was washed with NaHCO3 solution (3 ¥ 20 mL) and dried
over anhydrous Na2SO4 and filtered. The filtrate was concentrated
under reduced pressure. The residual mass was purified by column
chromatography over silica gel using ethyl acetate–hexane (3 : 2
v/v) as eluent to afford the desired compound 4 (0.820 mg, 86%
yield, mp 92 C). H NMR (CDCl3 containing few drops of d6-
DMSO, 400 MHz): d 9.85 (s, 2H, amide NH), 8.90 (d, 2H, J =
4 Hz), 8.37–8.34 (m, 4H), 8.18 (s, 1H), 7.73 (s, 2H), 7.32 (t, 2H, J =
8 Hz), 4.09 (t, 2H, J = 8 Hz), 1.86–1.79 (m, 2H), 1.49–1.44 (m, 2H),
1.35–1.25 (m, 8H), 0.91 (t, 2H, J = 8 Hz); 13C NMR (d6DMSO,
100 MHz): d 165.0, 158.7, 144.8, 142.1, 135.9, 135.6, 127.4, 123.5,
119.4, 116.8, 68.2, 31.2, 28.7, 28.7, 28.6, 25.5, 22.1, 13.9; FTIR
(KBr, n in cm-1): 3349, 2914, 2850, 1668, 1543, 1486, 1426, 1294,
1240, 802; Mass (LCMS): 447.2 (M + 1)+, 240.2, 224.2. Anal cal.
C26H30 N4O3: C, 69.93; H, 6.77; N, 12.55; Found: C, 69.81; H, 6.72;
N, 12.49.
Synthesis of receptor (1). To a solution of 4 (0.100 g,
0.223 mmol) in dry CH3CN (10 mL) containing dry DMF (2 mL),
compound 5 (0.091 g, 0.672 mmol) in dry CH3CN (20 mL) was
added. The reaction mixture was refluxed with stirring for 5
days under nitrogen atmosphere. On cooling the reaction mixture,
precipitate appeared. The precipitate was filtered off and washed
with CH3CN several times and then with diethyl ether to give pure
dichloride salt 6 (0.130 g, 81%). The pure dichloride salt 6 (0.130 g,
0.181 mmol) was dissolved in 5 mL hot CH3OH containing a
few drops of DMF and the volume was reduced to 2 mL. Then
aqueous solution of NH4PF6 (0.089 g, 0.54 mmol) was added
in one portion to carry out the anion exchange reaction. After
stirring the reaction mixture for 35 min, precipitate appeared.
Filtration of the precipitate followed by thorough washing with
diethyl ether afforded the receptor 1 in 65% yield (0.102 g), mp
116 ◦C. 1H NMR (CD3CN, 400 MHz): d 9.80 (s, 2H, amide NH),
9.53 (s, 2H), 8.65 (d, 2H, J = 8 Hz), 8.41 (d, 2H, J = 8 Hz), 8.23
(s, 1H), 8.07–8.03 (m, 2H), 7.75 (s, 2H), 6.99 (br s, 2H, amide
NH), 5.27 (s, 4H), 4.21 (t, 2H, J = 8 Hz), 3.25–3.21 (m, 4H), 1.95–
1.83 (m, 2H), 1.60–1.49 (m, 6H), 1.40–1.24 (m, 8H), 0.97–0.91
(m, 9H); 13C NMR (CDCl3, containing few drops of d6-DMSO,
100 MHz): 165.88, 163.37, 159.51, 140.01, 139.80, 136.73, 135.50,
134.68, 127.55, 119.24, 118.21, 68.75, 62.70, 41.74, 31.64, 29.18,
29.09, 28.98, 25.85, 22.48, 22.2, 14.0, 11.32; FTIR (KBr, n in cm-1):
3419, 2966, 2933, 2878, 1680, 1594, 1557, 1462, 838; Mass (ESI):
790.9 (M - PF6-1)+, 645.1 (M - 2PF6-1)+; Anal cal. C36H50 N6O5
(PF6)2: C, 46.16; H, 5.38; N, 8.97; Found: C, 46.12; H, 5.26; N,
8.83.
◦
1
2-Chloro-N-propylacetamide (5). To a stirred solution of n-
propylamine (2 g, 33.84 mmol) in CHCl3 (30 mL), chloroacetyl
chloride (4.2 g, 37.2 mmol) was added dropwise at 0 ◦C. Then water
(10 mL) was added to the reaction mixture followed by addition
of K2CO3 (7.01 g, 50.76 mmol). A catalytic amount of tetrabuty-
lammonium hydrogensulphate was added to the reaction mixture
and stirred for 4 h. After completion of the reaction, as monitored
by TLC, the organic layer was separated and dried over anhydrous
Na2SO4 and filtered. The filtrate was concentrated under reduced
pressure. The residual mass was purified by silica gel column
chromatography using 1% CH3OH in CHCl3 as eluent to afford
Synthesis of receptor (2). To a solution of 4 (0.08 g, 0.18 mmol)
in dry CH3CN (10 mL) containing dry DMF (2 mL), compound 7
(0.174 g, 0.59 mmol) dissolved in CH3CN–DMF (1 : 1 v/v, 20 mL)
was added. A catalytic amount of tetrabutylammonium iodide was
added to the reaction mixture and the mixture was refluxed for 5
days under nitrogen atmosphere. On cooling the reaction mixture,
precipitate appeared. The precipitate was filtered off and washed
with CH3CN several times and then with diethyl ether to give pure
dichloride salt 8 (0.147 g, 79%). The dichloride salt 8 (0.100 g,
0.09 mmol) was next dissolved in 5 mL hot CH3OH containing
a few drops of DMF and the volume was reduced to 2 mL.
