Ortho-phenylenediamine-based open and macrocyclic receptors in selective sensing of H2PO4-, ATP and ADP under different conditions

Article abstract of DOI:10.1039/c2ob26995c

Ortho-phenylenediamine-based open and macrocyclic receptors have been designed and synthesized. The open receptor 1 and the macrocyclic receptor 2 fluorimetrically distinguish H₂PO₄^- from the other anions examined in CH₃CN with appreciable binding constant values. As practical applications, they are also sensible to nucleotides in aq. CH₃CN (1:1, v/v). The receptor 1 shows significant emission change upon complexation of ATP and ADP. ADP is selectively distinguished by a ratiometric change in emission. In contrast, the macrocyclic receptor 2, under similar conditions, shows good binding with ATP over the others.

Full text of DOI:10.1039/c2ob26995c

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PAPER
Ortho-phenylenediamine-based open and macrocyclic receptors in selective
−
sensing of H2PO4 , ATP and ADP under different conditions†
Kumaresh Ghosh* and Indrajit Saha
Received 19th June 2012, Accepted 15th October 2012
DOI: 10.1039/c2ob26995c
Ortho-phenylenediamine-based open and macrocyclic receptors have been designed and synthesized. The
open receptor 1 and the macrocyclic receptor 2 fluorimetrically distinguish H2PO4− from the other anions
examined in CH3CN with appreciable binding constant values. As practical applications, they are also
sensible to nucleotides in aq. CH3CN (1 : 1, v/v). The receptor 1 shows significant emission change upon
complexation of ATP and ADP. ADP is selectively distinguished by a ratiometric change in emission. In
contrast, the macrocyclic receptor 2, under similar conditions, shows good binding with ATP over the
others.
nucleotide7 (e.g., ATP, ADP and AMP) recognition have been
reported in the literature.
Introduction
The design and synthesis of fluorescent receptors of simple
architectures for sensing anions of different shapes is an area of
interest to the supramolecular chemists.1 Anions are omnipresent
in biological systems and play important roles in biology, phar-
macy and environmental sciences.2 Among the different anions,
phosphate anions (both inorganic and organic) are important as
they take part in almost all metabolic processes.3 On the other
hand, phosphate derivatives encompassing nucleotides (ATP,
ADP and AMP) are equally important for their role in bioener-
getics, metabolism, and transfer of genetic information.4 For
instance, ATP is a multifunctional nucleotide, important as a
molecular currency of intracellular energy transfer, and is also
involved in DNA replication and transcription.5 ADP and AMP
are the hydrolysis products of ATP under cellular conditions and
therefore the ratio of ATP to AMP is useful in giving the infor-
mation on the metabolic rate of the cell. ATP also serves as a
phosphate donor in kinase catalysed protein phosphorylation.
Deficiency in ATP results in ischemia, Parkinson’s disease, and
hypoglycemia.5f Therefore, selective sensing of any one of these
nucleotides (ATP, ADP and AMP) and also the inorganic phos-
In continuation of our interest to develop ortho-phenylenedi-
amine-based synthetic clefts for the selective sensing of
anions,6k,7m,8 we report here two new receptor modules 1 and 2
that selectively recognize tetrabutylammonium dihydrogenphos-
phate in CH3CN over a series of other anions by exhibiting
characteristic emissions. In addition, both the receptors are sensi-
tive to the detection of ATP and ADP in aq. CH3CN (1 : 1, v/v)
with distinguishable features.
−
3−
phate either in the form of H2PO4 or PO4 is challenging.
Considerable effort has been devoted to recognising such species
Results and discussion
by synthetic fluororeceptors. To date various fluorescent recep-
The receptors 1 and 2 were accomplished according to Schemes
1 and 2. In Scheme 1, 2-naphthol was transformed into the
chloro compound 4b, which on reaction with benzimidazole in
the presence of NaH gave compound 5. Further reaction of 5
with the compound 3, obtained from the reaction of ortho-
phenylenediamine with chloroacetyl chloride, furnished dichlor-
ide salt 6. Exchange of Cl− in 6 with NH4PF6 yielded the com-
pound 1 in appreciable yield.
− 6
tors for dihydrogenphosphate (H2PO4
)
as well as for
Department of Chemistry, University of Kalyani, Kalyani-741235, India.
E-mail: ghosh_k2003@yahoo.co.in; Fax: +913325828282;
Tel: +913325828750
†Electronic supplementary information (ESI) available: Figures showing
the change in fluorescence and UV-vis titrations of receptors 1 and 2
with various anions, Job plot, NMR and mass spectra and other figures
of 1 and 2. See DOI: 10.1039/c2ob26995c
The macrocycle 2 was synthesized following the reaction
in Scheme 2. Initially, (rac)-1,1′-binaphthol (BINOL) 7 was
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Scheme 1 (a) i. Chloroacetyl chloride, Et3N, dry CH2Cl2, 1 h, 91%;
(b) ii. 2-Chloroethanol, K2CO3, acetone, reflux 24 h, 67%; iii. SOCl2,
dry pyridine (cat.), dry CHCl3, 12 h, 91%; iv. Benzimidazole, NaH,
TBAI, dry THF, reflux 10 h, 86%; v. 3, dry CH3CN, reflux, 5 days,
64%; vi. NH4PF6, DMF–H2O (1 : 1, v/v), 12 h, 90%.
Fig. 1 Change in emission of receptor 1 (c = 3.75 × 10−5 M) in
CH3CN with increase in concentration of (a) H2PO4−, (b) ClO4−, (c) F−
and (d) AcO− upon excitation at 300 nm.
are isoenergetic to each other, we see one broad excimer peak.
