Selective calcium ion sensing with a bichromophoric squaraine foldamer

Article abstract of DOI:10.1021/ja045760e

Several squaraine tethered bichromophoric podand systems 1a-d and a monochromophoric analogue 2 were prepared and characterized. Among these, the bichromophore, 1b, containing five oxygen atoms in the flexible podand moiety was found to specifically bind Ca²⁺ in the presence of other metal ions such as K⁺, Na⁺, and Mg²⁺. The selective binding of Ca²⁺ is clear from the absorption and emission spectral changes as well as by the visual color change of 1b from light-blue to an intense purple-blue. Benesi-Hildebrand and Job plots confirmed a 1:1 binding between 1b and Ca²⁺. Signaling of the binding event is achieved by the cation-induced folding of the bichromophore and the resultant exciton coupling between the squaraine chromophores. The monochromophoric squaraine dye 2 failed to give optical signals upon Ca²⁺ binding, due to the absence of exciton interaction in the bound complex. Titration of the folded complex 9 with EDTA released the metal ion from the complex, thereby regaining the original absorption and emission properties of the bichromophore. The squaraine foldamer 1 b reported here is the first example of a selective chromogenic Ca²⁺ sensor, which works on the principle of exciton interaction in the folded Ca²⁺ complex of a bichromophore, the optical properties of which are similar to those of the "H"-type aggregates of analogous squaraine dyes.

Full text of DOI:10.1021/ja045760e

Published on Web 02/16/2005
Selective Calcium Ion Sensing with a Bichromophoric
Squaraine Foldamer
Easwaran Arunkumar,† Ayyappanpillai Ajayaghosh,*,† and Jo¨rg Daub‡
Contribution from the Photosciences and Photonics DiVision, Regional Research Laboratory
(CSIR), TriVandrum - 695019, India, and Institut fu¨r Organische Chemie, UniVersita¨t
Regensburg, D-93040 Regensburg, Germany
Received July 15, 2004; E-mail: aajayaghosh@rediffmail.com
Abstract: Several squaraine tethered bichromophoric podand systems 1a-d and a monochromophoric
analogue 2 were prepared and characterized. Among these, the bichromophore, 1b, containing five oxygen
atoms in the flexible podand moiety was found to specifically bind Ca2+ in the presence of other metal ions
such as K+, Na+, and Mg2+. The selective binding of Ca2+ is clear from the absorption and emission spectral
changes as well as by the visual color change of 1b from light-blue to an intense purple-blue. Benesi-
Hildebrand and Job plots confirmed a 1:1 binding between 1b and Ca2+. Signaling of the binding event is
achieved by the cation-induced folding of the bichromophore and the resultant exciton coupling between
the squaraine chromophores. The monochromophoric squaraine dye 2 failed to give optical signals upon
Ca2+ binding, due to the absence of exciton interaction in the bound complex. Titration of the folded complex
9 with EDTA released the metal ion from the complex, thereby regaining the original absorption and emission
properties of the bichromophore. The squaraine foldamer 1b reported here is the first example of a selective
chromogenic Ca2+ sensor, which works on the principle of exciton interaction in the folded Ca2+ complex
of a bichromophore, the optical properties of which are similar to those of the “H”-type aggregates of
analogous squaraine dyes.
