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Cite this: New J. Chem., 2012, 36, 1231–1245

www.rsc.org/njc PAPER
Design and synthesis of anthracene-based bispyridinium amides: anion
binding, cell staining and DNA interaction studiesw
Kumaresh Ghosh,*a Avik Ranjan Sarkar,a Atanu Ghoraib and Utpal Ghoshb
Received (in Montpellier, France) 9th December 2011, Accepted 20th February 2012
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

DOI: 10.1039/c2nj21024j

The design and synthesis of anthracene labeled bispyridinium amides 1–4 along with their anion
binding, cell staining and DNA interaction studies are reported. All the chemosensors exhibit
significant response towards H2PO4 in CH3CN. Furthermore, sensors 2 and 3 are quite
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interesting for the selective sensing of aliphatic dicarboxylates. Among these compounds, 1 is
found to be useful in cell staining. Also all of them exhibit significant interaction with DNA. All
these properties are found to be dependent on the nature of the spacer that holds the pyridinium
binding sites.

Introduction CH proton donors.12 Monocationic carboxamide-1-alkyl-

Selective sensing of anions of biological and environmental
relevance by synthetic hydrogen bonding receptors is an
important research topic in supramolecular chemistry.1 In
relation to this, the binding unit, which is capable of binding
anions strongly or moderately, has the crucial role in giving
the selectivity to the designed receptor. Examples in this field
are available in the literature.2 Among the various types,
amide,3 urea,4 guanidinium,5 amidinium,6 imidazolium,7
benzimidazolium,8 polyammonium cations,9 etc. are widely
used in anion binding and they were found to be worthy in
functioning. In this context, the pyridinium ion is another
motif, which was first used by Jeong and Cho in the binding of
carboxylates.10 The pyridinium motif provides hydrogen-
bonding donors (both conventional and unconventional) for
complexation of anions and the complex is further stabilized
by charge–charge interaction. Keeping this view in mind it
was explored later on by other groups. Steed et al. used this
motif in the binding of chloride ions in a tripodal cavity.11
Tricationic N-alkylpyridinium receptors containing sulfonamide
or pyrrole moieties were found to provide highly efficient anion
complexation via simultaneous binding to NH and pyridinium
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 Biochemistry and Biophysics, University of Kalyani,
Kalyani-741235, India
w Electronic supplementary information (ESI) available: Spectral data
of receptors 1–4. Figures showing the change in fluorescence and
UV-vis spectra of receptors 1–4, Job plots of 1–4 with anions from
fluorescence and UV-vis. Selected binding constant curves from
fluorescence and UV-vis. ROSEY spectra of receptors 1–4.
1
H NMR spectra for interaction study. 31P NMR of H2PO4 with
1–4 in d6-DMSO. MOs of 1–4 and their 1 : 1 complexes with H2PO4.
Gel retardation and cell staining study. Interaction profile in a semi-
aqueous system. CD study. See DOI: 10.1039/c2nj21024j
pyridinium receptors have been used for anion-templated
pseudorotaxane formation.13 During the course of our work
in the area of supramolecular chemistry, we also used this motif
in various designs for sensing of anions such as H2PO4,14
dicarboxylates,15 ATP,16 etc. Recently, Dorazco-Gonzalez et al.
have reported the recognition behaviour of dicationic pyridine-2,6-
dicarboxamide receptors for anions and neutral guests indicating
our approach on the dicationic bisamide receptor derived from
isophthalic acid and two 3-aminopyridine.17 In their recent report,
they have compared the binding ability of the dicationic receptors
with their neutral precursors.
In continuation of our previous work on fluorescent dicationic
bisamide receptor 1 derived from isophthalic acid for anions,18
we report here the synthesis and anion sensing properties of a
series of dicationic receptors 2–4 (Fig. 1) where the cationic
binding sites are hooked on the different synthetic spacers. The
DNA binding and cell expression properties of these molecules
have also been evaluated.
Results and discussion
The dicationic receptors are all anthracene-based and they
were obtained according to Scheme 1. The neutral precursors
of the cationic salts were synthesized in a single step by
coupling 3-aminopyridine with the different diacidchlorides.
They were reacted with 9-chloromethylanthracene in dry
CH3CN under reflux to give the dichloride salts. The dichloride
salts were treated with NH4PF6 in aq. CH3OH to afford the
desired products.
Anion binding studies in organic solvents
The anion binding ability of receptor 1 among the series was
reported in our earlier publication.18 A series of anions such as
tetrabutylammonium salts of F, Cl, Br, I, AcO and H2PO4

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 1231–1245 1231

Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
Fig. 1 Chemical structures for 1–4.
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Scheme 1 (i) For 5: isophthaloyl dichloride, dry DCM, dry Et3N, rt;
(ii) For 6: pyridine-2,6-dicarbonyl dichloride, dry DCM, dry Et3N, rt;
(iii) For 7: glutaroyl dichloride, dry DCM, dry Et3N, rt; (iv) For 8:
adipoyl dichloride, dry DCM, dry Et3N, rt; (v) 9-(chloromethyl)-
anthracene, dry CH3CN containing few drops of dry DMF, stirring
and heating for 36 h; (vi) aq. NH4PF6, CH3OH, stirring for 2 h.
were undertaken in the study. Receptor 1 exhibited selective
sensing of H2PO4 in CH3CN. In the presence of H2PO4
ions, the monomer emission of 1 was greatly increased along
with the appearance of a new peak at 500 nm, ascribed to the
excimer formation between the closely spaced anthracene
moieties. This excimer formation allowed H2PO4 ions to be
distinguished from the other anions studied. Receptor 1
showed a modest affinity to AcO and F and is weakly
bound to other halide anions. Fig. 2 describes the change in
emission of 1 with the addition of 10 equiv. amounts of a
particular anion. The possible reason for the low affinity of
this receptor was the unfavourable conformation19 of the
1232 New J. Chem., 2012, 36, 1231–1245 This journal is
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Fig. 2 Fluorescent emission changes of 1 (6.56  105 M) upon
addition of various anions (10 equiv.) in CH3CN.
isophthaloyl moiety. In the present study, we took the same
anions for receptors 2–4 to see the effect of the synthetic
spacers on the conformation of the molecules as a result of
which sensitivity and selectivity towards the anions are changed.
As we move from compound 1 to the others such as 2, 3 and 4,
the change in photophysical properties of them was found to
vary with the input anions. Figs. 2–5 are the representative plots
where the fluorescence selectivity of the individual receptor is
clearly understood. Irrespective of the nature of the spacer that
holds the anthracene coupled pyridinium moieties, the change
in emission of the receptors is found to be significant for
H2PO4 ions. Figs. 6a–d display the change in emission of
1–4 upon complexation with H2PO4.
In the complexation event, while the monomer emissions of
2–4 are little perturbed, the excimer emissions are amplified to
a significant extent. The appearance of such intense peak
at B500 nm is attributed to the complexation induced closeness
of the anthracene moieties either intermolecularly or intra-
molecularly. Depending on the nature of the linker in between
the pyridinium moieties the intensity of the excimer emission
varies. As we proceed from 1 to 2, the intensity of the excimer
emission is found to increase many fold. This is presumably due
to less conformational mobility of 2 than 1. Isophthaloyl
diamides can attain different conformations A–C (Fig. 7).19
Of these, conformation A is less stable in solution due to steric
repulsion between the amide and peri hydrogens. This is
avoided in 2 where the isophthaloyl group is replaced by the
Fig. 3 Fluorescent emission changes of 2 (6.56  105 M) upon
addition of tetrabutylammonium salts of various anions up to 10 equiv.
in CH3CN.
c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
 View Article Online

Fig. 4 Fluorescent emission changes of 3 (6.56  105 M) upon

Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

addition of tetrabutylammonium salts of various anions up to 10 equiv.

in CH3CN.
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Fig. 5 Fluorescent emission changes of 4 (6.56  105 M) upon

addition of tetrabutylammonium salts of various anions up to 10 equiv.
in CH3CN.