However, the major contribution to the broad peak is due to the
relaxation from different vibrational levels of the excited state to
the ground state. In this case, there could be different distinct
excimer complexes which may give rise to structured broad
emission.9 Such a structured nature of the excimer emission was
more clearly observed in titration spectra of 1 with ClO4−, F−
and AcO− (Fig. 1b–1d), keeping the monomer maxima almost
unaltered at 345 nm, except for F−. A nearly 5 nm red shift of
the monomer emission was noticed with F−. The change in flu-
orescence intensity of 1 with the other anions was too small to
consider effective complexation (Fig. S1 in ESI†). As can be
seen from Fig. 2, the change in fluorescence ratio at 345 nm in
the presence of 3 equivalent amounts of anions is significant
only for H2PO4−. Upon addition of 3 equivalent amounts of the
anions, the excimer emission was not much intense except in the
case of H2PO4−. Therefore, the titration was further continued up
to the addition of 10 equivalent amounts of the guests. The
growth of the excimer was noted to be significant upon the
Scheme 2 i. 2-Chloroethanol, K2CO3, acetone, reflux 24 h, 65%; ii.
SOCl2, pyridine (cat.), dry CHCl3, 12 h, 81%; iii. Benzimidazole, NaH,
TBAI, dry THF, reflux 10 h, 42%; iv. 3, dry CH3CN (high dilution),
reflux, 4 days, 69%; (v) CH3OH–H2O, NH4PF6, 30 min, 87%.
refluxed with 2-chloroethanol in dry acetone in the presence of
K2CO3 to afford compound 8. It was next treated with SOCl2 in
dry CHCl3 to obtain the dichloro compound 9, which on reac-
tion with benzimidazole in the presence of NaH gave com-
pounds 10. High dilution reaction of 10 with the compound 3 in
dry CH3CN under refluxing conditions produced the dichloride
salt 11. The anion exchange of 11 using NH4PF6 in CH3OH
afforded compound 2 in 87% yield.
−
addition of F−, ClO4−, and H2PO4 (Fig. S5, ESI†). The associ-
ated change in ratio of the excimer emission to monomer emis-
sion with [G]/[H] is highlighted in Fig. S6 (ESI†). The
downward running of the curve for H2PO4 in Fig. S6† is attri-
Anion binding studies
−
buted either to decomplexation or the deprotonation of the
Anion binding properties of 1 towards a series of anions were
evaluated using UV-vis, and fluorescence titration experiments.
Additionally, H NMR spectra were recorded in the presence of
bound H2PO4− ions.10
1
The complexation induced enhancement of fluorescence in 1
is explained due to the deactivation of the thermodynamically
favorable PET process occurring in between the binding site and
the excited state of naphthalene.
However, the sharp break of the titration curves at [G]/[H] = 1
for AcO− and C6H5COO− with 1 (Fig. S7, ESI†) and at [G]/[H]
= 2 with H2PO4−, fumarate demonstrates the formation of 1 : 1
and 2 : 1 (guest : host) complexes, respectively. Job plots11
further confirmed this except in the case of H2PO4−. The exact
stoichiometry of the complex of H2PO4− with 1 was evaluated to
be 1 : 1 from Job’s plot (Fig. S8, ESI†).
the anionic guests to identify the interacting protons in the
binding domain of 1. In fluorescence, the receptor 1 (c = 3.75 ×
10−5 M) showed emission at 345 nm when excited at 300 nm in
CH3CN. Upon titration with H2PO4−, the emission intensity at
345 nm increased with a red shift of ∼15 nm (Fig. 1a) followed
by the appearance of broad structured emission centered at
420 nm. We believe that this broad, structured emission arises
from the energetically and structurally distinct isomers of the
excimer complex (that probably would exist only at the excited
state). In general, as most of the possible excited state isomers
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for 1 with the anions using the fluorescence data (Table 2).12a It
is evident from Table 2 that the open cleft of 1 has a preference
−
for the H2PO4 ion, although other anions exhibit moderate
binding.
−
The selectivity in the binding of H2PO4 by 1 was confirmed
from the change in emission of 1 in the presence of other anions.
Fig. 3 describes this aspect. Only F− shows some interference in
the binding process.
Fig. 2 Fluorescence ratio (I − I0/I0) of receptor 1 (c = 3.75 × 10−5 M)
at 345 nm upon addition of 3.0 equivalent amounts of a particular anion
in CH3CN.
Table 1 Fluorescence decay times (τ), and preexponential factors for 1
in CH3CN
Receptor in the presence and
absence of guest
Fluorescence decay time τ
(preexponential factor)
Time resolved fluorescence measurement of 1 (λexc = 300 nm)
1 at 345 nm
τ1 = 2.24 × 10−10 (91.11%),
τ2 = 7.07 × 10−9 (8.89%)
(χ2 = 1.04)
was carried out in the presence and absence of anions such as
−
H2PO4 and ClO4−. The emission decay profile of 1 monitored
1 + H2PO4− at 345 nm
1 + H2PO4− at 420 nm
1 + ClO4− at 345 nm
τ1 = 2.47 × 10−10 (47.63%),
τ2 = 1.78 × 10−9 (52.37%)
(χ2 = 1.03)
at 345 nm could be fitted bi-exponentially with two constants
τ1 = 0.224 ns (91.11%), τ2 = 7.07 ns (8.89%). The faster decay
component (0.224 ns) is due to the benzimidazolium moiety and
a relatively stable component with greater lifetime (7.07 ns) is
attributed to the naphthalene motif of 1. In the presence of 1
equivalent amount of H2PO4−, while the lifetime of the benzimi-
dazolium moiety marginally increases (0.247 ns, 47.63%), the
lifetime for the naphthalene motif decreases significantly
(1.78 ns, 52.37%). Between these two, the component 1.78 ns
for naphthalene with nearly the same preexponential factor as
that of the benzimidazolium motif is substantially destabilized
τ1 = 3.87 × 10−10 (8.58%),
τ2 = 5.14 × 10−9 (91.42%)
(χ2 = 1.11)
τ1 = 2.5 × 10−10 (90.79%),
τ2 = 5.9 × 10−9 (9.21%)
(χ2 = 1.08)
Table 2 Association constant values for receptor 1
−
due to complexation of H2PO4 into the benzimidazolium sites
Anions
Ka in M−1 at 422 nm
of 1. Complexation at this site induces a conformational change
for which the naphthalene groups come close to form the
excimer. This is established from the emission decay profile of 1
at the excimer wavelength (420 nm) in the presence of an equiv-
alent amount of H2−PO4−. The bi-exponential emission decay
profile of 1·H2PO4 monitored at the excimer wavelength
(420 nm) reveals that the contribution of this component towards
the total emission is significantly greater (5.14 ns, 91.42%). Like
−
H2PO4
1.23 × 104
nd
Fumarate
ClO4
3.65 × 103
4.97 × 103
1.85 × 103
−
F−
CH3COO−
nd = not determined.