Introduction
which pertain to the use of various chromophore appended
systems as Ca2+ sensors.5 In several other reports, chromophore-
Design of chemosensors for the selective detection of a
specific analyte is a topic of considerable interest, due to their
wide ranging application in the broad areas of chemistry and
biology.1 Selective detection of biologically important cations
such as Na+, K+, Mg2+, and Ca2+, particularly the detection of
Ca2+ in the presence of Na+ and K+, is important because many
physiological processes are triggered, regulated, or influenced
by calcium ion.2 Because of the importance of ionic calcium in
biological processes, chemists have been interested in the design
of chemosensors for the specific detection of calcium ions. The
early work by Tsien and co-workers has contributed significantly
to the design of Ca2+ sensors under biological conditions.3 Later,
de Silva et al. have reported the synthesis of a few Ca2+ specific
sensors using the principle of photoinduced electron transfer
(PET).4 In addition, there are several reports in the literature
linked pseudocyclic polyether systems (podands) have been
shown to bind cations.6 Among these, the early reports by Vo¨gtle
and co-workers and the subsequent work by Nakamura and co-
workers are noteworthy.7 In the majority of the chemosensors
reported so far, the signaling of the binding event is achieved
by electron transfer,8 energy or charge-transfer processes,9 and/
or chromophore (excimer) interaction.10
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† Regional Research Laboratory.
‡ Universita¨t Regensburg.
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Selective Ca2 Sensing
A R T I C L E S
One of the important criteria for the designing of chemosen-
sors is the selection of the signaling unit, which should produce
a strong signal, preferably as a visual color change (chromoge-
nic), upon an analyte binding. Organic dyes, particularly
squaraine dyes whose optical properties are sensitive to the
surrounding media, are ideal for this purpose. Although
squaraine dyes have been used for the design of chemosensors,11
the selectivity and sensitivity of most of these sensors are poor
except in a very few cases.11b,c Earlier, we have shown that
conjugated squaraine oligomers containing podand side chains
have the ability to specifically bind Li+ and K+ with high
sensitivity.12 Recently, in a novel but simple approach, we have
shown selective sensing of calcium and other alkaline earth
metal ions using the principle of metal ion-induced folding of
squaraine tethered podands.13 In this approach, we introduced
the concept of exciton interaction in squaraine “H”-aggregate
as an alternate signaling mechanism for a cation binding process.
The rationale behind the design of such chemosensors is based
on the phenomena of “H” and “J” aggregation of organic dyes
such as cyanines, merocyanines, and squaraines, which show
distinctly different optical properties between aggregates and
monomers.14,15 Using our strategy of exciton coupled signaling
through cation-induced conformational folding, Block and Hecht
Figure 1. A chromogenic chemosensor based on a specific cation-driven,
exciton interaction in a dye-linked podand.
have recently reported the synthesis of a few polysquaraine-
based sensors.16 Our sustained interest in designing a selective
chemosensor for Ca2+, a biologically significant cation, which
is difficult to detect in the presence of other similar ions, leads
to the design of a new chromogenic probe based on a squaraine
foldamer.17 To our surprise, squaraines when tethered to a
podand with five oxygen atoms, in a bichromophoric fashion,
become insensitive to alkali metal ions but show remarkable
selectivity and response toward Ca2+ as shown in Figure 1.
Results and Discussion
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Synthesis and Characterization of the Bichromophores
1a-d and the Monochromophore 2. Synthesis of 1a-d was
achieved as shown in Scheme 1. 3-(4-(N,N-Dimethylamino)-
phenyl)-4-hydroxycyclobut-3-ene-1,2-dione (8) was prepared
according to reported procedures.18 The bisaniline derivatives
5a-d were prepared by the reaction of the aniline derivative 3
with the corresponding ditosylates (4a-d) in 45-55% yields.
Reaction of 8 with 5a-d in 2-propanol/tributyl orthoformate
under refluxing yielded the bichromophores 1a-d in 20-25%
yield. FT-IR spectra of 1a-d showed a strong absorption at
1590 cm-1 characteristic of the resonance stabilized zwitterionic
structure of squaraine dyes. 1H NMR spectra of 1a-d showed
a set of peaks corresponding to the aromatic protons. Four of
the aromatic protons together appeared as two doublets, which
upon merging looked like a triplet at δ 6.8 ppm. The remaining
four aromatic protons appeared as two doublets around δ 8.3
ppm. The 13C NMR spectra, MALDI-TOF mass spectra, HRMS
data, and elemental analysis data of 1a-d were in agreement
with the structures. Similarly, the monochromophore 2 was
characterized using FT-IR, 1H NMR, 13C NMR, and elemental
analyses.
