Вы находитесь на странице: 1из 15

View Article Online / Journal Homepage / Table of Contents for this issue

NJC Dynamic Article Links

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
Downloaded by Kalyani University on 08 January 2013

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-


pyridinium receptors have been used for anion-templated
Selective sensing of anions of biological and environmental pseudorotaxane formation.13 During the course of our work
relevance by synthetic hydrogen bonding receptors is an in the area of supramolecular chemistry, we also used this motif
important research topic in supramolecular chemistry.1 In in various designs for sensing of anions such as H2PO4,14
relation to this, the binding unit, which is capable of binding dicarboxylates,15 ATP,16 etc. Recently, Dorazco-Gonzalez et al.
anions strongly or moderately, has the crucial role in giving have reported the recognition behaviour of dicationic pyridine-2,6-
the selectivity to the designed receptor. Examples in this field dicarboxamide receptors for anions and neutral guests indicating
are available in the literature.2 Among the various types, our approach on the dicationic bisamide receptor derived from
amide,3 urea,4 guanidinium,5 amidinium,6 imidazolium,7 isophthalic acid and two 3-aminopyridine.17 In their recent report,
benzimidazolium,8 polyammonium cations,9 etc. are widely they have compared the binding ability of the dicationic receptors
used in anion binding and they were found to be worthy in with their neutral precursors.
functioning. In this context, the pyridinium ion is another In continuation of our previous work on fluorescent dicationic
motif, which was first used by Jeong and Cho in the binding of bisamide receptor 1 derived from isophthalic acid for anions,18
carboxylates.10 The pyridinium motif provides hydrogen- we report here the synthesis and anion sensing properties of a
bonding donors (both conventional and unconventional) for series of dicationic receptors 2–4 (Fig. 1) where the cationic
complexation of anions and the complex is further stabilized binding sites are hooked on the different synthetic spacers. The
by charge–charge interaction. Keeping this view in mind it DNA binding and cell expression properties of these molecules
was explored later on by other groups. Steed et al. used this have also been evaluated.
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 Results and discussion
complexation via simultaneous binding to NH and pyridinium
The dicationic receptors are all anthracene-based and they
were obtained according to Scheme 1. The neutral precursors
a
Department of Chemistry, University of Kalyani, Kalyani-741235, of the cationic salts were synthesized in a single step by
India. E-mail: ghosh_k2003@yahoo.co.in; Fax: +91 3325828282; coupling 3-aminopyridine with the different diacidchlorides.
Tel: +91 3325828750 They were reacted with 9-chloromethylanthracene in dry
b
Department of Biochemistry and Biophysics, University of Kalyani,
Kalyani-741235, India CH3CN under reflux to give the dichloride salts. The dichloride
w Electronic supplementary information (ESI) available: Spectral data salts were treated with NH4PF6 in aq. CH3OH to afford the
of receptors 1–4. Figures showing the change in fluorescence and desired products.
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.
Anion binding studies in organic solvents
1
H NMR spectra for interaction study. 31P NMR of H2PO4 with The anion binding ability of receptor 1 among the series was
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- reported in our earlier publication.18 A series of anions such as
aqueous system. CD study. See DOI: 10.1039/c2nj21024j 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
View Article Online

Fig. 2 Fluorescent emission changes of 1 (6.56  105 M) upon


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

addition of various anions (10 equiv.) in CH3CN.

isophthaloyl moiety. In the present study, we took the same


Fig. 1 Chemical structures for 1–4. anions for receptors 2–4 to see the effect of the synthetic
spacers on the conformation of the molecules as a result of
Downloaded by Kalyani University on 08 January 2013

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
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; avoided in 2 where the isophthaloyl group is replaced by the
(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 Fig. 3 Fluorescent emission changes of 2 (6.56  105 M) upon
particular anion. The possible reason for the low affinity of addition of tetrabutylammonium salts of various anions up to 10 equiv.
this receptor was the unfavourable conformation19 of the in CH3CN.

1232 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. 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.
Downloaded by Kalyani University on 08 January 2013

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
View Article Online

Fig. 7 The possible three major conformations of 1.


Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
Downloaded by Kalyani University on 08 January 2013

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
Downloaded by Kalyani University on 08 January 2013

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.

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,
Fig. 11 Fluorescence ratio [(I  I0)/I0] of receptors 1–4 at 412 nm other receptors 2–4 displayed interactions with H2PO4 in the
upon addition of 10 equivalents of a particular anion in CH3CN. 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
Downloaded by Kalyani University on 08 January 2013

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
View Article Online

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
Downloaded by Kalyani University on 08 January 2013

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
View Article Online

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.
Downloaded by Kalyani University on 08 January 2013