−
H2PO4−, ClO4 anion binding also affected the decay profile of
1. The bi-exponential fluorescence decay profile of the complex
1·ClO4− at the monomer emission wavelength (345 nm) corrobo-
rates that the contribution of the component for the naphthalene
motif towards total emission is considerably small (5.9 ns,
9.21%) compared to the short-lived component assigned to the
benzimidazolium motif (0.25 ns, 90.79%). This is quite different
−
from the situation as observed for the complex of 1·H2PO4
.
The results are accumulated in Table 1 and the decay profiles are
represented in Fig. S9 and S10 (ESI†).
In order to investigate the ground state binding of 1, UV-vis
titration experiments were carried out in CH3CN (Fig. S3, ESI†).
Except for H2PO4− and F−, the anion binding induced change in
absorbance for the naphthalene moiety (∼320 nm) in 1 was
minor with anions such as Cl− and NO3 (Fig. S3, ESI†). As
−
noted in Fig. S3,† the greater change in absorbance at 269 nm
for the benzimidazolium moiety relative to the naphthalene
moiety explains the weak interaction of the cleft of 1 with the
anions in the ground state. Accordingly, we were unable to deter-
mine the binding constant values for the anions considering the
absorbance data. Indeed, we determined the binding constants
Fig. 3 Change in fluorescence ratio of 1 (c = 3.75 × 10−5 M) upon
addition of 10 equivalent amounts of H2PO4 in the presence of other
−
anions in CH3CN.
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The anion binding was also realized from 1H NMR in
CD3CN. In this regard, Fig. S11 (ESI†) shows the change in H
the event. On complexation the pendant naphthalene groups
under flexible linkers are stacked either in edge-to-face or face-
to-face mode. This is in accordance with the appearance of the
excimer in Fig. 1.
1
NMR of 1 in the presence and absence of equivalent amounts of
AcO− and F− anions. Upon addition of AcO−, the amide
protons (Ha), which appeared at 8.75 ppm, became too broad to
detect. This may be due to either deprotonation or the hydrogen
bonding effect. But the benzimidazolium protons (Hc) suffered a
considerable downfield chemical shift (Δδ = 0.54 ppm) indicat-
ing their intimate hydrogen bonding contact with the carboxylate
moiety. A similar finding was noted on addition of F− to the
solution of 1 in CD3CN. Interestingly, the methylene protons of
type b underwent downfield shifts by 0.13 to 0.21 ppm upon
complexation of both AcO− and F− ions. However, addition of
To improve the selectivity in binding, the macrocyclic struc-
ture 2 (c = 3.43 × 10−5 M) which showed emission at 365 nm in
CH3CN on excitation at 300 nm was considered. Fig. 6 rep-
resents the change in emission of 2 upon addition of 2.0 equival-
ent amounts of various anions in CH3CN and the emission
intensity of the macrocycle is significantly perturbed in the pres-
−
ence of H2PO4 and fumarate only. Both the anions followed
2 : 1 (guest : host) binding stoichiometry, evident from the break
of the titration curves in Fig. S13 (ESI†). Fig. 7a and 7b illustrate
−
−
equivalent amounts of H2PO4 to 1 in CD3CN resulted in pre-
the emission titration spectra of 2 with H2PO4 and fumarate
1
cipitation. But we were successful in recording H NMR of the
anions, respectively. During titration, the emission at 365 nm
was red shifted by ∼13 nm and ∼11 nm with H2PO4− and fuma-
rate anions, respectively. Here also the complexation induced
−
complex of 1 with H2PO4 in DMSO-d6 which is known as a
hydrogen bonding interfering solvent in host–guest complexa-
−
tion.13 In this regard, the spectral change for 1 with H2PO4 is
shown in Fig. 4. The results for AcO− and F− can be found in
−
Fig. S12 (ESI†). With H2PO4 the change in the chemical shift
of the interacting protons of 1 was considerable and the binding
constant value was determined to be 1.63 × 103 M−1 12b
This is
.
less compared to the value obtained by the fluorescence method
in less polar CH3CN. For other anions, small shifting of the
interacting amide and benzimidazolium protons due to signifi-
cant interference of DMSO was the main hindrance to determin-
ing the binding constant values accurately.