Solvent-Induced Aggregation Behavior of 1a-d and 2. The
bichromophores 1a-d showed a strong absorption maximum
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A R T I C L E S
Scheme 1
Arunkumar et al.
around 630 nm in acetonitrile with an emission maximum
around 655 nm. It is interesting to note that 1a-d have low
quantum yields of emission that gradually increased from 0.024
to 0.04 with an increase in the length of the podand chain.
However, in acetonitrile, 2 showed a strong emission at 662
nm with a relatively high quantum yield of 0.21. This is in
analogy to the report of Liang et al. who showed that squaraines,
which are linked to a flexible alkyl chain in a bichromophoric
fashion, show exciton coupled spectral changes, depending upon
the length of the alkyl chain.19
characteristic of an intermolecular interaction. Interestingly, in
the case of 1b, the solvent-induced aggregation resulted in a
visual color change from light-blue to purple-blue (Figure 2a,
Interestingly, in DMSO-H2O mixtures of different composi-
tions, 1a-d showed a blue-shifted absorption band at 586 nm
in addition to the monomer band at 649 nm as shown in the
case of 1b (Figure 2a). As the percentage of water in DMSO is
increased, the intensity of the 649 nm band decreased with the
concomitant increase in the intensity of the band at 586 nm
through an isosbestic point at 623 nm. The monochromophore
2 showed similar but weak changes in the absorption spectrum
in DMSO-H2O as shown in Figure 2b. These observations are
attributed to the “H”-aggregation, which are analogous to the
report by Whitten et al.20 In the case of 1a-d, the detailed
studies indicated that the aggregation is independent of con-
centration with an aggregation number of unity, characteristic
of an intramolecular folding of the bichromophoric podands.
However, the monochromophore 2 showed a concentration-
dependent aggregation with an aggregation number of two,
(19) Liang, K.; Farahat, M. S.; Perstein, J.; Law, K.-Y.; Whitten, D. G. J. Am.
Chem. Soc. 1997, 119, 830.
(20) (a) Chen, H.; Herkstroeter, W. G.; Perlstein, J.; Law, K.-Y.; Whitten, D.
G. J. Phys. Chem. 1994, 98, 5138. (b) Chen, H.; Law, K.-Y.; Perlstein, J.;
Whitten, D. G. J. Am. Chem. Soc. 1995, 117, 7257. (c) Chen, H.; Farahat,
M. S.; Law, K.-Y.; Whitten, D. G. J. Am. Chem. Soc. 1996, 118, 2584. (d)
Chen, H.; Law, K.-Y.; Whitten, D. G. J. Phys. Chem. 1996, 100, 5949.
Figure 2. Absorption changes in different water-DMSO compositions of
(a) 1b (7.57 µM) and (b) 2 (5.9 µM). Inset picture shows 1b in (1) 100%
DMSO; (2) 50% DMSO/50% H2O; and (3) 10% DMSO/90% H2O.
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Selective Ca2 Sensing
A R T I C L E S
Figure 3. Visible color change of 1b (5.1 µM) in acetonitrile upon addition
of Ca2+: (1) in the absence of metal salts, (2) Mg2+, (3) Na+, (4) K+, and
(5) Ca2+
.
inset), indicating a strong chromophore interaction, whereas in
the case of 2, the color change was weak. In light of these
observations, we were keen to know the changes in the optical
properties of 1a-d and 2 in the presence of various added metal
ions.