pyridine moiety and accordingly conformation of type A20 will

dominate in solution over the others.
Glutaroyl and adipoyl linkers in 3 and 4 introduce greater
flexibility. As a result, the complexation induced repositioning
of the anthracenyl groups will be easier, in principle. This is
reflected in the emission profiles of 3 and 4 in the presence of
H2PO4 ions. In both cases, the growth of the excimer
emission is mentionable (Fig. 6c and d). Between 3 and 4,
only the growth of excimer in 4 is significantly greater than 1.
In the case of 3 this is less compared to 1. Such difference in
intensity of the excimer emission is presumably partly due to
the odd/even number of the methylene groups present in the
linker chain which control the orientation of the pyridinium
binding sites. Like H2PO4, HSO4, ClO4 being tetrahedral
in shape showed a comparatively weak change in monomer as
well as in excimer emission. However, receptor 2 behaved similarly
Fig. 6 Fluorescence titration of (a) 1 (c = 6.56  105 M), (b) 2
toward the other anions like 1. AcO, F, propanoate, etc.
(c = 6.56  105 M), (c) 3 (c = 6.56  105 M) and (d) 4 (c = 6.56 
perturbed the emission of 2 weakly. Interaction of these 105 M) with H2PO4 in CH3CN at 25 1C. (lex = 365 nm); inset:
anions with 3 and 4 was negligible and thereby suggested the fluorogenic colour changes of 1–4 upon addition of 10 equiv. of
necessarily noncooperative interaction of the pyridinium H2PO4 and irradiation under UV-light.
moieties in the binding event under the guidance of the
spacers used. by UV Job plots (Fig. S2c in ESIw).21 Such difference in
In the interaction process, receptors 1–4 exhibited 2 : 1 stoichiometry in the excited and ground states is presumably
stoichiometries with H2PO4 as proved by the fluorescence due to the difference in polarity in the two states. In this
Job’s plots (Fig. 8).21 It is interesting to note that in the ground regard, our observation is in accordance with the report by
state all the receptors attained 1 : 1 stoichiometries, confirmed Dorazco-Gonzalez et al.17

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 1231–1245 1233
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Fig. 7 The possible three major conformations of 1.

Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
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Fig. 8 Fluorescence Job plots for receptors 1–4 with H2PO4 in

CH3CN at 412 nm, where [G] = [H] = 6.56  105 M.

After determining the stoichiometry of the complexes of

receptors 1–4 with H2PO4 in the excited state, analysis of the
emission data gave the binding constant values which were
calculated with the help of a nonlinear 2 : 1 binding isotherm.22
The binding constant values of receptors 1–4 for H2PO4
were calculated to be (for 1 K11 = 1.48  0.3  104 M1,
K12 = 3.29  0.4  104 M1), (for 2 K11 = 8.57  1.4 
103 M1, K12 = 2.29  0.3  104 M1), (for 3 K11 = 1.18 
0.2  104 M1, K12 = 2.08  0.4  104 M1) and (for 4 K11 =
1.29  0.1  104 M1, K12 = 1.48  0.1  104 M1).
Previously it was reported that receptor 1 forms a 1 : 1 complex
with the F (1.94  0.3  103 M1) and AcO (7.07  1 
103 M1) in the excited state. Now this is also true for receptor
2 with F (6.15  103 M1) and AcO (6.97  103 M1). For
receptors 3 and 4, due to the presence of a flexible linker the
binding of other anions except H2PO4 was found to be Fig. 9 Change in the fluorescence ratio of receptor (a) 1 (c = 6.56 
insignificant as indicated by the very minor changes in emission 105 M), (b) 2 (c = 6.56  105 M), (c) 3 (c = 6.56  105 M), (d) 4
spectra (Figs. 4 and 5). Therefore we were unable to determine (c = 6.56  105 M) upon addition of 2 equivalent amounts of H2PO4
in the presence of other anions in CH3CN.
the binding constants for them accurately. Thus the experi-
mental findings illustrate that the receptors are very selective to
H2PO4 in the recognition process. As can be seen from While the emission of the anthracene unit in the receptors was
Fig. 9a–d, the change in emission of the receptors upon addition changed marginally in the presence of H3PO4, it was significantly
of H2PO4 was almost unperturbed in the presence of other changed upon addition of an equivalent amount of tetrabutyl-
anions expect F. In the presence of F, H2PO4 anion binding ammonium hydroxide (TBAOH) to the solution containing
induced change in emission of the receptors was found to be less receptor and H3PO4. Even the excimer emission, in each case,
and thus demonstrated its interference in the binding process. was noticed. As previously explained, TBAOH causes
Interestingly receptors 1–4 were almost non-interacting deprotonation of H3PO4 to give anions (H2PO4, HPO42
with H3PO4. A control experiment for this was performed. or PO43, etc.), which bind to the cleft of the receptors more

1234 New J. Chem., 2012, 36, 1231–1245 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
 View Article Online

Fig. 10 Emission profile for sensitivity of receptor 1 with H2PO4

Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

over H3PO4.

efficiently than H3PO4. Fig. 10 explains this phenomenon with

receptor 1. Other receptors 2–4 behaved similarly and the
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observations are available in the ESIw (Figs. S4a–c).

In a wider aspect receptors 1–4 were further employed in the
binding studies of aliphatic dicarboxylates of different chain
lengths although the binding of monocarboxylate (i.e. AcO,
propanoate, etc.) did not bring any meaningful change in
emission of the receptors.
During titrations of 1–4 with the different dicarboxylates
(taken as the tetrabutylammonium salt of malonic, succinic,
glutaric and adipic acids) in CH3CN, the emission of receptors
1 and 2 remained almost unchanged. Only 3 and 4, with flexible Fig. 12 Fluorescence ratio [(I  I0)/I0] of receptor (a) 3 at 412 nm and
550 nm, (b) 4 at 412 nm and 550 nm with a particular dicarboxylate
linkers present in between two-pyridinium units, responded to
anion in CH3CN.
complexation as observed from the significant change in emission
during titration. Fig. 11, in this regard, represents the change
different conformations of receptors 3 and 4 in which the
in the fluorescence ratio at the monomer emission wavelength
pyridinium amide binders assume different orientations
(lmax = 412 nm) in the presence of dicarboxylates. Indeed, the
around the flexible linkers for complexation.
excimer/exciplex emission (may be formed either inter- or
In order to ascertain the selectivity in the binding process of
intramolecularly) at 550 nm was also noticed during
these dicarboxylates with 3 and 4, we initially determine the
complexation.
stoichiometry of the complexes. In each case 1 : 1 stoichiometry
The changes in intensities of the monomer and excimer
of the receptor–dicarboxylate complexes was confirmed by
emissions for 3 and 4 are displayed in Fig. 12. It is interesting
fluorescence Job plots.22 Figs. 13a and b demonstrate the Job
to note that the excimer/exciplex emission intensity for 3 and 4
plots for glutarate with receptor 3 and glutarate, succinate,
varies with the chain length of the dicarboxylates. While the
adipate with 4, respectively. The binding constants,22 calculated
excimer/exciplex emission for 3 is marked with glutarate, the
for 3 and 4 with the dicarboxylates considering 1 : 1 stoichio-
same for 4 is found to be of equal in magnitude with succinate,
metry, are summarized in Table 1. In the case of 1 and 2 we
glutarate and adipate. This is presumably attributed to the
were unable to evaluate the binding constant values as the
perturbation of the emission was minimum for them during
titration. The values in Table 1 reveal that receptors 3 and 4 are
selective for glutarate and adipate, respectively.

Fig. 11 Fluorescence ratio [(I  I0)/I0] of receptors 1–4 at 412 nm
upon addition of 10 equivalents of a particular anion in CH3CN.
UV-vis study
To see the ground state interaction properties of 1–4 towards
the anions, UV-vis experiments were performed in the same
solvent. Earlier we noticed that 1 showed weak interactions
with the anions as established from UV-vis spectral changes.18
Only H2PO4 exhibited a strong interaction by showing a
marked change in absorbance of anthracene with a considerable
bathochromic shift. The stoichiometry of its complex with 1
was complicated. Two inflection points in the Job plot indicated
both 1 : 1 and 2 : 1 (G : H) stoichiometries.18 In a similar way,
other receptors 2–4 displayed interactions with H2PO4 in the
ground state indicating gradual decrease in absorbance of

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 1231–1245 1235
 View Article Online

1.13  0.07  104 M1, 1.15  0.09  104 M1 for 1, 2, 3 and 4,
respectively. The binding constant values were found to follow
the similar trend as in fluorescence. In the case of 1 the Ka for
other anions was of B103 to B102 M1 order.18 Due to the
irregular change, we were unable to determine Ka values for
2–4 with other anions examined.