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 Cell staining. All the four synthetic compounds 1–4 have the
addition of 50 equiv. amounts of a particular anion (dissolved in H2O property to stain the cultured cells (Fig. 19). For this, we used
containing 10 mM HEPS buffer, pH = 7.2). HeLa cells and followed a standard method of cell staining
(as described in the Experimental section) with compounds
receptors 1 and 3 show significant changes in monomer 1–4. Fixation of cultured cells enhances their membrane
emission at 412 nm upon addition of 50 equivalent amounts permeability and the compounds were able to enter into the
of H2PO4 and HPO42 over PO43. During titration, no peak cells and bind cellular materials. Possibly, they are involved in
at higher wavelength for excimer or exciplex formation was binding of DNA molecules as evident from our DNA binding
observed. Bisamides 2 and 4 showed a weak change in emission studies. In cells, there are a huge number of molecules and we
in the presence of 50 equiv. amounts of phosphate anions. did not investigate the binding with other components of cells
A similar study was performed in aq. CH3CN (CH3CN : such as protein. From the photographs it is clear that all the
H2O = 4 : 1 v/v) without using buffer solution (ESIw, Fig. S8k) four compounds 1–4 can stain whole cells including nucleus
and here also we noticed similar observations. Analysis of (i.e., DNA). Comparing with the cells stained with Hoechst
fluorescence data was considered to determine the binding dye (Fig. 19e), it can be inferred that all the compounds can
constant values for H2PO4, HPO42 with 1 and 2 using the stain whole cells not only DNA as Hoechst dye does. The cell
Benesi–Hildebrand plot.27 The linear nature of the plot clearly staining pictures in the bright-field mode are available in the
indicated the formation of 1 : 1 complexes of 1 and 2 with ESIw (Fig. S14). From the experimental findings qualitatively
H2PO4, and HPO42. The binding constant values for 1 with it is concluded that all compounds 1–4 (Figs. 19a–d) have
H2PO4 and HPO42 were found to be 742 and 86 M1, almost same staining property when cells were fixed.
respectively. In comparison, for 3 with H2PO4 and HPO42
the binding constant values were 78.2 and 84.1 M1, respectively. DNA interaction study
Importantly, the binding constant values are of lower magnitude Gel retardation assay. If any compound binds to DNA, the
due to strong hydration of the anions. Due to a minor or mobility of that DNA through gel will be different and it
irregular change in emission, the binding constant values for 2 should be reflected in the band pattern of that DNA with
and 4 were not determined. respect to the control one where only free DNA is present. Size
In addition, the ground state interaction of the receptors and conformation of the compounds can also influence the
was understood by UV-vis titrations under similar conditions. mobility of DNA through gel. It is clearly seen from Fig. 20
Indeed, the change in absorbance of anthracene at 370 nm in that there is a distinguishable change in the band pattern of
1–4 with all the phosphate anions was negligible and thereby plasmid DNA (pEGFP-C1, Mol wt = 3102 kDa) complexed
indicated weak interactions (Figs. S8g–j in the ESIw). with the compounds with respect to the corresponding free
Due to the significant interaction of the receptors with the plasmid DNA. In Fig. 20, lane 1 is for only free plasmid
phosphates in both organic and aqueous organic systems, we DNA and lanes 2, 3, 4, 5 are for DNA bound with receptors 1,
planned to investigate their interactions with cells and DNA, 3, 4 and 2, respectively. Here the plasmid DNA shows two
the important components of biological systems. A cell which 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
View Article Online
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.
Downloaded by Kalyani University on 08 January 2013

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 Fig. 21 Emission spectra of (a) receptor 1, (b) receptor 2, (c) receptor
than 3 and 4, the hydrocarbon chains present in compounds 3 3 and (d) receptor 4 in the presence of different ctDNA concentrations
(lexc = 365 nm). Insets show the variation of the respective fluores-
and 4 impart conformational flexibility as a result of which the
cence intensities with DNA concentrations.
molecular volumes of 3 and 4 are larger than compounds 1 and 2.
The super-coiled form of DNA has more compact structure than
bands with maxima at around 415 nm. Addition of calf-thymus
the relaxed one and is thus restricted or provides few ligand
DNA to all the compounds results in the increase of fluores-
binding sites.28 Receptors 3 and 4 being more structurally
cence intensity at B418 nm along with a slight red shift
flexible, bind effectively to the relaxed circular DNA (upper
(B15 nm) for compound 2. The increase in fluorescence
band) than the super-coiled form (lower band) of plasmid
intensity varies with the compounds studied (Fig. 21). The
DNA. Accordingly, we got a greater shift for binding with
concentration of DNA required for the saturation of the
relaxed DNA. Conversely, in the case of receptors 1 and 2, a
fluorescence intensities is found to be in the order compound
larger shift of the super-coiled form (lower intense band) of
1 4 compound 3 4 compound 4 4 compound 2 (insets of
plasmid DNA was observed than in the case of receptors 3 and 4.
Fig. 21). The observed fluorescence data reveal the binding
If we compare the three dimensional structures of super-coiled
interaction between the compounds and the ctDNA. The ratio
and relaxed circular DNAs, the grooves (major/minor) and
of peak fluorescence intensity (F0/dF) in the presence and in the
intercalating spaces in the relaxed DNA are more accessible
absence of ctDNA has been plotted as a function of DNA
to the ligand than the case with super-coiled DNA. Hence,
concentration for all the four compounds (Fig. 22). A best
receptors 3 and 4 having much flexibility relative to receptors 1
linear least-squares fit gives the slope and intercept for all the
and 2 may undergo intercalation or be accommodated into DNA
compounds. Hence, association constant29 (Ka) values for all
grooves (induced fit) in addition to the binding of phosphate of
the four compounds were determined (Table 4).
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 CD study. The binding interactions of the compounds with
is why 3 and 4 preferentially bind to the relaxed DNA. More- DNA were further realized from the CD study. Binding of a
over, receptors 1 and 2 having stronger affinity to phosphate ions compound with DNA may stabilize or destabilize the latter.
than that of 3 and 4 preferentially bind to super-coiled DNA Circular dichroism (CD) is an effective method to examine the
where majority of phosphate bonds are exposed and some of its structural modification of DNA, including changes in the
grooves and intercalating spaces are buried. Thus in the present secondary and tertiary structures of DNA caused by an
experiment, while receptors 1 and 2 having the same binding interaction with some compounds. Literature reports suggest
pattern favours super-coiled DNA binding, under similar condi- that the secondary structures of DNAs are perturbed by
tions receptors 3 and 4 with the identical binding pattern favours interaction with small molecules.30,31
the binding of relaxed DNA. We also performed the gel retarda- Binding of small molecules to DNA may drive the entire
tion assay for compounds 1–4 with calf-thymus DNA (ctDNA) assembly of the multistranded DNA structure. In the present
whose molecular weight is much greater than plasmid DNA. For study, we exploited circular dichroism to identify the perturbation
calf thymus the shift was less (Fig. S15 in the ESIw) compared to in the secondary structure of ctDNA due to interaction with
plasmid DNA after binding with compounds. the four compounds. Before investigating the interaction of
the compounds with the DNA through the change in the CD
Steady-state fluorescence study. The fluorescence spectra of signal, we initially recorded the CD spectra of the compounds
Tris-EDTA (TE) buffer of 1, 2, 3, and 4 show quite alike broad 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
View Article Online
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J
Downloaded by Kalyani University on 08 January 2013