1
Based on observations in H NMR and emission studies (see
Fig. 1), we proposed binding structures of 1 with AcO−,
−
H2PO4−, F− and ClO4 ions which were optimized by the
PM6 method14 in CH3CN solvent (Fig. 5). As can be seen from
−
Fig. 5, F− and ClO4 ions are complexed into the cavity invol-
ving less hydrogen bonds. The fluoride ion being smaller in the
series does not fit into the cavity properly. Even though the
change in emission of 1 in the presence of F− is attributed to its
more basic character that regulates hydrogen bond formation and
deprotonation of the benzimidazolium and amide protons during
Fig. 5 PM6 optimized structures for 1 with (a) H2PO4− [a = 1.79 Å, b
= 2.80 Å, c = 1.72 Å, d = 2.05 Å, e = 1.92 Å, f = 2.28 Å, g = 2.55 Å;
dnap–nap = 3.12 Å], (b) AcO− [a = 1.88 Å, b = 2.19 Å, c = 1.71 Å, d =
1.69 Å, e = 2.18 Å, f = 1.90 Å, dnap–nap = 2.76 Å], (c) F− [a = 1.022 Å,
−
dnap–nap = 3.89 Å] and (d) ClO4 [a = 1.99 Å, b = 2.16 Å, c = 2.07 Å,
d = 2.44 Å, 2.55 Å, dnap–nap = 3.20 Å], in CH3CN.
Fig. 4 Change in 1H NMR (400 MHz) of 1 (c = 2.67 × 10−3 M) upon
successive addition of tetrabutylammonium dihydrogenphosphate
(c = 6.76 × 10−2 M) in d6-DMSO.
Fig. 6 Change in fluorescence intensity of 2 (c = 3.43 × 10−5) at
365 nm (λEx = 300 nm) in the presence of 2 equivalent amounts of
anions.
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Table 3 Fluorescence decay times (τ), and preexponential factors for 2
in CH3CN
Receptor in the presence and
absence of guest
Fluorescence decay time τ
(preexponential factor)
2 at 365 nm
τ1 = 1.36 × 10−10 (20.19%),
τ2 = 5.1 × 10−9 (79.81%)
(χ2 = 1.09)
2 + H2PO4− at 365 nm
τ1 = 2.53 × 10−10 (17.68%),
τ2 = 3.0 × 10−9 (82.32%)
(χ2 = 0.98)
Fig. 7 Change in emission of receptor (a) 2 (c = 3.43 × 10−5 M) with
increase in concentrations H2PO4− and (b) fumarate (λexc = 300 nm).
Fig. 8 Fluorescence ratio (I − I0/I0) of receptors 1 (c = 3.75 × 10−5 M)
and 2 (c = 3.43 × 10−5 M) nm upon addition of 3.0 equivalent amounts
of a particular anion in CH3CN (grey color = receptor 2 and black
color = receptor 1).
Fig. 9 Change in 1H NMR (400 MHz) of 2 (c = 2.85 × 10−3 M) upon
addition of tetrabutylammonium dihydrogenphosphate (c = 6.76 ×
10−2 M) in d6-DMSO.
increase in emission is assumed to be due to the deactivation of
the PET (photo-induced electron transfer) process occurring in
between the macrocyclic binding domain and the excited state of
82.32%) (Fig. S16b, ESI†) and contributes to the total fluor-
escence with a greater preexponential factor compared to that of
the benzimidazolium motif which is stabilized due to complexa-
−
−
the BINOL fluorophore. However, other anions, except H2PO4
tion of H2PO4 via hydrogen bonding and charge–charge
and fumarate, interacted weakly with the macrocycle 2 (Fig. S2,
ESI†) in CH3CN and it was difficult to determine the stoichio-
metries and association constants for them accurately. Fig. S14
(ESI†) describes the change in fluorescence ratio of 2 upon
addition of 4.0 equivalent amounts of various anions. Fig. 8
illustrates the comparative views on the change in emission of
the receptors 1 and 2 upon complexation of 3 equivalent
amounts of each anion. The comparison interprets that the
macrocycle 2 shows greater sensitivity and selectivity towards
H2PO4− and fumarate anions in CH3CN than the acyclic receptor 1.
In the interaction process, the stoichiometries of the com-
interactions.
The ground-state binding of 2 was understood from UV-vis
titration experiments in CH3CN (Fig. S4, ESI†). The change in
absorbance of 2 with increase in concentration of a particular
anion was negligible or irregular suggesting weak interaction in
the ground state. Therefore, we determined the association con-
−
stant values12a for 2 with the anions (Ka for H2PO4 = 7.47 ×
103 M−1 and Ka for fumarate = 3.71 × 103 M−1) considering the
−
emission data. The selectivity in the binding of H2PO4 by 2
was realized by observing the change in fluorescence ratio in the
presence and absence of other anions (Fig. S17, ESI†). Only
fumarate showed interference in the selectivity profile.
However, with a view to identifying the interacting protons of
the macrocycle 2, we tried to record H NMR of 2 in the pres-
−
plexes of 2 with both H2PO4 and fumarate were established
from Job’s plots. In the case of H2PO4−, an equilibrium mixture
of both 1 : 1 and 1 : 2 (host : guest) types of complexes was
noticed (Fig. S15a, ESI†). On the other hand, fumarate attained
a sharp 1 : 1 stoichiometry (Fig. S15b, ESI†).
1
−
ence of equivalent amounts of H2PO4 and fumarate (taken as
their tetrabutylammonium salts) in CD3CN in the concentration
range ∼10−3 M. But we failed to record due to precipitation.