1
H NMR study
To assess the conformational behaviour of the molecules, we
recorded the ROESY spectrum in d6-DMSO. Careful analysis
shows that the molecules 1–4 assume the different conforma-
tions in solution and as a result of which the pyridinium
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

moieties as the binders of anions follow different orientations

(Fig. S10 in the ESIw). In addition, we also recorded the
1
H NMR of the receptors individually and in the presence of
anions such as H2PO4, AcO and F in CDCl3 containing 6%
d6-DMSO (Fig. S11a–d in the ESIw). In the presence of H2PO4
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among the said anions, the signals for amide protons moved
downfield and in few cases were not found due to broadening or
precipitation upon addition of guests. The pyridinium protons
Ho and Ho0 indicated a strong downfield chemical shift and
thereby corroborated their participation in hydrogen bonding
with the anions. In this context, receptor 1 followed a coopera-
tive binding mode with H2PO4 as reported previously.18
Fig. 13 (a) Fluorescence Job plot for 3 with glutarate in CH3CN at Addition of H2PO4 to the solution of 2 resulted in precipita-
412 nm, where [G] = [H] = 6.56  105 M1. (b) Fluorescence Job tion (Fig. S11a, ESIw). In the presence of an equiv. amount of
plots for 4 with (i) succinate, (ii) glutarate and (iii) adipate at 412 nm, F and AcO, the Ho proton showed a downfield chemical shift
where [G] = [H] = 6.56  105 M. by 0.38 and 0.53 ppm, respectively (Fig. S11a, ESIw). In the case
of 3, the amide protons underwent a 1.21 ppm downfield shift in
Table 1 Binding constant values (Ka, M1) of receptors 3 and 4 with the presence of an equiv. amount of H2PO4 ions (Fig. S11b,
the dicarboxylates as calculated by the 1 : 1 binding isotherm at 412 nm ESIw). The Ho and Ho0 protons moved downfield by 0.54 and
in CH3CN 0.38 ppm, respectively. However, further addition of H2PO4
Guesta 3 4 did not bring any change in chemical shift values of the key
2
protons of 3. The presence of AcO and F in equiv. amounts
Malonate (5.68  0.6)  10 (5.42  0.5)  102
induced a downfield shift of the Ho protons of 3 by 1.02 and
Succinate (4.97  0.9)  102 (8.55  0.2)  102
Glutarate (1.09  0.1)  103 (1.29  0.4)  102 0.40 ppm, respectively (Fig. S11c, ESIw). A similar situation
Adipate (9.79  1.0)  102 (4.58  0.5)  103 aroused for 4 when equiv. amounts of AcO and F were
a
Anions added as their tetrabutylammonium salt. added to the solution of 4 in CDCl3 containing 6% d6-DMSO
(Fig. S11d, ESIw). In this case, although amide protons vanished,
the Ho and Ho 0 protons showed a significant downfield
anthracene with a red shift of 5 nm. In the case of other tested chemical shift. While the Ho and Ho 0 in 4 exhibited downfield
anions the change in absorbance was insignificant (Figs. S1b, shifts of 0.99 and 0.47 ppm, respectively, in the presence of
d, f and h in the ESIw). For 2, the absorption intensity at AcO, the same protons moved to the downfield direction by
372 nm upon addition of 5 equivalent amounts of F was 0.42 and 0.31 ppm, respectively, in the presence of F ions.
negligibly changed but, in the presence of excess F, the Addition of H2PO4 gave a precipitate. Thus, the change in
absorbance increased to a considerable extent. A similar result chemical shift values of the pyridinium protons of the receptors
was obtained when TBAOH solution was added to the signifies that anions stick to this site involving hydrogen bonding
solution of receptor 2. So the change in absorbance of 2 with and charge–charge interaction. Due to greater changes in
F in its higher concentration is due to deprotonation. For fluorescence spectra of 3 and 4 in the presence of glutarate
receptors 3 and 4 the change in absorption intensity was found and adipate, respectively, we intended to record their 1H NMR
to be negligible. This was due to the presence of flexible linkers in CDCl3 containing 6% d6-DMSO (DMSO was used due to the
between two pyridinium units as a result of which the insolubility problem). Upon addition of an equiv. amount of
pyridinium moieties are not cooperatively involved in binding glutarate to 3, the Ho and Ho0 protons moved to the downfield
of smaller sized anions. This was further evident from the almost directions to the extent of 0.91 and 0.48 ppm, respectively
linear response of absorption change with guest concentration (Fig. S11e, ESIw). For adipate with 4, the same protons moved
(Figs. S6a and b in the ESIw). The binding constant22 (Ka) values downfield by 1.32 and 0.60 ppm, respectively (Fig. S11f, ESIw).
for 1–4 with H2PO4, evaluated by analysing the absorption data In both cases, signals for amide protons were too broad to detect
at 372 nm, are 2.15  0.2  104 M1, 9.21  0.5  103 M1, their accurate positions.

1236 New J. Chem., 2012, 36, 1231–1245 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
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Table 2 Complex-induced chemical shift of P in H2PO4 from 31P

NMR (H3PO4 was used as internal standard) in d6-DMSO in its
corresponding 1 : 1 and 2 : 1 complexes with receptors 1–4

Receptor 1 : 1 ([G]/[H]) 2 : 1 ([G]/[H])

1 0.47 0. 72
2 0.07 0.51
3 0.15 0.75
4 0.14 0.19
Fig. 15 DFT optimized geometries of (a) 2 (E = 2235.88 a.u.) and
For further confirmation about the interaction of H2PO4 in (b) the complex of 2 with H2PO4 (E = 2864.00 a.u.; bond distances:
the clefts of 1–4 31P NMR spectra in d6-DMSO were recorded. a = 1.71 Å, b = 1.78 Å, c = 1.92 Å, d = 2.06 Å, e = 2.08 Å, f = 2.10 Å,
In the presence of the receptors the signal for P in H2PO4 g = 2.55 Å, h = 2.46 Å, i = 2.13 Å and j = 4.31 Å).
underwent a downfield shift to different extent (Table 2).
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

Molecular modelling
The hydrogen bonding features of the pyridinium motifs
in 1–4 were further understood from the DFT optimized
structures23 of the compounds with a single H2PO4 ion. In
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all the cases, the cavities provide space for complexation of