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
View Article Online

negative peak is almost unaffected. A plausible explanation N1,N3-di(pyridin-3-yl) isophthalamide (5). The symmetrical
for the change in the normal CD spectra of ctDNA is the bis-amide 5 was obtained by coupling of 3-aminopyridine
disruption of stacking interaction of the bases, which is required (1.2 g, 12.8 mmol) with isophthaloyl diacidchloride (1.3 g,
to optimize the binding interactions.33 These changes may thus 6.4 mmol) in dry CH2Cl2 (50 mL) followed by addition of
reflect local untwisting of the DNA helical backbone and triethylamine (1.4 mL) under a nitrogen atmosphere. The
changes in relative orientation of the bases to accommodate reaction mixture was stirred overnight. After completion of
the intercalating compounds within a particular base pair.33,35 the reaction, the solvent was removed under vacuum. The
The extent of increase in the ellipticity is different for the four residual mass was extracted with the CHCl3/CH3OH mixture
compounds (Fig. 23), being maximum for 1 (Fig. 23a) and (3  20 mL). The organic layer was washed with NaHCO3
minimum for 2 (Fig. 23b), which implies that binding of solution (3  15 mL) and dried over anhydrous Na2SO4. The
compound 1 causes the largest perturbation to the DNA solvent was removed and the residual mass was purified by
strands and the most extensive destacking and destabilization column chromatography over silica gel using chloroform :
of the DNA helix. methanol (4 : 1) as eluent to afford the desired compound 5
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

(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
Conclusion (s, 1H), 8.33 (d, J = 4 Hz, 2H), 8.23–8.18 (m, 4H), 7.74
In conclusion, we have reported in this full account that (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,
Downloaded by Kalyani University on 08 January 2013

anthracene labeled different bispyridinium amides 1–4 are


useful in the selective sensing of dihydrogenphosphate36 in 136.5, 135.5, 131.8, 129.6, 128.2, 128.0, 124.4 ppm. FTIR (KBr):
organic solvent. The sensing ability is dependent on the nature n (cm1): 3418, 3054, 1676, 1613, 1585, 1556, 1481, 1429. Mass
of the spacers that hold the anion binders. In a semi-aqueous (ESI): 319.1 [M + H]+, 225.2, 130.2.
system the sensors are weakly bound to dihydrogenphosphate
and hydrogenphosphate ions rather than phosphates. Depending N2,N6-di(pyridin-3-yl) pyridine-2,6-dicarboxamide (6). The
on the flexible nature of the spacer in between the pyridinium symmetrical bisamide 6 was obtained by coupling of 3-amino-
amides the dicarboxylate ions can be sensed effectively. In the pyridine (0.76 g, 5.39 mmol) with pyridine-2,6-dicarbonyl
present case, receptors 3 and 4 exhibit selectivity for glutarate dichloride (0.512 g, 2.695 mmol) in dry CH2Cl2 (50 mL)
and adipate, respectively, by showing excimers of significant followed by addition of triethylamine (1.4 mL) under a nitrogen
intensities. With the information of binding of phosphate atmosphere. The reaction mixture was stirred overnight. After
derivatives, a detailed study on the receptors in cell staining completion of the reaction, solvent was removed under vacuum.
and DNA interaction has been performed. The receptors are The residual mass was extracted with the CHCl3/CH3OH
quite impressive in cell staining. They stain not only DNA mixture (3  20 mL). The organic layer was washed with
(as Hoechst dye does) but also the entire cell. It has also been NaHCO3 solution (3  15 mL) and dried over anhydrous
established, for the first time, that the pyridinium based Na2SO4. The solvent was removed and the residual mass was
receptors have the strong propensity towards interaction with purified by column chromatography over silica gel using
DNA. The gel retardation and CD studies have proved the chloroform : methanol (4 : 1) as eluent to afford the desired
strong DNA interaction and the effectiveness is related to compound 6 (0.734 g, 85% yield, mp 240 1C). 1H NMR
the nature of the spacer that holds the pyridinium binding (d6-DMSO, 400 MHz): d 11.2 (s, 2H, amide NH), 9.11
sites. Further work along this direction is underway in our (s, 2H), 8.43–8.40 (m, 4H), 8.35–8.31 (m, 3H), 7.50 (d, 1H,
laboratory. 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.
Experimental Mass (ESI): 320.1 (M + H)+, 209.2, 130.3.
General methods for anion binding studies in organic solvents
N1,N5-di(pyridin-3-yl) glutaramide (7). The symmetrical
Materials were obtained from commercial suppliers and were bisamide 7 was obtained by coupling of 3-aminopyridine
used without further purification. Thin layer chromatography (1.145 g, 12.2 mmol) with glutaroyl dichloride (1 g, 6.1 mmol)
(TLC) was carried out using Merck 60 F254 plates with a in dry CH2Cl2 (50 mL) followed by addition of triethylamine
thickness of 0.25 mm. Melting points were determined on a (1.4 mL) under a nitrogen atmosphere. The reaction mixture
hot-plate melting point apparatus in an open-mouth capillary was stirred overnight. After completion of the reaction,
and are uncorrected. 1H, 13C and 31P NMR spectra were solvent was removed under vacuum. The residual mass was
recorded on Brucker 400 MHz and 500 MHz instruments. extracted with the CHCl3/CH3OH mixture (3  20 mL). The
For NMR spectra, CDCl3, CD3CN and d6-DMSO were used organic layer was washed with NaHCO3 solution (3  15 mL)
as solvents using TMS as an internal standard. Chemical shifts and dried over anhydrous Na2SO4. The solvent was removed
are expressed in d units and 1H–1H and 1H–C coupling and the residual mass was purified by column chromatography
constants in Hz. IR spectra were recorded on a Perkin Elmer over silica gel using chloroform : methanol (4 : 1) as eluent to
Spectrum One, using KBr discs. Fluorescence spectra were afford the desired compound 7 (0.644 g, 39% yield, mp
recorded on a Perkin Elmer Model LS 55 spectrophotometer 188 1C). 1H NMR (d6-DMSO, 400 MHz): d 10.13 (s, 2H, amide
and UV-vis spectra recorded on a Perkin Elmer Model NH), 8.72 (d, 2H, J = 2 Hz), 8.23 (d, 2H, J = 4.40 Hz), 8.02 (d,
Lambda 25, respectively. 2H, J = 8 Hz), 7.34–7.30 (m, 2H), 2.42 (t, 4H, J = 7.30 Hz),