Metal Ion Binding Studies of 1a-d. To find out whether
the bichromophores 1a-d induce any visual color change, a
series of experiments were conducted in the presence of different
metal salts under various conditions. These studies revealed a
visible color change from light-blue to an intense purple-blue
in the presence of calcium perchlorate similar to the color change
noticed in DMSO-H2O mixtures. Surprisingly, in the case of
1b, the color change was intense and specific toward Ca2+
,
whereas addition of Na+, K+, and Mg2+ did not show any
change at all (Figure 3). Interestingly, in acetonitrile (5.1 µM),
significant changes in the absorption and emission spectra of
1b were noticed with the addition of Ca(ClO4)2 as shown in
Figure 4a and b, respectively. Upon gradual addition of Ca-
(ClO4)2, the intensity of the absorption maximum at 630 nm is
decreased with the concomitant formation of a hypsochromically
shifted band at 552 nm through an isosbestic point at 580 nm.
Similarly, a fluorescence emission maximum of 1b at 652 nm
when excited at 580 nm (5.1 µM, CH3CN) underwent consider-
able quenching upon gradual addition of Ca(ClO4)2 (Figure 4b).
The fluorescence quantum yield of 1b, which was 0.032 before
the addition of Ca(ClO4)2, was decreased to a minimum value
of 0.008. Singlet excited-state lifetime measurements of the
bichromophore 1b in acetonitrile showed monoexponential
decay with a short lifetime of 200 ps. Upon addition of Ca-
(ClO4)2, the lifetime is enhanced to 240 ps with a monoexpo-
nential decay (see Supporting Information). Although the
fluorescence quantum yield of 1b in the presence of Ca(ClO4)2
is lower than that in the absence of Ca(ClO4)2, the observed
excited-state lifetime is 40 ps higher in the former case. This
result is in accordance with some of the previous reports
pertaining to the excited-state dynamics of cyanine dyes.14e,21
Because of the importance of the selective detection of Ca2+
under aqueous physiological conditions, we attempted the
titration of Ca(ClO4)2 against 1b in acetonitrile-water (1:1)
using N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid
(TES, 0.01 M) buffer at a pH of 7.2. However, in the buffer
solution, two absorption maxima at 636 and 583 nm could be
observed even before the addition of Ca(ClO4)2, indicating the
aggregation of the chromophores. This observation reveals that
Figure 4. Changes in the (a) absorption and (b) emission spectra of 1b
(5.1 µM) in acetonitrile with the addition of Ca(ClO4)2. Insets show variation
of (a) absorbance at 552 and 630 nm and (b) fluorescence intensity of 1b
with increasing concentration of Ca(ClO4)2.
the dye is folded to form the H-type dimer as in the case of
DMSO-H2O mixtures. Therefore, further changes in the
absorption and emission spectra in buffer solutions were
marginal upon addition of Ca(ClO4)2 (see Supporting Informa-
tion).
Plots of the decrease in the absorbance at 630 nm and the
increase in absorbance at 552 nm reached the saturation point
when the ratio of Ca(ClO4)2 concentration reached the equivalent
dye concentration, indicating a 1:1 complexation (inset, Figure
4a). The inset of Figure 4b shows the variation of fluorescence
intensity of 1b with increasing Ca(ClO4)2 concentration, where
Io and I are the fluorescence intensities before and after the
addition of the metal perchlorates. The fluorescence intensity
at 652 nm is gradually decreased until the Ca2+ concentration
reached the dye concentration, thereby supporting a 1:1 com-
plexation. The Job plot showed a maximum value for the
absorbance at 552 nm when the mole fraction of 1b reached
0.5, which is a signature of a 1:1 binding between the dye and
Ca2+ (Figure 5).22 The stability constants Ks were determined
from the Benesi-Hildebrand plot of Ao/(Ao - A) against [M]-1
,
which showed a linear relationship, characteristic of a 1:1
(22) (a) Job, P. Ann. Chim. 1928, 9, 113. (b) Gil, V. M. S.; Oliveira, N. C. J.