H2PO4 ions. The amide and pyridinium protons are intimately
involved in hydrogen bonding with H2PO4. The pendant Fig. 16 DFT optimized geometries of (a) 3 (E = 2106.73 a.u.) and
anthracenes move closer upon binding. Figs. 14–17 demonstrate (b) the complex of 3 with H2PO4 (E = 2735.55 a.u.; bond distances:
the hydrogen bonding features with receptors 1–4. a = 1.52 Å, b = 2.37 Å, c = 2.28 Å, d = 1.68 Å, e = 1.96 Å, f = 2.10 Å,
The flexible aliphatic chains in 3 and 4 fold and adopt g = 2.10 Å, h = 4.60 Å and i = 4.75 Å).
conformations to complex H2PO4. The –CH2– groups in the
linker chain take part in hydrogen bonding along with the
pyridinium amide motifs (Figs. 16 and 17). The distribution of
the MOs of the receptors itself and in their complexes is shown
in the ESIw (Fig. S13a–d). It is clearly observed that the
HOMO, LUMO, HOMO  1 and LUMO + 1 in all the
cases are spread over the different moieties of the molecules. In
each case, the HOMO and HOMO  1 are found to exist on
the anthracene part and the LUMO/LUMO + 1 is on the
pyridinium part of the molecules. A similar trend is also found
for the complexes. Thus in all the receptors, anthracene acts as Fig. 17 DFT optimized geometries of (a) 4 (E = 2146.05 a.u.) and
a donor and the pyridinium moiety acts as an acceptor of (b) the complex of 4 with H2PO4 (E = 2789.84 a.u.; bond distances:
electrons. We believe that this donation of electrons (secondary a = 1.61 Å, b = 2.36 Å, c = 2.34 Å, d = 0.62 Å, e = 2.04 Å, f = 2.94 Å,
g = 2.09 Å, h = 2.03 Å, i = 2.96 Å and j = 4.13, k = 3.30 Å, l = 4.33 Å
PET process)24 is inhibited upon complexation with H2PO4. We
and m = 2.30 Å).
further presume that this situation will remain unaltered when
the second H2PO4 ion is to be complexed to the receptors.
(see Table 3) during complexation. The values in Table 3 are
To better understand the complexation of another H2PO4, we
found to be reduced almost by half from the values for
calculated various global parameters25 such as electronegativity
receptors only. This underlines the truth that 1 : 1 complexes
(w), hardness (Z) and electrophilicity (o) for the receptors and their
of the receptors are further prone to receive another H2PO4.
1 : 1 complexes with H2PO4 ions (shown in Figs. 14–17). Among
There is precedence in the literature, in this regard.26
the different parameters, the change in global electrophilicity
(power of acceptance of the nucleophile) is more marked Anion binding studies in an semi-aqueous system
We also investigated the anion binding affinity of 1–4 in a semi-
aqueous system. For this purpose, we recorded the interaction
properties of 1–4 with the sodium salts of phosphate anions
such as H2PO4, HPO42 and PO43 by fluorescence and
UV-vis methods. Use of an organic solvent was unavoidable
due to limited solubility of these receptors. In order to obtain
more optimal conditions for studying the interaction with the
anions we solubilised the compounds in aq. CH3CN (CH3CN :
Fig. 14 DFT optimized geometries of (a) 1 (E = 2219.83 a.u.) and H2O = 4 : 1 v/v, pH 7.2), using 10 mM HEPES buffer. Fig. 18
(b) the complex of 1 with H2PO4 (E = 2848.05 a.u.; bond distances: shows the fluorescence intensity changes of the monomer
a = 2.12 Å, b = 1.74 Å, c = 2.26 Å, d = 2.34 Å, e = 2.99 Å, f = 1.74 Å, emission of 1–4 in the presence of anions such as H2PO4,
g = 2.12 Å, h = 2.16 Å, i = 2.18 Å, j = 2.23 Å and k = 2.35 Å). HPO42 and PO43 (taken as their sodium salts). As can be seen,

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 1231–1245 1237
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Table 3 Dipole moment (D) and global electrophilicity index (o) of

the receptors 1–4 and their complexes ([G]/[H] = 1 : 1) with tetra-
butylammonium H2PO4

Entry Dipole moment/D w Z o

1 1.897 0.304 0.0831 0.556
1:H2PO4 (1 : 1) 0.5335 0.218 0.1062 0.223
2 0.04221 0.303 0.0826 0.555
2:H2PO4 (1 : 1) 0.7921 0.219 0.1104 0.217
3 1.674 0.296 0.0915 0.479
3:H2PO4 (1 : 1) 1.215 0.220 0.1091 0.221
4 4.205 0.292 0.0868 0.491
4:H2PO4 (1 : 1) 1.586 0.215 0.091 0.253

Fig. 19 Fixed HeLa cells stained with (a) receptor 1, (b) receptor 2,
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

(c) receptor 3, (d) receptor 4 and (e) Hoechst dye.

can be considered as higher order complex entity consists of

phosphate ions along with others such as proteins, nucleic
acids, lipids, carbohydrates, water and different metal ions.
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Similarly DNA, primarily responsible for genetic information,

is a polyanionic substrate with phosphate groups in the backbone.
Therefore, an insight on the mode of interaction of our pyridinium-
based receptors with such phosphate group containing
biomolecules is essentially important.

Cell staining and DNA interaction studies

Fig. 18 Fluorescence ratio [(I  I0)/I0] of receptors 1–4 in CH3CN :
H2O (4 : 1 v/v, 10 mM HEPES buffer, pH = 7.2) at 412 nm upon
addition of 50 equiv. amounts of a particular anion (dissolved in H2O
containing 10 mM HEPS buffer, pH = 7.2).
receptors 1 and 3 show significant changes in monomer
emission at 412 nm upon addition of 50 equivalent amounts
of H2PO4 and HPO42 over PO43. During titration, no peak
at higher wavelength for excimer or exciplex formation was
observed. Bisamides 2 and 4 showed a weak change in emission
in the presence of 50 equiv. amounts of phosphate anions.
A similar study was performed in aq. CH3CN (CH3CN :
H2O = 4 : 1 v/v) without using buffer solution (ESIw, Fig. S8k)
and here also we noticed similar observations. Analysis of
fluorescence data was considered to determine the binding
constant values for H2PO4, HPO42 with 1 and 2 using the
Benesi–Hildebrand plot.27 The linear nature of the plot clearly
indicated the formation of 1 : 1 complexes of 1 and 2 with
H2PO4, and HPO42. The binding constant values for 1 with
H2PO4 and HPO42 were found to be 742 and 86 M1,
respectively. In comparison, for 3 with H2PO4 and HPO42
the binding constant values were 78.2 and 84.1 M1, respectively.
Importantly, the binding constant values are of lower magnitude
due to strong hydration of the anions. Due to a minor or
irregular change in emission, the binding constant values for 2
and 4 were not determined.
In addition, the ground state interaction of the receptors
was understood by UV-vis titrations under similar conditions.
Indeed, the change in absorbance of anthracene at 370 nm in
1–4 with all the phosphate anions was negligible and thereby
indicated weak interactions (Figs. S8g–j in the ESIw).
Due to the significant interaction of the receptors with the
phosphates in both organic and aqueous organic systems, we
planned to investigate their interactions with cells and DNA,
the important components of biological systems. A cell which
Cell staining. All the four synthetic compounds 1–4 have the
property to stain the cultured cells (Fig. 19). For this, we used
HeLa cells and followed a standard method of cell staining
(as described in the Experimental section) with compounds
1–4. Fixation of cultured cells enhances their membrane
permeability and the compounds were able to enter into the
cells and bind cellular materials. Possibly, they are involved in
binding of DNA molecules as evident from our DNA binding
studies. In cells, there are a huge number of molecules and we
did not investigate the binding with other components of cells
such as protein. From the photographs it is clear that all the
four compounds 1–4 can stain whole cells including nucleus
(i.e., DNA). Comparing with the cells stained with Hoechst
dye (Fig. 19e), it can be inferred that all the compounds can
stain whole cells not only DNA as Hoechst dye does. The cell
staining pictures in the bright-field mode are available in the
ESIw (Fig. S14). From the experimental findings qualitatively
it is concluded that all compounds 1–4 (Figs. 19a–d) have
almost same staining property when cells were fixed.
DNA interaction study
Gel retardation assay. If any compound binds to DNA, the
mobility of that DNA through gel will be different and it
should be reflected in the band pattern of that DNA with
respect to the control one where only free DNA is present. Size
and conformation of the compounds can also influence the
mobility of DNA through gel. It is clearly seen from Fig. 20
that there is a distinguishable change in the band pattern of
plasmid DNA (pEGFP-C1, Mol wt = 3102 kDa) complexed
with the compounds with respect to the corresponding free
plasmid DNA. In Fig. 20, lane 1 is for only free plasmid
DNA and lanes 2, 3, 4, 5 are for DNA bound with receptors 1,
3, 4 and 2, respectively. Here the plasmid DNA shows two
predominant forms as represented by two intense bands (L1).