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 1241
View Article Online

1.96–1.88 (m, 2H) ppm. 13C NMR (d6-DMSO, 100 MHz): d diethyl ether to give pure dichloride salt 10 (0.076 g, 32%
172.5, 144.8, 141.5, 136.7, 126.8, 124.4, 36.9, 25.5. ppm. FTIR yield). The pure dichloride salt 10 (0.076 g, 0.098 mmol) was
(KBr): n (cm1): 3289, 3107, 2935, 2855, 1681, 1483, and 1459. dissolved in 5 mL of hot CH3OH and the volume was reduced
Mass (ESI): 285.3 (M + H)+, 190.9, 143.1. to 2 mL. Then aqueous solution of NH4PF6 (0.048 g,
0.290 mmol) was added in one portion to carry out the anion
N1,N6-di(pyridin-3-yl) adipamide (8). The symmetrical bisamide exchange reaction. After stirring the reaction mixture for
8 was obtained by coupling of 3-aminopyridine (1 g, 10.6 mmol) 50 min, the precipitate appeared. Filtration of the precipitate
with adipoyl dichloride (1g, 5.32 mmol) in dry CH2Cl2 (50 mL) followed by thorough washing with diethyl ether afforded
followed by addition of triethylamine (1.4 mL) under a nitrogen receptor 2 in 92% yield (0.089 g, mp 178 1C). 1H NMR
atmosphere. The reaction mixture was stirred overnight. After (d6-DMSO, 400 MHz): d 11.6 (s, 2H, amide NH), 9.53 (s, 2H),
completion of the reaction, solvent was removed under vacuum. 8.97 (s, 2H), 8.87 (brs, 2H), 8.69 (d, 2H, J = 8 Hz), 8.45 (d, 4H,
The residual mass was extracted with the CHCl3/CH3OH J = 8 Hz), 8.29–8.24 (m, 9H), 7.71 (t, 4H, J = 8 Hz), 7.64 (t, 4H,
mixture (3  20 mL). The organic layer was washed with J = 6.8 Hz), 7.03 (s, 4H) ppm. 13C NMR (d6-DMSO, 100 MHz):
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

NaHCO3 solution (3  15 mL) and dried over anhydrous d 162.3, 146.9, 140.6, 139.4, 138.5, 135.0, 134.2, 131.4, 131.1,
Na2SO4. The solvent was removed and the residual mass was 131.0, 129.5, 128.3, 126.2, 125.7, 123.2, 121.6, 56.5 (one aromatic
purified by column chromatography over silica gel using carbon is unresolved) ppm. FTIR (KBr): n (cm1): 3348,
chloroform : methanol (4 : 1) as eluent to afford the desired 3103, 1693, 1626, 1542. (ES+): m/z: 846.2 [(M–PF6)]+, 700.2
compound 8 (0.870 g, 55% yield, mp 222 1C). 1H NMR [(M–2PF6-1)]+.
Downloaded by Kalyani University on 08 January 2013

(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), Synthesis of receptor 3. To a solution of 7 (0.100 g, 0.35 mmol)
8.02 (d, 2H, J = 8.3 Hz), 7.33–7.30 (m, 2H), 2.37 (t, 4H, in 15 mL of dry CH3CN, 9-chloromethylanthracene (0.241 g,
J = 7.3 Hz), 1.64–1.68 (m, 4H) ppm. 13C NMR (d6-DMSO, 1.06 mmol) in 10 mL of CH3CN was added. The reaction
100 MHz): d 172.2, 144.8, 141.6, 136.7, 126.8, 124.4, and 36.19, mixture was refluxed with stirring for 2 days under a nitrogen
21.48 ppm. FTIR (KBr): n (cm1): 3304, 3176, 2949, 2875, atmosphere. On cooling the reaction mixture, the precipitate
1690, 1581, 1463. Mass (ESI): 299.3 (M + H)+, 237.3, 130.1. appeared. The precipitate was filtered off and washed with
Synthesis of receptor 1. To a solution of 5 (0.07 g, CH3CN several times and then with diethyl ether to give pure
0.220 mmol) in dry CH3CN (15 mL) containing dry DMF dichloride salt 11 (0.151 g, 58% yield). The pure dichloride salt
(2 mL), 9-chloromethyl anthracene (0.149 g, 0.657 mmol) in 11 (0.073 g, 0.099 mmol) was dissolved in 5 mL of hot CH3OH
dry CH3CN (20 mL) was added. The reaction mixture was and the volume was reduced to 2 mL. Then aqueous solution
refluxed with stirring for 2 days under a nitrogen atmosphere. of NH4PF6 (0.049 g, 0.297 mmol) was added in one portion to
On cooling the reaction mixture, the precipitate appeared. The carry out the anion exchange reaction. After stirring the reaction
precipitate was filtered off and washed with CH3CN several mixture for 50 min, the precipitate appeared. Filtration of the
times and then with diethyl ether to give pure dichloride salt 9 precipitate followed by thorough washing with diethyl ether
(0.115 g, 68%). The pure dichloride salt 9 (0.059 g, afforded receptor 3 in 77% yield (0.073 g, mp 192 1C).
1
0.077 mmol) was dissolved in 5 mL of hot CH3OH and the H NMR (d6-DMSO, 400 MHz): d 10.73 (s, 2H, amide NH),
volume was reduced to 2 mL. Then aqueous solution of 9.0 (s, 2H), 8.94 (s, 2H), 8.76 (d, 2H, J = 8 Hz), 8.45 (d, 2H, J = 8
NH4PF6 (0.038 g, 0.231 mmol) was added in one portion to Hz), 8.37 (d, 4H, J = 8 Hz), 8.27 (d, 4H, J = 8 Hz), 8.06–8.02 (m,
carry out the anion exchange reaction. After stirring 2H), 7.70–7.62 (m, 8H), 6.94 (s, 4H), 2.42 (t, 4H, J = 8 Hz),
the reaction mixture for 50 min, the precipitate appeared 1.66–1.77 (m, 2H) ppm. 13C NMR (d6-DMSO, 100 MHz): d
Filtration of the precipitate followed by thorough washing 172.0, 139.3, 138.5, 133.7, 132.8, 131.3, 131.1, 130.9, 129.5, 128.4,
with diethyl ether afforded the receptor 1 in 88% yield 128.2, 125.7, 123.1, 121.5, 56.27, 34.9, 19.6 ppm. FTIR (KBr):
(0.067 g), mp 216 1C; 1H NMR (d6-DMSO, 400 MHz): d n (cm1): 3390, 3278, 3116, 3060, 1685, 1556, 1449. (ES+):
11.15 (s, NH, 2H), 9.19 (s, 2H), 8.97 (s, 2H), 8.89 (d, 2H, J = 8 m/z: 811.2 [(M–PF6)]+, 665.2 [(M–2PF6-1)]+.
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 Synthesis of receptor 4. To a solution of 8 (0.100 g,
(d, 2H, J = 8 Hz), 7.71–7.63 (m, 9H), 6.99 (s, 4 H) ppm; 0.34 mmol) in 15 mL of dry CH3CN, 9-chloromethylanthracene
13
C NMR (DMSO-d6, 125 MHz): d 161.0, 134.9, 130.7, 130.1, (0.228 g, 1.01 mmol) in 10 mL of CH3CN was added. The
129.6, 129.5, 128.8, 127.3, 126.9, 126.7, 126.5, 125.1, 124.5, reaction mixture was refluxed with stirring for 2 days under a
123.9, 122.9, 121.3, 118.7, 117.1, 52.0 ppm; FTIR (KBr): nitrogen atmosphere. On cooling the reaction mixture, the
n (cm1): 3444, 1685, 1633, 1522, 1501, 1449 ppm. (ES+): precipitate appeared. The precipitate was filtered off and
m/z: 845.3 [(M–PF6)]+, 813.3, 735.3, 699.4, 509.2. washed with CH3CN several times and then with diethyl ether
to give pure dichloride salt 12 (0.122 g, 49% yield). The pure
Synthesis of receptor 2. To a solution of 6 (0.100 g, dichloride salt 12 (0.073 g, 0.097 mmol) was dissolved in 5 mL
0.31 mmol) in 15 mL of dry CH3CN containing dry DMF of hot CH3OH and the volume was reduced to 2 mL. Then
(2 mL), 9-chloromethylanthracene (0.210 g, 0.93 mmol) in 10 mL aqueous solution of NH4PF6 (0.048 g, 0.292 mmol) was added
of CH3CN was added. The reaction mixture was refluxed with in one portion to carry out the anion exchange reaction. After
stirring for 2 days under a nitrogen atmosphere. On cooling the stirring the reaction mixture for 50 min, the precipitate
reaction mixture, the precipitate appeared. The precipitate was appeared. Filtration of the precipitate followed by thorough
filtered off and washed with CH3CN several times and then with washing with diethyl ether afforded receptor 4 in 86% yield