Chem. Educ. 1990, 67, 473. (c) Specht, A.; Bernard, P.; Goeldner, M.;
Peng, L. Angew. Chem., Int. Ed. 2002, 41, 4706. (d) Huang, F.; Gibson,
H. W.; Bryant, W. S.; Nagvekar, D. S.; Fronczek, F. R. J. Am. Chem. Soc.
2003, 125, 9367. (e) Huang, F.; Fronczek, F. R.; Gibson, H. W. Chem.
Commun. 2003, 1480.
(21) (a) Khairutdinov, R. F.; Serpone, N. J. Phys. Chem. B 1997, 101, 2602.
(b) Seifert, J. L.; Connor, R. E.; Kushon, S. A.; Wang, M.; Armitage, B.
A. J. Am. Chem. Soc. 1999, 121, 2987.
9
J. AM. CHEM. SOC. VOL. 127, NO. 9, 2005 3159

A R T I C L E S
Arunkumar et al.
Figure 5. Job plot obtained for a 1:1 complexation of Ca2+ and 1b.
Table 1. Binding Constantsa (log K) of 1a-d with Various Metal
Ions in Acetonitrile
Figure 7. Plot of binding constants of 1a-d with various metal cations.
+
+
+
+
compound
Mg2
Ca2
Sr2
Ba2
Na+
K+
(see Supporting Information). Addition of alkali metal ions such
as Li+, Na+, and K+ did not show any measurable response
with 1a, 1c, and 1d. Binding constants of 1a-d with various
metal cations are calculated from the changes in the absorption
spectra, and these values are shown in Table 1. The highest
binding constant is obtained for the binding of Ca2+ to the
bichromophore 1b. 1a showed weak binding constants with all
of the cations added, whereas 1c and 1d showed weak binding
toward Mg2+ and Ca2+. The binding constants of other metal
cations with 1b could not be determined because the corre-
sponding changes in the absorption or emission properties were
too weak. Figure 7 shows the bar diagram of the binding
constants of each bichromophore against various alkaline earth
metal ions.
1a
1b
1c
1d
3.86
b
3.61
3.75
3.95
4.29
3.98
3.93
3.80
3.62
c
c
c
c
c
c
c
c
b
b
b
b
b
b
a Reported binding constants are based on the assumption that the
concentration of the free ions is equal to the total salt concentration.
b Changes were too small to calculate the binding constant. c No response.
The difference in the binding affinity of 1a-d with various
cations under investigation can be attributed to the length of
the podand chains. Unlike in macrocyclic crown ethers, the ionic
size of the cation is not the only parameter that determines the
binding affinity of pseudocyclic polyether systems. The number
of oxygen atoms, the size of the pseudo crown cavity, the charge
density, and the coordination number of the cation all play a
considerable role in the binding interactions.24 Surprisingly, the
polyether chain length of 1b with five oxygen atoms is found
to be ideal for the binding of Ca2+. This observation is clear
from a comparison of the plots of the fluorescence quantum
yields of 1a-d against Ca2+ binding (Figure 8). The maximum
quenching of fluorescence is obtained for Ca2+ binding when
the number of oxygen atoms becomes five in the podand chain
as observed with 1b. Our finding is in analogy to the previous
reports in the literature on selective binding of Ca2+ to similar
podand chains.6d,7a-c
Cyclic Voltammetric Measurements. The cyclic voltam-
mogram (CV) of 1b in acetonitrile using tetrabutylammonium
hexafluorophosphate as the supporting electrolyte at a scan speed
of 100 mV/s shows two redox waves (Figure 9). The first wave
at 270 mV was reversible and the second one at 586 mV was
irreversible at slow scan rate under thin layer electrochemical
conditions. The first and the second formal redox potentials were
obtained by averaging the anodic and cathodic peak potentials
of the CV. It was earlier reported by Law et al.25 that
substituents, both at the nitrogen atom and in the phenyl ring,
have a strong influence on the oxidation potential, which
Figure 6. Plot of Φf versus the ratio between metal ion and 1b, which
illustrates the selectivity for Ca2+ (2) over Na+ (6), K+ (b), Mg2+ (9),
Sr2+ (f), and Ba2+ (1).
binding.23 The stability constants were calculated from the ratio
of intercept/slope, and their values are shown in Table 1.