1238 New J. Chem., 2012, 36, 1231–1245 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
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Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

Fig. 20 Changes in the agarose gel electrophoretic pattern of plasmid

DNA induced by all the four compounds. Lane 1, DNA alone; lane 2,
receptor 1 (26.5 mM); lane 3, receptor 3 (26.5 mM); lane 4, receptor 4
(26.5 mM); lane 5, receptor 2 (26.5 mM) respectively.
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The upper intense band is for the relaxed circular form of

plasmid DNA and the lower intense one is for super-coiled form
of plasmid DNA having same molecular weight (Fig. 20, L1).
Although molecular weights of compounds 1 and 2 are greater
than 3 and 4, the hydrocarbon chains present in compounds 3
and 4 impart conformational flexibility as a result of which the
molecular volumes of 3 and 4 are larger than compounds 1 and 2.
The super-coiled form of DNA has more compact structure than
the relaxed one and is thus restricted or provides few ligand
binding sites.28 Receptors 3 and 4 being more structurally
flexible, bind effectively to the relaxed circular DNA (upper
band) than the super-coiled form (lower band) of plasmid
DNA. Accordingly, we got a greater shift for binding with
relaxed DNA. Conversely, in the case of receptors 1 and 2, a
larger shift of the super-coiled form (lower intense band) of
plasmid DNA was observed than in the case of receptors 3 and 4.
If we compare the three dimensional structures of super-coiled
and relaxed circular DNAs, the grooves (major/minor) and
intercalating spaces in the relaxed DNA are more accessible
to the ligand than the case with super-coiled DNA. Hence,
receptors 3 and 4 having much flexibility relative to receptors 1
and 2 may undergo intercalation or be accommodated into DNA
grooves (induced fit) in addition to the binding of phosphate of
DNA. In contrast, receptors 1 and 2 having comparatively rigid
structures may not be accommodated into those DNA grooves
or intercalating spaces as much as that of 3 and 4. Possibly, that
is why 3 and 4 preferentially bind to the relaxed DNA. More-
over, receptors 1 and 2 having stronger affinity to phosphate ions
than that of 3 and 4 preferentially bind to super-coiled DNA
where majority of phosphate bonds are exposed and some of its
grooves and intercalating spaces are buried. Thus in the present
experiment, while receptors 1 and 2 having the same binding
pattern favours super-coiled DNA binding, under similar condi-
tions receptors 3 and 4 with the identical binding pattern favours
the binding of relaxed DNA. We also performed the gel retarda-
tion assay for compounds 1–4 with calf-thymus DNA (ctDNA)
whose molecular weight is much greater than plasmid DNA. For
calf thymus the shift was less (Fig. S15 in the ESIw) compared to
plasmid DNA after binding with compounds.
Steady-state fluorescence study. The fluorescence spectra of
Tris-EDTA (TE) buffer of 1, 2, 3, and 4 show quite alike broad
Fig. 21 Emission spectra of (a) receptor 1, (b) receptor 2, (c) receptor
3 and (d) receptor 4 in the presence of different ctDNA concentrations
(lexc = 365 nm). Insets show the variation of the respective fluores-
cence intensities with DNA concentrations.
bands with maxima at around 415 nm. Addition of calf-thymus
DNA to all the compounds results in the increase of fluores-
cence intensity at B418 nm along with a slight red shift
(B15 nm) for compound 2. The increase in fluorescence
intensity varies with the compounds studied (Fig. 21). The
concentration of DNA required for the saturation of the
fluorescence intensities is found to be in the order compound
1 4 compound 3 4 compound 4 4 compound 2 (insets of
Fig. 21). The observed fluorescence data reveal the binding
interaction between the compounds and the ctDNA. The ratio
of peak fluorescence intensity (F0/dF) in the presence and in the
absence of ctDNA has been plotted as a function of DNA
concentration for all the four compounds (Fig. 22). A best
linear least-squares fit gives the slope and intercept for all the
compounds. Hence, association constant29 (Ka) values for all
the four compounds were determined (Table 4).
CD study. The binding interactions of the compounds with
DNA were further realized from the CD study. Binding of a
compound with DNA may stabilize or destabilize the latter.
Circular dichroism (CD) is an effective method to examine the
structural modification of DNA, including changes in the
secondary and tertiary structures of DNA caused by an
interaction with some compounds. Literature reports suggest
that the secondary structures of DNAs are perturbed by
interaction with small molecules.30,31
Binding of small molecules to DNA may drive the entire
assembly of the multistranded DNA structure. In the present
study, we exploited circular dichroism to identify the perturbation
in the secondary structure of ctDNA due to interaction with
the four compounds. Before investigating the interaction of
the compounds with the DNA through the change in the CD
signal, we initially recorded the CD spectra of the compounds
itself (Fig. S16 in the ESIw). The spectra look alike and all the

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 1231–1245 1239
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Fig. 22 Relative extent of fluorescence increase (F0/dF) for (a) receptor 1,

(b) receptor 2, (c) receptor 3 and (d) receptor 4 as a function of 1/Cg
(Cg = ctDNA concentration, nM).

Table 4 Comparison of slope, intercept and Ka values for all the four
compounds in ct DNA environments

Receptor Slope Intercept Ka/nM1

1 71.1 0.03297 4.637  104
2 115.11 0.07568 6.574  104
3 19.22 0.02304 0.119  104
4 24.529 0.05259 0.214  104

compounds 1–4 exhibit a negative Cotton effect at B255 nm.

The backbone conformation of ctDNA shows a CD spectrum
characteristic of the right-handed B form in the far-UV region
(220–320 nm). Structural alterations of the DNA caused by its
interaction with synthetic compounds are reflected in changes
in this intrinsic CD spectrum.32 Fig. 23 shows the CD spectrum
of free DNA in TE buffer at pH 8, having a positive Cotton
effect at B280 nm (p) and a negative Cotton effect at B246 nm (n).
These bands are due to stacking interactions between the
bases and the helical suprastructure of the polynucleotide that
provide an asymmetric environment for the bases.33 The spectra
are characteristic of the B form with 10.4 base pairs per turn,
which is the normal conformation of DNA in aqueous solution
at moderate salt concentration.34 Gradual addition of each of
the four compounds to the DNA solution led to remarkable
perturbation of both the positive and negative bands (Fig. 23).
The band intensities or ellipticities as well as the position of
bands are changed drastically. These changes are significant Fig. 23 Intrinsic CD spectra of ctDNA with varying concentrations
and highly related to the concentration of the compounds and (5 mM, 10 mM and 20 mM) of (a) receptor 1, (b) receptor 2, (c) receptor 3
and (d) receptor 4. DNA concentration in each case was 30 mM. (p) and
the structural features of the compounds. The degree of
(n) are the positive and negative peaks of normal ctDNA, respectively.
perturbation of the DNA secondary structure by our four
compounds is in the order: compound 1 4 compound 3 4
compound 4 4 compound 2. It is interesting to note that for 1. On the other hand, compound 4 (Fig. 23d) did not reverse
compound 1 dramatically alters the helical ellipticity of normal much the helical form at lower concentration but at 20mM the
B-DNA into a reverse form. This form of DNA or the actual negative peak of B-DNA was observed to be reduced to
perturbation of DNA structure is increased with the increase almost zero ellipticity. In the case of compound 2 (Fig. 23b)
of the compound concentration. Compound 3 (Fig. 23c) was band intensities are enhanced and the positive peak is shifted
observed to follow the similar feature of CD spectrum as found from B280 nm to B262 nm, whereas the position of the