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
View Article Online

(0.063 g, mp 182 1C). 1H NMR (d6-DMSO, 400 MHz): d 10.73 corresponding concentrations of the host and the guest; K1 and
(s, 2H, amide NH), 9.03 (s, 2H), 8.94 (s, 2H), 8.75 (d, 2H, J = 8 Hz), K2 are two binding constants for two successive steps in eqn (1) and
8.45 (d, 2H, J = 8 Hz), 8.37 (d, 4H, J = 8 Hz), 8.26 (d, 4H, J = 8 K is the association constant in eqn (2) and (3), respectively [this
Hz), 8.05 (t, 2H, J = 8 Hz), 7.70–7.61 (m, 8H), 6.94 (s, 4H), 2.24 equation also works in absorption; I to be replaced by absorption
(brt, 4H, J = 7.2 Hz), 1.5–1.43 (brm, 4H) ppm. 13C NMR intensity (A)]. The association constants and correlation coefficients
(d6-DMSO, 100 MHz): d 172.2, 139.3, 138.4, 1337, 132.8, 131.3, (R) are obtained by a non-linear least-square analysis of I vs. CG
131.1, 130.9, 129.4, 128.4, 128.2, 125.7, 123.1, 121.5, 56.7, 35.7, 23.8 for eqn (1) and (3). In the case of eqn (2) the association constant
ppm. FTIR (KBr): n (cm1): 3394, 3097, 2957, 1697, 1593, 1464, and correlation coefficients (R) are obtained by a non-linear least-
1450. (ES+): m/z: 825.2 [(M–PF6)]+, 679.2 [(M–2PF6-1)]+. square analysis of I vs. CH and CG.

General procedure for fluorescence and UV-vis titrations Materials and methods for cell staining and DNA interaction studies
Stock solutions of the receptors were prepared in CH3CN in Chemicals. Hoechst dye was purchased from Sigma Chemicals
the concentration range B105 M. 2.5 mL of the receptor (USA). Cell culture medium (MEM) was procured from
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 HiMedia, Mumbai, India. Agarose, calf thymus DNA were
the concentration range B104 M were prepared in the same obtained from SRL, Mumbai, India. Other molecular biology
solvents and were individually added in different amounts to the grade fine chemicals were procured locally.
receptor solution. Upon addition of guests, the change in emission
of the receptor was noted. The same stock solutions for receptors Cell culture. Human cervical cancer cell line (HeLa) was
Downloaded by Kalyani University on 08 January 2013