Addition of other alkaline earth metal salts such as Mg-
(ClO4)2, Sr(ClO4)2, and Ba(ClO4)2 showed relatively weak
changes in the absorption and emission spectra (see Supporting
Information). On the other hand, addition of a 3-fold excess
each of Na+, K+, and Li+ to a solution of 1b in CH3CN could
not induce any change in the absorption or emission properties.
These observations show the unique ability of 1b to selectively
bind Ca2+, which is clear from the plot of the variation of Φf
against the ratio of the metal ion and 1b (Figure 6). The
maximum decrease in Φf is obtained for the binding of Ca2+
,
revealing the high sensitivity and selectivity of 1b to Ca2+
.
Cation binding studies of other bichromophores 1a, 1c, and
1d with Mg2+, Ca2+, Sr2+, and Ba2+ showed relatively weak
response when compared to those of 1b. However, similar to
1b, Ca2+ showed maximum response toward 1a, 1c, and 1d
(24) Watanabe, S.; Ikishima, S.; Matsuo, T.; Yoshida, K. J. Am. Chem. Soc.
2001, 123, 8402.
(25) Law, K.-Y.; Facci, J. S.; Balley, F. C.; Yanus, J. F. J. Imaging Sci. 1990,
34, 31.
(23) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703.
9
3160 J. AM. CHEM. SOC. VOL. 127, NO. 9, 2005

+
Selective Ca2 Sensing
A R T I C L E S
Figure 8. Plots of I/I0 versus the ratio of [Ca(ClO4)2] to [dye], illustrating
the fluorescence response of various bichromophores, 1a (9), 1b (b), 1c
(2), 1d (1), and 2 ([), with increasing concentration of Ca(ClO4)2.
Figure 10. 1H NMR spectra in 80% CDCl3-20% CD3CN of 1b (3.1 mM)
(a) before addition of metal perchlorates, (b) with 3.1 mM NaClO4, and (c)
with 3.1 mM Ca(ClO4)2.
as a result of the Ca2+-induced folding and stacking of 1b, which
is in equilibrium between the folded and the unfolded confor-
mations. On the other hand, addition of Na+ or K+ did not cause
any considerable change in the 1H NMR spectrum of 1b (Figure
10b). Any influence of the perchlorate counteranion on the NMR
peak broadening is ruled out because the addition of NaClO4
or KClO4 did not show any change in the NMR spectrum of
1b. Similarly, any role of a permanent damage of the dye in
the presence of Ca(ClO4)2 and the associated NMR signal
broadening is ruled out by adding EDTA to the solution of 9
which brought back the original optical properties of the dye.
Metal Ion Decomplexation Studies. EDTA is known to be
a strong chelator of Ca2+ and Mg2+. Because of the high stability
constant of the EDTA-Ca2+ complex, it was anticipated that
addition of EDTA will induce the decomplexation of the Ca2+
from the complex 9, thereby restoring the original photophysical
properties of 1b. With this intention, 1 equiv of EDTA solution
is added to the Ca2+ complex of 1b, which visibly turned the
intense purple-blue color of the 1b-Ca2+ complex into the initial
pale-blue color of 1b. The change in the absorption spectrum
of 1b-Ca2+ complex upon addition of EDTA solution is shown
in Figure 11a, which is compared to the original spectrum of
1b. The short wavelength absorption band of the 1b-Ca2+
complex at 552 nm completely disappeared, and the spectrum
matched that of 1b where the original intensity of the band at
630 nm is regained. The fluorescence emission intensity is also
restored upon addition of the EDTA to the Ca2+ complexed
dye (Figure 11b). These results clearly indicate that the observed
change in the absorption and emission properties of 1b with
Ca2+ is due to the formation of a reversible complex between
the two.