1240 New J. Chem., 2012, 36, 1231–1245 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

negative peak is almost unaffected. A plausible explanation
for the change in the normal CD spectra of ctDNA is the
disruption of stacking interaction of the bases, which is required
to optimize the binding interactions.33 These changes may thus
reflect local untwisting of the DNA helical backbone and
changes in relative orientation of the bases to accommodate
the intercalating compounds within a particular base pair.33,35
The extent of increase in the ellipticity is different for the four
compounds (Fig. 23), being maximum for 1 (Fig. 23a) and
minimum for 2 (Fig. 23b), which implies that binding of
compound 1 causes the largest perturbation to the DNA
strands and the most extensive destacking and destabilization
of the DNA helix.
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
Conclusion
In conclusion, we have reported in this full account that
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anthracene labeled different bispyridinium amides 1–4 are
useful in the selective sensing of dihydrogenphosphate36 in
organic solvent. The sensing ability is dependent on the nature
of the spacers that hold the anion binders. In a semi-aqueous
system the sensors are weakly bound to dihydrogenphosphate
and hydrogenphosphate ions rather than phosphates. Depending
on the flexible nature of the spacer in between the pyridinium
amides the dicarboxylate ions can be sensed effectively. In the
present case, receptors 3 and 4 exhibit selectivity for glutarate
and adipate, respectively, by showing excimers of significant
intensities. With the information of binding of phosphate
derivatives, a detailed study on the receptors in cell staining
and DNA interaction has been performed. The receptors are
quite impressive in cell staining. They stain not only DNA
(as Hoechst dye does) but also the entire cell. It has also been
established, for the first time, that the pyridinium based
receptors have the strong propensity towards interaction with
DNA. The gel retardation and CD studies have proved the
strong DNA interaction and the effectiveness is related to
the nature of the spacer that holds the pyridinium binding
sites. Further work along this direction is underway in our
laboratory.
Experimental
General methods for anion binding studies in organic solvents
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, 13C and 31P NMR spectra were
recorded on Brucker 400 MHz and 500 MHz instruments.
For NMR spectra, CDCl3, CD3CN and d6-DMSO were used
as solvents using TMS as an internal standard. Chemical shifts
are expressed in d units and 1H–1H and 1H–C coupling
constants in Hz. 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 recorded on a Perkin Elmer Model
Lambda 25, respectively.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
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N1,N3-di(pyridin-3-yl) isophthalamide (5). The symmetrical
bis-amide 5 was obtained by coupling of 3-aminopyridine
(1.2 g, 12.8 mmol) with isophthaloyl diacidchloride (1.3 g,
6.4 mmol) in dry CH2Cl2 (50 mL) followed by addition of
triethylamine (1.4 mL) under a 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 the CHCl3/CH3OH mixture
(3  20 mL). The organic layer was washed with NaHCO3
solution (3  15 mL) and dried over anhydrous Na2SO4. The
solvent was removed and the residual mass was purified by
column chromatography over silica gel using chloroform :
methanol (4 : 1) as eluent to afford the desired compound 5
(0.730 g, 36% yield, mp 210 1C). 1H NMR (d6-DMSO,
400 MHz): d 10.69 (s, 2H, amide NH), 8.96 (s, 2H), 8.59
(s, 1H), 8.33 (d, J = 4 Hz, 2H), 8.23–8.18 (m, 4H), 7.74
(t, J = 8 Hz, 1H), 7.43 (d, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H)
ppm. 13C NMR (DMSO-d6, 125 MHz): d 166.2, 145.6, 142.9,
136.5, 135.5, 131.8, 129.6, 128.2, 128.0, 124.4 ppm. FTIR (KBr):
n (cm1): 3418, 3054, 1676, 1613, 1585, 1556, 1481, 1429. Mass
(ESI): 319.1 [M + H]+, 225.2, 130.2.
N2,N6-di(pyridin-3-yl) pyridine-2,6-dicarboxamide (6). The
symmetrical bisamide 6 was obtained by coupling of 3-amino-
pyridine (0.76 g, 5.39 mmol) with pyridine-2,6-dicarbonyl
dichloride (0.512 g, 2.695 mmol) in dry CH2Cl2 (50 mL)
followed by addition of triethylamine (1.4 mL) under a nitrogen
atmosphere. The reaction mixture was stirred overnight. After
completion of the reaction, solvent was removed under vacuum.
The residual mass was extracted with the CHCl3/CH3OH
mixture (3  20 mL). The organic layer was washed with
NaHCO3 solution (3  15 mL) and dried over anhydrous
Na2SO4. The solvent was removed and the residual mass was
purified by column chromatography over silica gel using
chloroform : methanol (4 : 1) as eluent to afford the desired
compound 6 (0.734 g, 85% yield, mp 240 1C). 1H NMR
(d6-DMSO, 400 MHz): d 11.2 (s, 2H, amide NH), 9.11
(s, 2H), 8.43–8.40 (m, 4H), 8.35–8.31 (m, 3H), 7.50 (d, 1H,
J = 8 Hz), 7.48 (d, 1H, J = 8 Hz) ppm. 13C NMR (d6-DMSO,
100 MHz): d 162.9, 149.2, 146.1, 143.5, 140.9, 135.5, 129.0,
126.4, 124.5 ppm. FTIR (KBr): n (cm1): 3511, 3063, 1685, 1.
Mass (ESI): 320.1 (M + H)+, 209.2, 130.3.
N1,N5-di(pyridin-3-yl) glutaramide (7). The symmetrical
bisamide 7 was obtained by coupling of 3-aminopyridine
(1.145 g, 12.2 mmol) with glutaroyl dichloride (1 g, 6.1 mmol)
in dry CH2Cl2 (50 mL) followed by addition of triethylamine
(1.4 mL) under a nitrogen atmosphere. The reaction mixture
was stirred overnight. After completion of the reaction,
solvent was removed under vacuum. The residual mass was
extracted with the CHCl3/CH3OH mixture (3  20 mL). The
organic layer was washed with NaHCO3 solution (3  15 mL)
and dried over anhydrous Na2SO4. The solvent was removed
and the residual mass was purified by column chromatography
over silica gel using chloroform : methanol (4 : 1) as eluent to
afford the desired compound 7 (0.644 g, 39% yield, mp
188 1C). 1H NMR (d6-DMSO, 400 MHz): d 10.13 (s, 2H, amide
NH), 8.72 (d, 2H, J = 2 Hz), 8.23 (d, 2H, J = 4.40 Hz), 8.02 (d,
2H, J = 8 Hz), 7.34–7.30 (m, 2H), 2.42 (t, 4H, J = 7.30 Hz),
New J. Chem., 2012, 36, 1231–1245 1241