and guests were used to perform the UV-vis titration experiment. obtained from National Centre for Cell Sciences, Pune, India.
Guest solution was successively added in different amounts to the HeLa cells were routinely grown in MEM (HiMedia, Mumbai,
receptor solution (2.5 mL) taken in the cuvette and the absorption India) supplemented with 10% bovine serum (complete medium)
spectra were recorded. Both fluorescence and UV-vis titration in a CO2 incubator (Heal Force, Shanghai Lishen, China) at 37 1C
experiments were carried out at 25 1C. All the experiments were in a humidified atmosphere containing 5% CO2.
repeated thrice to check the reproducibility.
Measurement of DNA concentration. Stock solutions were
Job plots prepared by dissolving the solid calf-thymus DNA in Tris-EDTA
(TE) buffer (pH = 8) and stored at 4 1C. Freshly prepared DNA
The stoichiometry was determined by the continuous variation
solution was used for all the experiments. The purity of DNA
method (Job plot).21 In this method, solutions of host and
was verified by monitoring the ratio of absorbance at 260 nm to
guests of equal concentrations were prepared in the solvents
that at 280 nm, which was in the range of 1.8–1.9. The
used in the experiment. Then host and guest solutions were
concentration of DNA was determined spectrophotometrically,
mixed in different proportions maintaining a total volume of
using ADNA = 13 600 M1 cm1 at 258 nm.37,38 DNA
3 mL of the mixture. All the prepared solutions were kept for
concentration mentioned in fluorescence quenching and CD
1 h with occasional shaking at room temperature. Then emission
study is in terms of base pair (bp).
and absorbance of the solutions of different compositions
were recorded. The concentration of the complex i.e., [HG] Cell staining with synthetic compounds. The HeLa cells were
was calculated using the equation [HG] = DI/I0  [H] or cultured over cover slips in complete MEM medium. After
[HG] = DA/A0  [H] where DI/I0 and DA/A0 indicate the 24 h incubation in a CO2 incubator, the cover slips were taken
relative emission and absorbance intensities. [H] corresponds to out, washed with phosphate buffered saline (PBS) twice. The
the concentration of pure host. Mole fraction of the host (XH) cells were then fixed with methanol–acetone (1 : 1) at 4 1C for
was plotted against concentration of the complex [HG]. In the 1 h. After fixation cells were again washed with PBS buffer
plot, the mole fraction of the host at which the concentration of twice and incubated with 5.0 mM of receptor 1, 5.0 mM of
the host–guest complex concentration [HG] is maximum gives receptor 3, 5.0 mM of receptor 4 and 5.0 mM of receptor 2 in
the stoichiometry of the complex. PBS separately at room temperature for 5 minutes. After
incubation in the dark the cover slips were washed twice and
Determination of binding constants
placed upside down over a glass slide, so that cells were in
Binding constant values were determined by fluorescence and touch with the glass slide. The slide was observed under
absorption methods using eqn (1)–(3). fluorescence microscope (Carl Zeiss) with a UV excitation
filter as well as in normal light mode. Another set was done
I = (I0 + K1  CG  I[1 : 1] + K1  K2  Ilim  C2G)/
for live cell imaging without fixation. After grown over cover
(1 + K1  CG + K1  K2  C2G) (1) slips cells were washed with PBS twice and stained with the
I = I0 + (I  I0)/2CH{CH + CG + 1/K compounds as described earlier in PBS at room temperature
for 5 minutes. Cells were observed and photographed under a
 [(CG + CH + 1/K)2  4CGCH]0.5} (2) fluorescence microscope (Carl Zeiss) with a UV excitation
I = (I0 + I  K  CG)/(1 + K  CG) (3) filter as well as in normal light mode. All the photographs
were taken at the same microscopic field in both bright-field
where I represents fluorescence intensity; I0 represents (Fig. S14 in the ESIw) and fluorescence mode.
the intensity of pure host; I[1 : 1] represents the intensity of
the 1 : 1 complex between host and guest and Ilim represents the Bacterial transformation and plasmid isolation. E. coli DH5a
intensity at infinite guest concentration. CH and CG are the cells (Clontech, USA) were made competent using calcium

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 1243
View Article Online

chloride following the standard method as described in Cohen departments under DST-FIST program. We are also grateful
et al. (1972)39 with slight modification. 50 ng of pEGFP-C1 to Prof. Soumen Basak, Chemical Sciences Division, Saha
(Clontech, USA) plasmid (3102 kDa) was used for transforma- Institute of Nuclear Physics, Kolkata, India, for giving us
tion and the transformed cells were grown in the presence of an opportunity to use the JASCO J-720 spectropolarimeter
50 mg mL1 kanamycin (HiMedia, India) in LB broth medium for CD spectra.
(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
References
SDS as described in Sambrook et al. (1989).40 Isolated 1 (a) Chemical Sensor and Biosensors for Medical and Biological
plasmid was treated with RNase A (20 mg mL1) to avoid the Application, ed. U. E. Spichiger-Keller, Wiely-VCH, Weinheim,
Germany, 1998; (b) C. F. Mason, Biology of Freshwater Pollution,
RNA contamination. Finally the plasmid DNA was purified by a Longman, New York, 2nd edn, 1991; (c) P. A. Gale, Acc. Chem.
standard phenol–chloroform extraction procedure and checked Res., 2006, 39, 465; (d) J. W. Steed, Chem. Commun., 2006, 2637.
through 1% agarose gel electrophoresis.40 The concentration of 2 (a) P. S. Corbin and S. C. Zimmermann, J. Am. Chem. Soc., 2000,
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

122, 3779; (b) M. Barboiu and J.-M. Lehn, Proc. Natl. Acad. Sci.
the isolated plasmid DNA was checked by a spectrophotometer.
U. S. A., 2002, 99, 5201; (c) R. Martinez- Manez and F. Sancenon,
This plasmid DNA was used for the gel retardation assay. Chem. Rev., 2003, 103, 4419; (d) F. P. Schmidtchen and M. Berger,
Chem. Rev., 1997, 97, 1609; (e) P. D. Beer and P. A. Gale, Angew.
DNA binding Chem., Int. Ed., 2001, 40, 486; (f) J. Yoon, S. K. Kim, N. J. Sing
1. Gel retardation assay. 26.5 mM of each compound (1–4) and K. S. Kim, Chem. Soc. Rev., 2006, 35, 355;
Downloaded by Kalyani University on 08 January 2013