Thus, like 1, 1H NMR titration of 2 with the anions was
attempted in d6-DMSO. Strong interference of DMSO hindered
the interaction of anions into the macrocyclic cavity as
confirmed by the small change in chemical shift in Fig. S18
Time resolved fluorescence measurement was also performed
−
with 2 (λexc = 300 nm) in the presence and absence of H2PO4
(Table 3). The emission decay profile of 2 monitored at 365 nm
could be fitted bi-exponentially with two constants τ1 = 0.136 ns
(20.19%), τ2 = 5.1 ns (79.81%) (Fig. S16, ESI†). The faster
decay component (0.136 ns) is due to the benzimidazolium
moiety and a relatively stable component with greater lifetime
(5.1 ns) is attributed to the BINOL motif of 2. However, in the
presence of 1 equivalent amount of H2PO4−, while the lifetime
of the benzimidazolium moiety increases (0.253 ns, 17.68%),
the lifetime for the BINOL motif decreases significantly (3 ns,
−
(ESI†). Fig. 9 displays the spectral change for 2 with H2PO4 in
d6-DMSO where only a minor change in chemical shift values
of benzimidazolium and amide protons is observed and the
binding constant value was evaluated12b to be 4.0 × 102 M−1
which is less than the value obtained by the fluorescence method
in less polar CH3CN.
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Fig. 11 Change in fluorescence ratio (I − I0/I0) of receptor 1 (c = 4.92
× 10−5 M) at 385 nm upon addition of 20 equivalents of a particular
anion in CH3CN–H2O (1 : 1, v/v).
−
Fig. 10 PM6 optimized structure for 2 with H2PO4 (a = 2.06 Å,
b = 1.77 Å, c = 1.71 Å, d = 2.17 Å) in CH3CN.
−
The hydrogen bonding capability of 2 with H2PO4 was
further understood from the PM6 optimized structure14 in
CH3CN. Only the benzimidazolium and amide protons are
involved in the complexation (Fig. 10).
In order to understand the recognition properties of com-
pounds 1 and 2 towards the same anions in a semi-aqueous
system, we further carried out the interaction study in CH3CN–
H2O (1 : 1 v/v). Both 1 and 2 showed minor changes in emission
spectra upon addition of the anions.
Fig. 12 Change in emission of receptor 1 (c = 4.92 × 10−5 M) with
increase in concentration of (a) ADP [inset: difference spectra obtained
upon addition of ADP against initial emission of 1] and (b) ATP upon
excitation at 300 nm in CH3CN–H2O (1 : 1, v/v).
Complexation studies with biologically relevant phosphate
anions
To explore the potential applications of both the receptors 1 and
2, we further investigated the behaviour of these molecules with
the sodium salt of seven biologically relevant phosphate anions
3−
−
such as ATP, ADP, AMP, H2PO4−, HPO42−, PO4 and P2O7
.
In this context, a higher content of water in aqueous measuring
solution was desirable, but such an approach was constrained
due to the limited solubility of the receptors in water. As a
reasonable negotiation, a 1 : 1 aqueous CH3CN solution was
used in the study.
The receptor 1 showed an emission band at 345 nm in
CH3CN–H2O (1 : 1, v/v) when excited at 300 nm. The com-
plexation induced change in fluorescence of 1 upon addition of
20 equivalent amounts of the anions is displayed in Fig. 11. It is
clearly understood that receptor 1 has a preference for ATP and
ADP over the other anions studied. Fig. 12a and 12b represent
the emission titration spectra for 1 with ADP and ATP,
respectively.
A closer look into the emission profiles reveals that on pro-
gression of the titration a new band at ∼383 nm appears signifi-
cant. We presume that this band, observed in the case of both
ATP and ADP, is due to the complexation-induced unique sand-
wich π–π stacking interaction of naphthalene–adenine–naphtha-
lene units as shown in Fig. 13 for ADP. Optimization of this
suggested complex by the PM6 method in pure CH3CN inti-
mated a deviation (Fig. S21, ESI†). One of the naphthalenes
resides close to the adenine nucleus of ADP. To know about the
actual organization of the components, we tried to crystallize 1
with ADP and ATP in several combinations of organic solvents.
But we were unsuccessful in obtaining good quality crystals for
X-ray diffraction. Interestingly, though the emission at the higher
Fig. 13 Suggested binding structure of 1 with ADP.
wavelength for the excimer was observed upon complexation of
both ADP and ATP, the change in monomer emission distin-
guished these two species effectively. In the case of fluorometric
titration of 2 with ADP the monomer emission decreases and the
excimer emission increases showing a ratiometric change with
an isosbestic point at ∼360 nm (inset Fig. 12a). To the best of
our knowledge, till date, no ratiometric chemosensor for ADP is
known in the literature. On the other hand, with increasing con-
centrations of ATP, both the monomer and excimer emissions of
1 gradually increase with a slight red shift. Stoichiometries of
the complexes of 1 with ADP and ATP were 1 : 1 as confirmed
by Job’s plot (Fig. S22, ESI†).11 Thus the receptor 1 shows
selectivity towards ADP and ATP, and more importantly, it can
fluorometrically discriminate ADP from other phosphates by
showing a ratiometric change in emission.
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Table 4 Binding constant (Ka) values for receptor 2
Receptor 1
Receptor 2
Anions
Ka (M−1
)
Ka (M−1
)
ATP
ADP
Pyrophosphate
7.15 × 102 M−1
9.86 × 102 M−1
nd
5.09 × 103 M−1
2.07 × 103 M−1
3.80 × 103 M−1
nd = not determined.
Fig. 14 Change in fluorescence ratio (I − I0/I0) of receptor 2 (c = 3.83
× 10−5 M) at 365 nm upon addition of 4.0 equivalents of a particular
anion in CH3CN–H2O (1 : 1, v/v).
On the other hand, macrocycle 2 showed a well defined emis-
sion band at 365 nm when excited at 300 nm in CH3CN–H2O
(1 : 1, v/v). Fig. 14 represents the change in fluorescence ratio of
2 upon addition of 4 equivalent amounts of various anions in
CH3CN–H2O (1 : 1, v/v). Only an ATP-induced large change in
emission was observed although pyrophosphate and ADP per-
turbed the emission of 2 moderately. Fig. S23 (ESI†) represents
the change in emission of 2 with ATP and ADP. Other phosphate
anions except pyrophosphate brought about a marginal change
(Fig. S19 and S20, ESI†).