Figure 9. Cyclic voltammogram of 1b in CH3CN using 0.1 M tetrabuty-
lammonium hexafluorophosphate as supporting electrolyte at a scan rate
of 100 mV/s: (a) before and (b) after the addition of Ca(ClO4)2.
decreases as the electron-donating ability of the substituent
increases. Addition of Ca(ClO4)2 into a solution of 1b caused
the shift of the redox potential to more positive values, that is,
from 270 to 401 mV and from 586 to 690 mV. This result is
attributed to the coordination of the nitrogen lone pair of
electrons with Ca2+ that decreases the charge-transfer character
of the chromophore. This finding is in accordance with the report
of Loutfy and Law, who observed that the oxidation potential
of D-A molecules generally increases as the CT character of
the chromophore decreases.26 Therefore, the observed increase
in the redox potential of 1b is attributed to the binding of Ca2+
.
1H NMR Binding Studies. Changes in the 1H NMR spectrum
of 1b before and after the addition of Ca(ClO4)2 in CDCl3/CD3-
CN (8:2) at 27 °C are shown in Figure 10. The resonance signal
due to the -NCH3 protons, which appeared at δ 3.18 ppm, is
shifted upfield upon addition of 1 equiv of Ca2+. Similarly, the
signals due to the -OCH2- and -NCH2- protons appearing
at δ 3.59-3.69 ppm changed to a broad multiplet. Aromatic
signals, which appeared at δ 6.73-6.79 ppm and δ 8.33-8.38
ppm, became weak and broad upon addition of Ca2+ (Figure
10c). These changes are indications of chromophore interaction
Figure 12 shows the effect of temperature on the absorption
properties of the 1b-Ca2+ complex. As the temperature is
increased from 25 to 70 °C, the intensity of the peak at 630 nm
gradually increased, whereas the intensity of the blue-shifted
band at 552 nm gradually decreased. This can be attributed to
the decomplexation of Ca2+ from the bound metal complex of
1b at higher temperatures. However, a complete change in the
absorption spectrum could not be obtained at 70 °C. A further
increase in temperature was not possible because the complex-
ation was carried out in acetonitrile.
(26) Loutfy, R. O.; Law, K.-Y. J. Phys. Chem. 1980, 84, 2803.
9
J. AM. CHEM. SOC. VOL. 127, NO. 9, 2005 3161

A R T I C L E S
Arunkumar et al.
Figure 11. Change in (a) absorption and (b) emission spectra of 9 (7.3 µM) with the addition of 7.3 µM EDTA. Spectra 1, 2, and 3 show the absorption
and emission of 1b, 1b + Ca2+, and 1b + Ca2+ + EDTA, respectively.
cation-driven folding of the bichromophores. This conclusion
is further confirmed by the Ca2+ titration studies of the
monochromophore 2, which contains only one squaraine chro-
mophore. In this case, although a 1:1 binding of Ca2+ is evident
1
from the H NMR spectral data (see Supporting Information),
no change either in the absorption or in the emission was
observed. These results support the proposed signaling mech-
anism of the bichromophores.
Conclusions
A chromogenic sensor for the selective detection of Ca2+ in
the presence of other cations such as Na+, K+, and Mg2+, based
on a squaraine foldamer, which works on the principle of exciton
coupled signal transduction is described. The specific binding
of Ca2+ is clear from the visual color change as well as the
Figure 12. Effect of temperature on the absorption spectrum of 1b after
change in the absorption and emission spectra, thereby allowing
adding 1 equiv of Ca(ClO4)2.
a three-way signaling. The Job plot and Benesi-Hildebrand plot
satisfy a 1:1 stoichiometry for the complexation of 1b with Ca2+
.