was mixed separately with 1.0 mg of pEGFP-C1 plasmid DNA (g) M. E. Moragues, R. Martinez- Manez and F. Sancenon, Chem.
Soc. Rev., 2011, 40, 2593.
(Clontech, USA) in Tris-EDTA (TE) buffer, pH 8 in micro- 3 (a) A. Szumna and J. Jurczak, Eur. J. Org. Chem., 2001, 4031;
centrifuge tubes and incubated for 25 minutes. All samples (b) C. P. Causey and W. E. Allen, J. Org. Chem., 2002, 67, 5963;
were loaded in 1.0% normal agarose gel after adding 1 gel (c) S. J. Coles, L. S. Evans, P. A. Gale, M. B. Hursthouse and
M. E. Light, Chem. Commun., 2003, 568; (d) L. S. Evans and P. A.
loading dye and run in 1 Tris-acetate EDTA (TAE) buffer at
Gale, Chem. Commun., 2004, 1286; (e) J. M. Kang and J. H. Kim,
constant 60 V for 3 h. After staining with ethidium bromide Tetrahedron Lett., 2005, 46, 1759; (f) S. J. Brooks, L. S. Evans,
(1.0 mg mL1) for 5 minutes, samples were observed using a P. A. Gale, M. B. Hursthouse and M. E. Light, Chem. Commun.,
Typhoon 9210 (Variable Mode Imager), GE Healthcare. 2005, 734; (g) S. H. Lee, H. J. Kim, Y. O. Lee, J. Vicens and
J. S. Kim, Tetrahedron Lett., 2006, 47, 4373; (h) W. Gong, S. Bao,
2. Steady-state fluorescence study. Steady-state fluorescence F. wang, J. Ye, G. Ning and K. Hiratani, Tetrahedron Lett., 2011,
52, 630.
measurements were carried out using a Perkin-Elmer Luminescence 4 (a) R. C. Jagessar, M. Shang, W. R. Scheidt and D. H. Burns, J. Am.
spectrometer (model LS50B) equipped with FLWINLAB software Chem. Soc., 1998, 120, 11684; (b) E. J. Cho, J. W. Moon, S. W. Ko,
with a rectangular quartz cuvette of path length 1 cm. By varying J. Y. Lee, S. K. Kim, J. Yoon and K. C. Nam, J. Am. Chem. Soc.,
2003, 125, 12376; (c) C. R. Bondy, P. A. Gale and S. J. Loeb, J. Am.
DNA concentration all the emission spectra were recorded for
Chem. Soc., 2004, 126, 5030; (d) D. Estean-Gómez, L. Fabbrizzi and
receptors 1–4 separately in Tris-EDTA (TE) buffer (pH = 8). After M. Licchelli, J. Org. Chem., 2005, 70, 5717; (e) K. Ghosh and
buffer subtraction fluorescence intensity was plotted as a function S. Adhikari, Tetrahedron Lett., 2006, 47, 8165.
of wavelength and DNA concentration separately for all the four 5 M. D. Best, S. L. Tobey and E. V. Anslyn, Coord. Chem. Rev.,
2003, 240, 3.
compounds. The association constant or Ka value was calculated by 6 (a) J. H. Hartley, T. D. James and C. J. Ward, J. Chem. Soc., Perkin
using eqn (1) where F0 and F denote the measured fluorescence Trans. 1, 2000, 3155; (b) R. J. T. Houk, S. L. Tobey and E. V. Anslyn,
intensity before and after adding DNA. Here fM and fp are Top. Curr. Chem., 2005, 255, 199; (c) R. J. Fitzmaurice, G. M. Kyne,
fluorescence quantum yields of the monomer and complex, respec- D. Douheret and J. D. Kilburn, J. Chem. Soc., Perkin Trans. 1, 2002,
841; (d) S. Kubik, C. Reyheller and S. J. Stüwe, J. Inclusion Phenom.
tively. Cg is the final concentration of DNA after each addition. Macrocyclic Chem., 2005, 52, 137; (e) J. Yoon, S. K. Kim, N. J. Singh
and K. S. Kim, Chem. Soc. Rev., 2006, 35, 355.
F0/F  F0 = [fMAM/fpAp  fMAM][(1/KaCg) + 1]. . .29 7 Z. Xu, S. K. Kim and J. Yoon, Chem. Soc. Rev., 2010, 39, 1457.
(4) 8 (a) K. Ghosh and I. Saha, Tetrahedron Lett., 2008, 49, 4591;
(b) K. Ghosh, I. Saha and A. Patra, Tetrahedron Lett., 2009,
50, 2392; (c) S. Kumar and S. Kumar, Tetrahedron Lett., 2009,
3. CD Study. CD spectra were recorded on a JASCO J-720 50, 4463; (d) V. K. Khatri, S. Upreti and P. S. Pandey, J. Org.
spectropolarimeter, using a rectangular quartz cuvette of path Chem., 2007, 72, 10224; (e) K. Ghosh and D. Kar, Beilstein J. Org.
Chem., 2011, 7, 254; (f) B. Wannalarse, T. Tuntulani and
length 1 cm. Spectra shown are averages of four successive B. Tomapatanaget, Tetrahedron Lett., 2008, 64, 10619;
scans recorded at a scan speed of 50 nm min1, from which the (g) K. Ghosh, D. Kar and P. Ray Chowdhry, Tetrahedron Lett.,
appropriate blanks have been subtracted. All the experiments 2011, 52, 5098.
were performed at ambient temperature (300 K) with 9 M. Boiocchi, M. Bonizzoni, L. Fabbrizzi, G. Piovani and
A. Taglietti, Angew. Chem., Int. Ed., 2003, 42, 647.
air-equilibrated solutions. We have used 30 mM of ctDNA 10 K.-S. Jeong and Y. L. Cho, Tetrahedron Lett., 1997, 38, 3279.
and three different concentrations of compounds like 5 mM, 11 K. J. Wallace, W. J. Belcher, D. R. Turner, K. F. Syed and
10 mM and 20 mM for each of the compounds. J. W. Steed, J. Am. Chem. Soc., 2003, 125, 9699.
12 V. Amendola, M. Boiocchi, L. Fabbrizi and A. Pachetti, Chem.–Eur. J.,
2005, 11, 5648.
Acknowledgements 13 (a) J. A. Wisner, P. D. Beer, N. G. Berry and B. Tomapatenaget,
Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 4983; (b) M. R.
KG thanks CSIR, New Delhi, India, for financial support. Sambrook, P. D. Beer, J. A. Wisner, R. L. Paul, A. R. Cowley,
ARS thanks University of Kalyani, India, for a fellowship. F. Szemes and M. G. B. Drew, J. Am. Chem. Soc., 2005, 127, 2292.
14 (a) K. Ghosh, A. R. Sarkar and A. Patra, Tetrahedron Lett., 2009,
KG and UG would like to thank DST, Govt. of India, for 50, 6557; (b) K. Ghosh and I. Saha, Supramol. Chem., 2011,
providing facilities in Chemistry and Biochemistry & Biophysics 23, 518.