UV-vis spectra of both 1 and 2 with phosphate anions in
CH3CN–H2O (1 : 1, v/v) showed negligible change and inti-
mated their weak interactions. Binding constant values, as deter-
mined by the Benesi–Hilderband plot using emission data,13 are
depicted in Table 4. It is clearly indicated that the macrocycle 2
binds ATP and ADP more strongly than the open structure 1. We
believe that this is due to strong chelation of the phosphate
group of ATP and ADP into the organized binding centre of the
macrocycle 2.
Fig. 15 Partial 1H NMR (DMSO-d6, 400 MHz) of 1 (c = 2.65 ×
10−3 M) with equivalent amounts of ATP and ADP [see structure 1
for labeling].
Conclusions
In conclusion, the experimental findings underline the fact that
the selective recognition of H2PO4−, ADP and ATP by the
ortho-phenylenediamine-based symmetrical clefts is possible.
Due to the interplay of hydrogen bonding and charge–charge
interaction while both the receptors 1 and 2 are prone to bind
1
The interaction of 1 with ATP/ADP was monitored using H
NMR in DMSO-d6. The signal at 10.24 ppm that pertains to the
–NH groups shifted downfield by 0.56 ppm in the presence of
equivalent amounts of ADP (dissolved in D2O). The benzimida-
zolium and methylene protons showed downfield chemical shifts
of 0.09 and 0.11 ppm, respectively. This indicates that the phos-
phate ion part of ADP interacts at the cavity of the receptor. The
upfield chemical shift of the adenine ring protons during inter-
action signifies the stacking of the adenine ring in between the
pendant naphthalene units. Similar findings were observed with
ATP. The corresponding changes in chemical shift values are rep-
resented in Fig. 15.
Like 1, macrocycle 2 showed measurable interaction with
ADP and ATP under similar conditions. The amide protons that
appeared at 10.33 ppm became too broad to detect its accurate
position in the presence of both ATP and ADP (Fig. S24, ESI†).
The benzimidazolium protons underwent downfield shifts
by 0.53 ppm and 0.59 ppm, in the presence of
equivalent amounts of ADP and ATP, respectively. Indeed,
the change in chemical shift of benzimidazolium protons in 2
is relatively greater than 1. This is attributed to the
tighter association of the phosphate group into the macrocyclic
cavity of 2. In addition, the upfield shift of the adenine
ring protons (0.02 to −0.16 ppm) of both ATP and ADP signifies
the close contact of the adenine part with the BINOL motif of
the receptor.
−
H2PO4 in CH3CN selectively, in aqueous CH3CN they exhibit
reasonable preference for nucleotide phosphates such as ATP
and ADP. Receptor 1 distinguishes ADP from ATP by showing
ratiometric change in emission which is unknown in the litera-
ture. In contrast, the macrocyclic receptor 2 shows the preferen-
tial binding of ATP over ADP.
Experimental
Syntheses
2-(Naphthalen-2-yloxy) ethanol 4a. To
a
mixture of
β-naphthol (4.00 g, 27.5 mmol) and potassium carbonate
(5.75 g, 41.62 mmol) in dry acetone (40 mL), 2-chloroethanol
(3.35 g, 41.6 mmol) in 5 mL acetone was added dropwise and
the reaction mixture was refluxed for 24 h. The reaction mixture
was concentrated under vacuum and 30 mL water was added to
the mixture. The aqueous layer was extracted with CHCl3 (3 ×
100 mL) and dried over anhydrous Na2SO4. After the evapor-
ation of solvent in vacuum the crude product was purified by
column chromatography using 5% ethyl acetate in petroleum
ether as an eluent to give a white crystalline product 4a (3.50 g,
This journal is © The Royal Society of Chemistry 2012
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yield 67%), m.p. 100 °C. 1H NMR (400 MHz, CDCl3): δ
7.77–7.71 (3H, m), 7.44 (1H, t, J = 8 Hz), 7.34 (1H, t, J =
8 Hz), 7.18–7.15 (2H, br m), 4.21 (2H, t, J = 8 Hz), 4.04–4.00
(2H, m), 2.08 (1H, t, J = 4Hz); FTIR (KBr, cm−1): 3368, 2949,
2931, 1629, 1600, 1079. m/z (ES+): 189.0 [M + H]+.
127.4, 126.6, 126.5, 125.8, 125.1, 123.9, 118.2, 114.0, 113.5,
107.0, 65.3, 49.1, 46.4 (one carbon in the aromatic region is
unresolved); FTIR (KBr, cm−1): 3392, 3158, 3100, 1709, 1629,
+
1601, 1542, 1218; HRMS (TOF MS ES+): C48H42N6O4PF6 ,
(M − PF6) requires 911.2904 found 911.4358.
2-(2-Chloroethoxy)naphthalene 4b. To a stirred solution of 4a
(3.00 g, 15.94 mmol) in CHCl3 (30 mL), SOCl2 (1.4 mL,
19.13 mmol) was added dropwise followed by addition of a cata-
lytic amount of pyridine. The reaction mixture was stirred for
12 h and the solvent was removed under reduced pressure. The
residue was neutralized with aqueous NaHCO3 solution and
extracted with CHCl3 (3 × 30 mL). The organic layer was dried
over anhydrous Na2SO4 and concentrated on a rotary evaporator.
The crude product was purified by silica gel column chromato-
graphy using 2% ethyl acetate in petroleum ether to give pure
compound 4b (3.00 g, yield 91%), m.p. 120 °C. This was
directly used in the next step without characterization.