1.96–1.88 (m, 2H) ppm. 13C NMR (d6-DMSO, 100 MHz): d
172.5, 144.8, 141.5, 136.7, 126.8, 124.4, 36.9, 25.5. ppm. FTIR
(KBr): n (cm1): 3289, 3107, 2935, 2855, 1681, 1483, and 1459.
Mass (ESI): 285.3 (M + H)+, 190.9, 143.1.
N1,N6-di(pyridin-3-yl) adipamide (8). The symmetrical bisamide
8 was obtained by coupling of 3-aminopyridine (1 g, 10.6 mmol)
with adipoyl dichloride (1g, 5.32 mmol) in dry CH2Cl2 (50 mL)
followed by addition of triethylamine (1.4 mL) under a nitrogen
atmosphere. The reaction mixture was stirred overnight. After
completion of the reaction, solvent was removed under vacuum.
The residual mass was extracted with the CHCl3/CH3OH
mixture (3  20 mL). The organic layer was washed with
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
NaHCO3 solution (3  15 mL) and dried over anhydrous
Na2SO4. The solvent was removed and the residual mass was
purified by column chromatography over silica gel using
chloroform : methanol (4 : 1) as eluent to afford the desired
compound 8 (0.870 g, 55% yield, mp 222 1C). 1H NMR
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(d6-DMSO, 400 MHz): d 10.11 (s, 2H, amide NH), 8.72
(d, 2H, J = 2 Hz), 8.22 (dd, 2H, J = 4.44 Hz, 1.10 Hz),
8.02 (d, 2H, J = 8.3 Hz), 7.33–7.30 (m, 2H), 2.37 (t, 4H,
J = 7.3 Hz), 1.64–1.68 (m, 4H) ppm. 13C NMR (d6-DMSO,
100 MHz): d 172.2, 144.8, 141.6, 136.7, 126.8, 124.4, and 36.19,
21.48 ppm. FTIR (KBr): n (cm1): 3304, 3176, 2949, 2875,
1690, 1581, 1463. Mass (ESI): 299.3 (M + H)+, 237.3, 130.1.
Synthesis of receptor 1. To a solution of 5 (0.07 g,
0.220 mmol) in dry CH3CN (15 mL) containing dry DMF
(2 mL), 9-chloromethyl anthracene (0.149 g, 0.657 mmol) in
dry CH3CN (20 mL) was added. The reaction mixture was
refluxed with stirring for 2 days under a nitrogen atmosphere.
On cooling the reaction mixture, the precipitate appeared. The
precipitate was filtered off and washed with CH3CN several
times and then with diethyl ether to give pure dichloride salt 9
(0.115 g, 68%). The pure dichloride salt 9 (0.059 g,
0.077 mmol) was dissolved in 5 mL of hot CH3OH and the
volume was reduced to 2 mL. Then aqueous solution of
NH4PF6 (0.038 g, 0.231 mmol) was added in one portion to
carry out the anion exchange reaction. After stirring
the reaction mixture for 50 min, the precipitate appeared
Filtration of the precipitate followed by thorough washing
with diethyl ether afforded the receptor 1 in 88% yield
(0.067 g), mp 216 1C; 1H NMR (d6-DMSO, 400 MHz): d
11.15 (s, NH, 2H), 9.19 (s, 2H), 8.97 (s, 2H), 8.89 (d, 2H, J = 8
Hz), 8.70 (d, 2H, J = 8 Hz), 8.40 (d, 4H, J = 8 Hz), 8.28
(d, 4H, J = 8 Hz), 8.23 (s, 1H), 8.14 (t, 2H, J = 8 Hz), 8.05
(d, 2H, J = 8 Hz), 7.71–7.63 (m, 9H), 6.99 (s, 4 H) ppm;
13
C NMR (DMSO-d6, 125 MHz): d 161.0, 134.9, 130.7, 130.1,
129.6, 129.5, 128.8, 127.3, 126.9, 126.7, 126.5, 125.1, 124.5,
123.9, 122.9, 121.3, 118.7, 117.1, 52.0 ppm; FTIR (KBr):
n (cm1): 3444, 1685, 1633, 1522, 1501, 1449 ppm. (ES+):
m/z: 845.3 [(M–PF6)]+, 813.3, 735.3, 699.4, 509.2.
Synthesis of receptor 2. To a solution of 6 (0.100 g,
0.31 mmol) in 15 mL of dry CH3CN containing dry DMF
(2 mL), 9-chloromethylanthracene (0.210 g, 0.93 mmol) in 10 mL
of CH3CN was added. The reaction mixture was refluxed with
stirring for 2 days under a nitrogen atmosphere. On cooling the
reaction mixture, the precipitate appeared. The precipitate was
filtered off and washed with CH3CN several times and then with
1242 New J. Chem., 2012, 36, 1231–1245 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
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diethyl ether to give pure dichloride salt 10 (0.076 g, 32%
yield). The pure dichloride salt 10 (0.076 g, 0.098 mmol) was
dissolved in 5 mL of hot CH3OH and the volume was reduced
to 2 mL. Then aqueous solution of NH4PF6 (0.048 g,
0.290 mmol) was added in one portion to carry out the anion
exchange reaction. After stirring the reaction mixture for
50 min, the precipitate appeared. Filtration of the precipitate
followed by thorough washing with diethyl ether afforded
receptor 2 in 92% yield (0.089 g, mp 178 1C). 1H NMR
(d6-DMSO, 400 MHz): d 11.6 (s, 2H, amide NH), 9.53 (s, 2H),
8.97 (s, 2H), 8.87 (brs, 2H), 8.69 (d, 2H, J = 8 Hz), 8.45 (d, 4H,
J = 8 Hz), 8.29–8.24 (m, 9H), 7.71 (t, 4H, J = 8 Hz), 7.64 (t, 4H,
J = 6.8 Hz), 7.03 (s, 4H) ppm. 13C NMR (d6-DMSO, 100 MHz):
d 162.3, 146.9, 140.6, 139.4, 138.5, 135.0, 134.2, 131.4, 131.1,
131.0, 129.5, 128.3, 126.2, 125.7, 123.2, 121.6, 56.5 (one aromatic
carbon is unresolved) ppm. FTIR (KBr): n (cm1): 3348,
3103, 1693, 1626, 1542. (ES+): m/z: 846.2 [(M–PF6)]+, 700.2
[(M–2PF6-1)]+.
Synthesis of receptor 3. To a solution of 7 (0.100 g, 0.35 mmol)
in 15 mL of dry CH3CN, 9-chloromethylanthracene (0.241 g,
1.06 mmol) in 10 mL of CH3CN was added. The reaction
mixture was refluxed with stirring for 2 days under a nitrogen
atmosphere. On cooling the reaction mixture, the precipitate
appeared. The precipitate was filtered off and washed with
CH3CN several times and then with diethyl ether to give pure
dichloride salt 11 (0.151 g, 58% yield). The pure dichloride salt
11 (0.073 g, 0.099 mmol) was dissolved in 5 mL of hot CH3OH
and the volume was reduced to 2 mL. Then aqueous solution
of NH4PF6 (0.049 g, 0.297 mmol) was added in one portion to
carry out the anion exchange reaction. After stirring the reaction
mixture for 50 min, the precipitate appeared. Filtration of the
precipitate followed by thorough washing with diethyl ether
afforded receptor 3 in 77% yield (0.073 g, mp 192 1C).
1
H NMR (d6-DMSO, 400 MHz): d 10.73 (s, 2H, amide NH),
9.0 (s, 2H), 8.94 (s, 2H), 8.76 (d, 2H, J = 8 Hz), 8.45 (d, 2H, J = 8
Hz), 8.37 (d, 4H, J = 8 Hz), 8.27 (d, 4H, J = 8 Hz), 8.06–8.02 (m,
2H), 7.70–7.62 (m, 8H), 6.94 (s, 4H), 2.42 (t, 4H, J = 8 Hz),
1.66–1.77 (m, 2H) ppm. 13C NMR (d6-DMSO, 100 MHz): d
172.0, 139.3, 138.5, 133.7, 132.8, 131.3, 131.1, 130.9, 129.5, 128.4,
128.2, 125.7, 123.1, 121.5, 56.27, 34.9, 19.6 ppm. FTIR (KBr):
n (cm1): 3390, 3278, 3116, 3060, 1685, 1556, 1449. (ES+):
m/z: 811.2 [(M–PF6)]+, 665.2 [(M–2PF6-1)]+.
Synthesis of receptor 4. To a solution of 8 (0.100 g,
0.34 mmol) in 15 mL of dry CH3CN, 9-chloromethylanthracene
(0.228 g, 1.01 mmol) in 10 mL of CH3CN was added. The
reaction mixture was refluxed with stirring for 2 days under a
nitrogen atmosphere. On cooling the reaction mixture, the
precipitate appeared. The precipitate was filtered off and
washed with CH3CN several times and then with diethyl ether
to give pure dichloride salt 12 (0.122 g, 49% yield). The pure
dichloride salt 12 (0.073 g, 0.097 mmol) was dissolved in 5 mL
of hot CH3OH and the volume was reduced to 2 mL. Then
aqueous solution of NH4PF6 (0.048 g, 0.292 mmol) was added
in one portion to carry out the anion exchange reaction. After
stirring the reaction mixture for 50 min, the precipitate
appeared. Filtration of the precipitate followed by thorough
washing with diethyl ether afforded receptor 4 in 86% yield