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

15 (a) K. Ghosh and A. R. Sarkar, Tetrahedron Lett., 2009, 50, 85; 28 (a) B. Lewin, Genes IX, Jones and Bartlett, 40 Tall Pine Drive,
(b) K. Ghosh, G. Masanta and A. P. Chattopadhyay, Eur. J. Org. Pearson Prentice Hall, Sundbury, MA 01776, pp. 476–482;
Chem., 2009, 4515; (c) K. Ghosh, I. Saha, G. Masanta, E. B. Wang (b) D. Voet and J. G. Voet, Biochemistry, John Wiley and Sons,
and C. A. Parish, Tetrahedron Lett., 2010, 51, 343. pp. 873–882.
16 K. Ghosh and I. Saha, New J. Chem., 2011, 35, 1397. 29 P. T. Chou, G. R. Wu, C. Y. Wei, C. C. Cheng, C. P. Chang and
17 A. Dorazco-Gonzalez, H. Hopfel, F. Medrano and A. K. Yatsimirsky, F. T. Hung, J. Phys. Chem. B, 2000, 104, 7818.
J. Org. Chem., 2010, 75, 2259. 30 S. S. Jain, A. Matjaz and N. V. Hud, Nucleic Acids Res., 2003,
18 K. Ghosh, A. R. Sarkar and G. Masanta, Tetrahedron Lett., 2007, 31, 4608.
48, 8725. 31 J. L. Mergny, G. Duval-Valentin, C. H. Nguyen, L. Perrouault,
19 (a) M. Herm, O. Mott and T. Schrader, Chem.–Eur. J., 2002, 8, 1485; B. Faucon, M. Rougee, T. MontenayGarestier, E. Bisagni and
(b) A. P. Davis and R. Wareham, Angew. Chem., 1998, 110, 2397. C. Helene, Science, 1992, 256, 1681.
20 (a) R. P. Bonar-Law, A. P. Davis and B. A. Murray, Angew. 32 A. Bonincontro, M. Falivene, C. LaMesa, G. Risuleo and
Chem., 1990, 102, 1497; (b) A. P. Davis and R. Wareham, M. RuizPena, Langmuir, 2008, 24, 1973.
Chem.–Eur. J., 1998, 110, 2270; (c) M. Herm, O. Molt and 33 Y. F. Long, Q. G. Liao, C. ZhiHuang, J. Ling and Y. F. Li,
T. Schrader, Chem.–Eur. J., 2002, 8, 1485. J. Phys. Chem. B, 2008, 112, 1783.
21 P. Job, Ann. Chim., 1928, 9, 113. 34 K. Van Holde, W. C. Johnson and P. S. Ho, Principles of Physical
Published on 09 March 2012 on http://pubs.rsc.org | doi:10.1039/C2NJ21024J

22 B. Valeur, J. Pouget, J. Bourson, M. Kaschke and N. P. Ernsting, Biochemistry, Prentice Hall, New York, 1998.
J. Phys. Chem., 1992, 96, 6545. 35 M. Monnot, O. Mauffret, E. Lescot and S. Fermandjian, Eur. J.
23 (a) M. J. Frisch, et al., Gaussian 03, Revision C.01, Gaussian, Inc., Biochem., 1992, 204, 1035.
Wallingford, CT, 2004; (b) M. W. Schmidt, et al., J. Comput. 36 (a) K. Shin-Ichi, H. Yuichi, K. Namiko and Y. Yumihiko, Chem.
Chem., 1993, 14, 1347; (c) Y. Podolyan and J. Leszczynski, Int. J. Commun., 2005, 1720; (b) S. I. Konodo, Y. Hiraoka,
Quantum Chem., 2009, 109, 8; (d) R. Krishnan, J. S. Binkeley, N. Kurumatani and Y. Yano, Chem. Commun., 2005, 1720;
Downloaded by Kalyani University on 08 January 2013

R. Seeger and J. A. Pople, J. Chem. Phys., 1980, 72, 650. (c) H. Ihm, S. Yun, H. G. Kim, J. K. Kim and K. S. Kim, Org.
24 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Lett., 2002, 4, 2897; (d) K. Choi and A. D. Hamilton, Angew.
Huxley, C. P. McCoy, J. T. Rademacherand and T. F. Rice, Chem. Chem., Int. Ed., 2001, 40, 3912; (e) S. Sasaki, D. Citterio, S. Ozawa
Rev., 1997, 97, 1515. and K. Suzuki, J. Chem. Soc., Perkin Trans. 2, 2001, 2309;
25 (a) P. K. Chattaraj, U. Sarkar and D. R. Roy, Chem. Rev., 2006, (f) C. Caltagirone, A. Mulas, F. Isaia, V. Lippolis, P. A. Gale
106, 2065; (b) R. G. Parr, L. V. Szentpaly and S. Liu, J. Am. Chem. and M. E. Light, Chem. Commun., 2009, 6279.
Soc., 1999, 121, 1922; (c) R. G. Pearson, Inorg. Chem., 1988, 37 N. K. Modukuru, K. J. Snow, B. S. Perrin and C. V. Kumar,
27, 734; (d) T. A. Koopmans, Physica, 1933, 1, 104. J. Phys. Chem. B, 2005, 109, 11810.
26 (a) D. M. Rudkevich, W. Verboom, Z. Brzozka, M. J. Palys, W. P. 38 D. Sarkar, P. Das, S. Basak and N. Chattopadhyay, J. Phys.
R. V. Stauthamer, G. J. VanHummel, S. M. Franken, S. Harkema, Chem. B, 2008, 112, 9243.
J. F. J. Engbersen and D. N. Reinhoudt, J. Am. Chem. Soc., 1994, 39 S. N. Cohen, A. C. Y. Chang and L. Hsu, Proc. Natl. Acad. Sci.
116, 4341; (b) P. Dydio, T. Zielinski and J. Jurczak, Org. Lett., U. S. A., 1972, 69, 2110.
2010, 12, 1076. 40 J. Sambrook, F. E. Fritsch and T. Mainiatis, Molecular cloning: a
27 H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, laboratory manual, Cold Spring Harbor Laboratory Press, Cold
71, 2703. Spring Harbor, New York, 2nd edn, 1989.

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 1245