2,2′-(1,1′-Binaphthyl-2,2′-diylbis(oxy))diethanol
8. To
a
mixture of (rac)-BINOL 7 (3.00 g, 10.48 mmol) and K2CO3
(4.34 g, 31.43 mmol) in dry acetone (40 mL), 2-chloroethanol
(2.53 g, 31.43 mmol) in 5 mL acetone was added dropwise and
the reaction mixture was refluxed for 24 h. The reaction mixture
was concentrated under vacuum and 30 mL water was added to
the mixture. The aqueous layer was extracted with CHCl3 (3 ×
40 mL) and dried over anhydrous Na2SO4. After evaporation of
the solvent in vacuum, the crude product was purified by column
chromatography using 5% ethyl acetate in petroleum ether as an
eluent to give a white crystalline product 8 (2.6 g, yield 65%),
m.p. 108 °C. 1H NMR (400 MHz, CDCl3 containing 1 drop
DMSO-d6): δ 7.93 (2H, d, J = 8 Hz), 7.84 (2H, d, J = 8 Hz),
7.40 (2H, d, J = 8 Hz), 7.30 (2H, t, J = 8 Hz), 7.19 (2H, t, J =
8 Hz), 7.07 (2H, d, J = 8 Hz), 4.19–4.15 (2H, m), 4.00–3.96
(2H, m), 3.57–3.48 (4H, m), 2.44 (2H, t, J = 4 Hz, –OH); FTIR
(KBr, cm−1): 3294, 2939, 2875, 1619, 1593, 1259, 1241, 1085,
1056; m/z (ES+): 375.1 [M + H]+.
1-(2-(Naphthalen-2-yloxy)ethyl)-1H-benzo[d]imidazole 5. To
a solution of benzimidazole (1.37 g, 11.61 mmol) in dry THF
(25 mL), NaH (0.278 mg. 11.61 mmol) was added and refluxed
for 1 h under a nitrogen atmosphere. The reaction mixture was
then cooled to room temperature and compound 4b (2.0 g,
9.68 mmol) in dry THF (15 mL) was added followed by addition
of tetrabutylammonium iodide (catalytic amount). Reflux was
continued for a further 10 h. After completion of the reaction,
THF was removed, water was added and the crude mass was
extracted with CHCl3 (3 × 30 mL). The organic layer was dried
over anhydrous Na2SO4 and concentrated on a rotary evaporator.
Purification of the crude mass by silica gel column chromato-
graphy using 20% ethyl acetate in petroleum ether yielded com-
2,2′-Bis(2-chloroethoxy)-1,1′-binaphthyl 9. To a stirred solu-
tion of 8 (1.00 g, 2.67 mmol) in CHCl3 (30 mL), SOCl2
(0.517 mL, 6.94 mmol) was added dropwise followed by
addition of pyridine (2 drops, catalytic amount). The reaction
mixture was stirred for 12 h and the solvent was removed under
reduced pressure. The residue was neutralized with aqueous
NaHCO3 solution and extracted with CHCl3 (3 × 30 mL). The
organic layer was dried over anhydrous Na2SO4 and concen-
trated on a rotary evaporator. The crude product was purified by
silica gel column chromatography with 2% ethyl acetate in pet-
roleum ether to give pure compound 9 (0.900 g, yield 81%),
1
pound 5 (2.4 g, yield 86%), m.p. 120 °C. H NMR (400 MHz,
CDCl3): δ 8.07 (1H, s), 7.81 (1H, d, J = 8 Hz), 7.73 (2H, t, J =
8 Hz), 7.66 (1H, d, J = 8 Hz), 7.50 (1H, d, J = 8 Hz), 7.41 (1H,
t, J = 8 Hz), 7.35–7.27 (3H, m), 7.10–7.06 (2H, m), 4.63 (2H, d,
J = 8 Hz), 4.42 (2H, t, J = 8 Hz); 13C NMR (100 MHz, CDCl3):
δ 155.8, 143.8, 143.5, 134.28, 133.8, 129.7, 129.2, 127.7, 126.7,
126.6, 124.0, 123.0, 122.2, 120.5, 118.5, 109.5, 106.7, 66.0,
44.3; FTIR (KBr, cm−1): 3050, 2931, 1627, 1601, 1510, 1257,
1218. m/z (ES+): 288.2 [M + H]+.
1
m.p. 64 °C. H NMR (400 MHz, CDCl3): δ 7.96 (2H, d, J =
8 Hz), 7.87 (2H, d, J = 8 Hz), 7.42 (2H, d, J = 8 Hz), 7.37–7.33
(2H, m), 7.23–7.21 (2H, m), 7.12 (2H, d, J = 8 Hz), 4.22–4.09
(4H, m), 3.40–3.36 (4H, m); FTIR (KBr, cm−1): 3053, 2953,
2925, 1621, 1593, 1235, 1046; m/z (ES+): 311.1 [M + H]+.
Receptor 1. To a stirred solution of 3 (0.256 g, 0.98 mmol) in
CH3CN, compound 5 (0.848 g, 2.94 mmol) in CH3CN (10 mL)
was added. The reaction mixture was refluxed with stirring for 5
days under a nitrogen atmosphere. After completion, the reaction
mixture was cooled to room temperature and filtered. The pre-
cipitate was washed with CH3CN several times to give pure
dichloride salt 6 (0.700 g, yield 64%). The dichloride salt 6
(0.92 g, 0.11 mmol) was dissolved in 2 mL hot DMF and
NH4PF6 (0.056 g, 0.34 mmol) was added to it in one portion.
After stirring the reaction mixture for 30 min water was added.