◦
1
pure compound 5 (3.65 g, 95%), bp. 88 C. H NMR (CDCl3,
400 MHz): d 6.84 (s, 1H), 4.05 (s, 2H), 3.27 (q, 2H, J = 8 Hz),
1.62–1.53 (m, 2H), 0.95 (t, 2H, J = 8 Hz); FTIR (KBr, n in cm-1):
3423, 3300, 2967, 2937, 1662, 1545; Mass (ESI): 136.1 (M + 1)+.
6556 | Org. Biomol. Chem., 2011, 9, 6551–6558
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Subsequently, aqueous solution of NH4PF6 (0.048 g, 0.29 mmol)
was added in one portion to carry out the anion exchange. After
stirring the reaction mixture for 35 min, precipitate appeared.
Filtration of the precipitate followed by thorough washing with
diethyl ether afforded the receptor 2 in 89% yield (0.102 g); mp.
188 ◦C (decomposition). 1H NMR (d6-DMSO, 400 MHz): d 11.48
(s, 2H, amide NH), 11.06 (s, 2H, amide NH), 9.75 (s, 2H), 8.96 (br
s, 2H), 8.79 (d, 2H, J = 8 Hz), 8.48 (d, 2H, J = 8 Hz), 8.33–8.27
(m, 14H), 8.18 (br s, 3H), 8.10 (t, 2H, J = 8 Hz), 7.95 (s, 2H),
6.0 (s, 4H), 4.17 (t, 2H, J = 6.4 Hz), 1.81–1.79 (m, 2H), 1.46–1.44
(m, 2H), 1.40–1.26 (m, 8H), 0.91 (t, 3H, J = 6.4 Hz); 13C NMR
(d6-DMSO, 100 MHz): d 165.39, 164.40, 162.30, 158.80, 141.50,
137.61, 136.08, 134.98, 130.76, 130.48, 130.40, 128.66, 127.60,
127.39, 127.14, 126.95, 126.55, 125.48, 125.15, 125.00, 124.31,
123.89, 123.73, 122.96, 122.17, 119.86, 117.63, 68.34, 62.77, 31.2,
28.69, 28.63, 28.53, 25.47, 22.04, 13.91; FTIR (KBr, n in cm-1):
3383, 2927, 2855, 1688, 1593, 1508, 1459, 1340; Mass (ESI): 1107.5
(M - PF6)+, 961.7 (M - 2PF6-1)+, 704.6, 481.6; Anal cal. C62H54
N6O5 (PF6)2: C, 59.43; H, 4.34; N, 6.71; Found: C, 59.53; H, 4.42;
N, 6.63.
I = (I0 + IKCG)/(1 + KCG) . . .
(1)
I = I0+ ((I - I0)/(2CH))(CG + CH + 1/K - ((CG + CH + 1/K)2
- 4CGCH)0.5) . . .
(2)
Where I represents fluorescence intensity; I0 represents the inten-
sity of pure host; CH and CG are the corresponding concentrations
of host and the guest; K is the association constant (this
equation also works in absorption). The association constants
and correlation coefficients (R) were obtained by a non-linear
least-square analysis of I vs. CG for eqn (1) and the in case of eqn
(2) the association constant and correlation coefficients (R) were
obtained by a non-linear least-square analysis of I vs. CH and CG.
Acknowledgements
We thank CSIR, New Delhi, India for financial support. ARS
thanks University of Kalyani, India for a fellowship.
References
General procedure for fluorescence and UV-vis titrations
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Stock solutions of the receptors were prepared in 4 : 1(v/v)
CH3CN–H2O containing 10 mM Tris/HCl buffer pH = 6.3 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 concentration
range ~10-4 M, were prepared in the same solvents and were
individually added in different amounts to the receptor solution.
Upon addition of guests, the change in emission of the receptor
was noted. The same stock solutions for receptors and guests were
used to perform the UV-vis titration experiment. Guest solution
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 ◦C. All the experiments were repeated thrice
to check the reproducibility.
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Job plots
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The stoichiometry was determined by the continuous variation
method (Job Plot).13 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 different
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 calculated using
the equation [HG] = DI/I0 ¥ [H] or [HG] = DA/A0 ¥ [H] where
DI/I0 and DA/A0 indicate the relative emission and absorbance
intensities. [H] corresponds to the concentration of pure host.
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 maximum, gives the stoichiometry of the complex.
Determination of binding constants
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Binding constant values were determined by fluorescence and
absorption methods using eqn (1) and 2.14
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6558 | Org. Biomol. Chem., 2011, 9, 6551–6558
This journal is
The Royal Society of Chemistry 2011
©