(0.063 g, mp 182 1C). 1H NMR (d6-DMSO, 400 MHz): d 10.73
(s, 2H, amide NH), 9.03 (s, 2H), 8.94 (s, 2H), 8.75 (d, 2H, J = 8 Hz),
8.45 (d, 2H, J = 8 Hz), 8.37 (d, 4H, J = 8 Hz), 8.26 (d, 4H, J = 8
Hz), 8.05 (t, 2H, J = 8 Hz), 7.70–7.61 (m, 8H), 6.94 (s, 4H), 2.24
(brt, 4H, J = 7.2 Hz), 1.5–1.43 (brm, 4H) ppm. 13C NMR
(d6-DMSO, 100 MHz): d 172.2, 139.3, 138.4, 1337, 132.8, 131.3,
131.1, 130.9, 129.4, 128.4, 128.2, 125.7, 123.1, 121.5, 56.7, 35.7, 23.8
ppm. FTIR (KBr): n (cm1): 3394, 3097, 2957, 1697, 1593, 1464,
1450. (ES+): m/z: 825.2 [(M–PF6)]+, 679.2 [(M–2PF6-1)]+.
General procedure for fluorescence and UV-vis titrations
Stock solutions of the receptors were prepared in CH3CN in
the concentration range B105 M. 2.5 mL of the receptor
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
solution was taken in the cuvette. Stock solutions of guests in
the concentration range B104 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
Downloaded by Kalyani University on 08 January 2013
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 1C. All the experiments were
repeated thrice to check the reproducibility.
Job plots
The stoichiometry was determined by the continuous variation
method (Job plot).21 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
Binding constant values were determined by fluorescence and
absorption methods using eqn (1)–(3).
I = (I0 + K1  CG  I[1 : 1] + K1  K2  Ilim  C2G)/
(1 + K1  CG + K1  K2  C2G)
I = I0 + (I  I0)/2CH{CH + CG + 1/K
 [(CG + CH + 1/K)2  4CGCH]0.5}
I = (I0 + I  K  CG)/(1 + K  CG)
where I represents fluorescence intensity; I0 represents
the intensity of pure host; I[1 : 1] represents the intensity of
the 1 : 1 complex between host and guest and Ilim represents the
intensity at infinite guest concentration. CH and CG are the
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
View Article Online
corresponding concentrations of the host and the guest; K1 and
K2 are two binding constants for two successive steps in eqn (1) and
K is the association constant in eqn (2) and (3), respectively [this
equation also works in absorption; I to be replaced by absorption
intensity (A)]. The association constants and correlation coefficients
(R) are obtained by a non-linear least-square analysis of I vs. CG
for eqn (1) and (3). In the case of eqn (2) the association constant
and correlation coefficients (R) are obtained by a non-linear least-
square analysis of I vs. CH and CG.
Materials and methods for cell staining and DNA interaction studies
Chemicals. Hoechst dye was purchased from Sigma Chemicals
(USA). Cell culture medium (MEM) was procured from
HiMedia, Mumbai, India. Agarose, calf thymus DNA were
obtained from SRL, Mumbai, India. Other molecular biology
grade fine chemicals were procured locally.
Cell culture. Human cervical cancer cell line (HeLa) was
obtained from National Centre for Cell Sciences, Pune, India.
HeLa cells were routinely grown in MEM (HiMedia, Mumbai,
India) supplemented with 10% bovine serum (complete medium)
in a CO2 incubator (Heal Force, Shanghai Lishen, China) at 37 1C
in a humidified atmosphere containing 5% CO2.
Measurement of DNA concentration. Stock solutions were
prepared by dissolving the solid calf-thymus DNA in Tris-EDTA
(TE) buffer (pH = 8) and stored at 4 1C. Freshly prepared DNA
solution was used for all the experiments. The purity of DNA
was verified by monitoring the ratio of absorbance at 260 nm to
that at 280 nm, which was in the range of 1.8–1.9. The
concentration of DNA was determined spectrophotometrically,
using ADNA = 13 600 M1 cm1 at 258 nm.37,38 DNA
concentration mentioned in fluorescence quenching and CD
study is in terms of base pair (bp).
Cell staining with synthetic compounds. The HeLa cells were
cultured over cover slips in complete MEM medium. After
24 h incubation in a CO2 incubator, the cover slips were taken
out, washed with phosphate buffered saline (PBS) twice. The
cells were then fixed with methanol–acetone (1 : 1) at 4 1C for
1 h. After fixation cells were again washed with PBS buffer
twice and incubated with 5.0 mM of receptor 1, 5.0 mM of
receptor 3, 5.0 mM of receptor 4 and 5.0 mM of receptor 2 in
PBS separately at room temperature for 5 minutes. After
incubation in the dark the cover slips were washed twice and
placed upside down over a glass slide, so that cells were in
touch with the glass slide. The slide was observed under
fluorescence microscope (Carl Zeiss) with a UV excitation
filter as well as in normal light mode. Another set was done
for live cell imaging without fixation. After grown over cover
(1) slips cells were washed with PBS twice and stained with the
compounds as described earlier in PBS at room temperature
for 5 minutes. Cells were observed and photographed under a
(2) fluorescence microscope (Carl Zeiss) with a UV excitation
(3) filter as well as in normal light mode. All the photographs
were taken at the same microscopic field in both bright-field
(Fig. S14 in the ESIw) and fluorescence mode.
Bacterial transformation and plasmid isolation. E. coli DH5a
cells (Clontech, USA) were made competent using calcium
New J. Chem., 2012, 36, 1231–1245 1243

chloride following the standard method as described in Cohen
et al. (1972)39 with slight modification. 50 ng of pEGFP-C1
(Clontech, USA) plasmid (3102 kDa) was used for transforma-
tion and the transformed cells were grown in the presence of
50 mg mL1 kanamycin (HiMedia, India) in LB broth medium
(HiMedia, India). Then the plasmid was isolated from the
overnight culture of transformed E. coli DH5a using the
standard method of mini-preparation by alkaline lysis with
SDS as described in Sambrook et al. (1989).40 Isolated
plasmid was treated with RNase A (20 mg mL1) to avoid the
RNA contamination. Finally the plasmid DNA was purified by a
standard phenol–chloroform extraction procedure and checked
through 1% agarose gel electrophoresis.40 The concentration of
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
the isolated plasmid DNA was checked by a spectrophotometer.
This plasmid DNA was used for the gel retardation assay.
DNA binding
1. Gel retardation assay. 26.5 mM of each compound (1–4)
Downloaded by Kalyani University on 08 January 2013
was mixed separately with 1.0 mg of pEGFP-C1 plasmid DNA
(Clontech, USA) in Tris-EDTA (TE) buffer, pH 8 in micro-
centrifuge tubes and incubated for 25 minutes. All samples
were loaded in 1.0% normal agarose gel after adding 1 gel
loading dye and run in 1 Tris-acetate EDTA (TAE) buffer at
constant 60 V for 3 h. After staining with ethidium bromide
(1.0 mg mL1) for 5 minutes, samples were observed using a
Typhoon 9210 (Variable Mode Imager), GE Healthcare.
2. Steady-state fluorescence study. Steady-state fluorescence
measurements were carried out using a Perkin-Elmer Luminescence
spectrometer (model LS50B) equipped with FLWINLAB software
with a rectangular quartz cuvette of path length 1 cm. By varying
DNA concentration all the emission spectra were recorded for
receptors 1–4 separately in Tris-EDTA (TE) buffer (pH = 8). After
buffer subtraction fluorescence intensity was plotted as a function
of wavelength and DNA concentration separately for all the four
compounds. The association constant or Ka value was calculated by
using eqn (1) where F0 and F denote the measured fluorescence
intensity before and after adding DNA. Here fM and fp are
fluorescence quantum yields of the monomer and complex, respec-
tively. Cg is the final concentration of DNA after each addition.
F0/F  F0 = [fMAM/fpAp  fMAM][(1/KaCg) + 1]. . .29
(4)
3. CD Study. CD spectra were recorded on a JASCO J-720
spectropolarimeter, using a rectangular quartz cuvette of path
length 1 cm. Spectra shown are averages of four successive
scans recorded at a scan speed of 50 nm min1, from which the
appropriate blanks have been subtracted. All the experiments
were performed at ambient temperature (300 K) with
air-equilibrated solutions. We have used 30 mM of ctDNA
and three different concentrations of compounds like 5 mM,
10 mM and 20 mM for each of the compounds.
Acknowledgements
KG thanks CSIR, New Delhi, India, for financial support.
ARS thanks University of Kalyani, India, for a fellowship.
KG and UG would like to thank DST, Govt. of India, for
providing facilities in Chemistry and Biochemistry & Biophysics
1244 New J. Chem., 2012, 36, 1231–1245 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
View Article Online
departments under DST-FIST program. We are also grateful
to Prof. Soumen Basak, Chemical Sciences Division, Saha
Institute of Nuclear Physics, Kolkata, India, for giving us
an opportunity to use the JASCO J-720 spectropolarimeter
for CD spectra.
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