The precipitate was filtered, and washed with water and dried to
2,2′-Bis(2-(1H-benzo[d]imidazol-1-yl)ethoxy)-1,1′-binaphthyl
10. To a solution of benzimidazole (0.747g, 6.32 mmol) in dry
THF (25 mL), NaH (0.151 mg, 6.32 mmol) was added and the
reaction mixture was refluxed for 1 h under a nitrogen atmos-
phere. The reaction mixture was then cooled to room temperature
and compound 9 (1.0 g, 2.43 mmol) in THF (15 mL) was added
followed by addition of tetrabutylammonium iodide (catalytic
amount). Reflux was continued for a further 10 h. After com-
pletion of the reaction, THF was removed, water was added and
extracted with CHCl3 (3 × 30 mL). The organic layer was dried
over anhydrous Na2SO4 and concentrated on a rotary evaporator.
Purification of the crude mass by silica gel column chromato-
graphy using 20% ethyl acetate in petroleum ether yielded com-
pound 10 (0.600 g, yield 42), m.p. 114 °C. 1H NMR (400 MHz,
CDCl3); δ 7.95 (2H, d, J = 8 Hz), 7.89 (2H, d, J = 8 Hz), 7.64
(2H, d, J = 8 Hz), 7.36 (2H, t, J = 8 Hz), 7.28 (2H, s), 7.22 (2H,
t, J = 8 Hz), 7.08–7.15 (4H, m), 6.97 (2H, t, J = 8 Hz), 6.85
(2H, d, J = 8 Hz), 6.69 (2H, s), 4.12–4.09 (2H, m), 3.83–3.96
1
give 1 (0.105 g, yield 90%), m.p. 142 °C. H NMR (400 MHz,
DMSO-d6): δ 10.19 (2H, s), 9.88 (2H, s), 8.19 (2H, d, J =
8 Hz), 7.85–7.74 (8H, m), 7.63 (2H, t, J = 8 Hz), 7.52 (2H, t,
J = 8 Hz), 7.45 (4H, t, J = 8 Hz), 7.36–7.30 (4H, m), 7.24 (2H,
br s), 7.09 (2H, dd, J1 = 8 Hz, J2 = 4 Hz), 5.55 (4H, s), 5.08
(4H, s), 4.56 (4H, s); 13C NMR (100 MHz, DMSO-d6): δ 163.6,
162.3, 155.3, 143.8, 133.9, 131.5, 130.8, 130.1, 129.3, 128.6,
9390 | Org. Biomol. Chem., 2012, 10, 9383–9392
This journal is © The Royal Society of Chemistry 2012

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(6H, m); 13C NMR (100 MHz, CDCl3): δ 153.0, 143.3, 143.0,
133.7, 133.2, 129.7, 129.4, 128.1, 126.6, 125.1, 124.0, 122.7,
121.8, 119.8, 119.7, 114.6, 109.0, 67.1, 44.2; FTIR (KBr,
cm−1): 3054, 2976, 2928, 1619, 1591, 1497, 1272, 1264; m/z
(ES+): 575.2 [M + H]+.
2.2 : 0.8, 2 : 1, 1.8 : 1.2, 1.5 : 1.5, 1 : 2, 0.8 : 2.2, 0.5 : 2.5,
0.2 : 2.8. All the prepared solutions were kept for 1 h with
occasional shaking at room temperature. Then emission and
absorbance of the solutions of different compositions were
recorded. The concentration of the complex, i.e. [HG], was cal-
culated using the equation [HG] = ΔI/I0 × [H] or [HG] = ΔA/A0
× [H] where ΔI/I0 and ΔA/A0 indicate the relative emission and
absorbance intensities. [H] corresponds to the concentration of
pure host. Mole fraction of the host (XH) was plotted against con-
centration 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.
Macrocycle 2. Compounds 10 (0.150 g, 0.26 mmol) and 3
(0.068 g, 0.26 mmol) were taken in dry CH3CN (60 mL) and the
reaction mixture was refluxed under high dilution conditions for
4 days under a nitrogen atmosphere. The reaction mixture was
then cooled to room temperature and the white precipitate was
filtered. The precipitate was washed with CH3CN several times
to give pure dichloride salt 11 (0.150, yield 69%). The dichloride
salt 11 (0.100 g, 0.12 mmol) without characterization was next
dissolved in 5 mL hot CH3OH and NH4PF6 (0.056 g,
0.34 mmol) was added to it in one portion. After stirring the
reaction mixture for 30 min water was added. The precipitate
was filtered, washed with water and dried to give pure salt 2
(0.110 g, yield 87%); m.p. 216 °C. 1H NMR (400 MHz, DMSO-
d6): δ 10.32 (2H, s), 9.41 (2H, s). 8.02–7.96 (4H, m), 7.74 (2H,
m), 7.55–7.46 (4H, m), 7.39–7. 12 (12H, m), 6.63 (2H, d, J =
8 Hz), 4.09–4.65 (4H, br m), 4.42–4.40 (4H, br m), 4.35–4.33
(4H, br m); 13C NMR (100 MHz, DMSO-d6): 163.5, 152.7,
142.6, 132.4, 130.7, 130.3, 130.0, 129.7, 128.8. 128.0, 126.3,
125.9, 125.0, 124.0, 123.9, 119.3, 115.7, 113.1, 112.9, 67.5,
48.8, 46.7 (two carbons in the aromatic region are unresolved).
FTIR (KBr, cm−1): 3385, 3060, 1699, 1619, 1598, 1568, 1238,
Acknowledgements
We thank DST, New Delhi, India for providing financial support
[project SR/S1/OC-76/2010(G); Date:23.08.2011]. IS thanks
CSIR, New Delhi, India for a fellowship.
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1088; HRMS (TOF MS ES+): C48H40N6O4PF6 , (M − PF6)+
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9392 | Org. Biomol. Chem., 2012, 10, 9383–9392
This journal is © The Royal Society of Chemistry 2012