Cation-Driven Folding and the Signal Transduction
Process. The changes observed in the absorption, emission, 1H
NMR, and redox properties upon the addition of Ca(ClO4)2 can
be rationalized on the basis of the Ca2+-induced folding of the
bichromophore to a folded complex 9 (Figure 13). The optical
properties of 1b-Ca2+ are similar to those of the solvent-
induced “H”-aggregate of 1b. These observations reveal that
the Ca2+-induced changes in the absorption and emission are
due to the exciton interaction of the folded complex.27 In the
case of the folded complex 9, the fluorescence is weaker when
compared to that of the unfolded 1b, because the internal
conversion from an upper excited state to a lower one occurs
immediately and the emission from a lower excited state is
theoretically forbidden (inset, Figure 13). The 1:1 complexation
mode obtained by the Job plots and the changes noted in the
absorption and emission spectra strongly support the proposed
The cation-steered folding of the bichromophore and the
consequent exciton coupling in the folded complex allowed the
signaling of the binding event. The optical silence of the
analogous monochromophore 2 with Ca2+ supports the sug-
gested signaling mechanism. Regeneration of the initial absorp-
tion and emission property of the 1b-Ca2+ complex 9 upon
addition of EDTA shows the reversibility of the suggested
complexation mode. In conclusion, we have shown that ag-
gregation, which is a drawback in the case of several of the
organic dyes, becomes a useful phenomenon in the design of
ion specific chemosensors. However, for a practical application,
it is necessary that the molecular probe described here should
be able to detect calcium ions under biological conditions. The
challenge here is the synthesis of highly water-soluble squaraine
foldamers that are stable and should not self-aggregate under
aqueous physiological conditions.
(27) The allowed and the forbidden transitions to the excitons are governed by
the tilt angle “R” of the transition moments with respect to the line of
centers. When “R” is less than 54°, transition to the lowest excited level is
allowed, resulting in a bathochromically shifted absorption due to head-
to-tail “J”-aggregates. If “R” is greater than 54°, transition to the highest
excited level is favored leading to hypsochromically shifted absorption
which corresponds to the head-to-head “H”-aggregates. (a) Kasha, M.;
Rawls, H. R.; EI-Bayoumi, M. A. Pure Appl. Chem. 1965, 11, 371. (b)
Davydov, A. S. Theory of Molecular Excitons; Plenum Press: New York,
1971. (c) McRae, E. G.; Kasha, M. Physical Process in Radiation Biology;
Academic Press: New York, 1964; p 17. (d) Hochstrasser, R. M.; Kasha,
M. Photochem. Photobiol. 1964, 3, 317. (e) Czikkely, V.; Forsterling, H.;
Kuhn, H. Chem. Phys. Lett. 1970, 6, 11. (f) Bucher, H.; Kuhn, H. Chem.
Phys. Lett. 1970, 6, 183.
Experimental Section
Unless otherwise stated, all reagents were purchased from com-
mercial suppliers and used without further purification. Solvents used
were purified and dried by standard methods prior to use. Melting points
were determined with a Mel-Temp-II melting point apparatus and are
uncorrected. High-resolution mass spectra were recorded in a Finnigan
MAT 95 instrument using Xenon as ionization gas. H and 13C NMR
spectra were measured on a 300 MHz Bruker Avance DPX spectrom-
eter. FT-IR spectra were recorded on a Nicolet Impact 400D infrared
1
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+
Selective Ca2 Sensing
A R T I C L E S
Figure 13. Ca2+-induced folding of 1b leading to an intramolecular “H”-dimer 9. Insets show the corresponding allowed and forbidden transitions in the
two conformations.
spectrophotometer. Elemental analyses were done using a Perkin-Elmer
series-II 2400 CHN analyzer. Cyclic voltammetry was performed in a
conventional undivided electrochemical cell with a three electrode
arrangement using a computer-controlled Amel 5000 or EG&G 283
system.28 The emission spectra were measured on a Spex-Fluorolog
F112X spectrofluorimeter. Fluorescence quantum yields were deter-
mined in spectroscopic grade CH3CN using optically matching solutions
of 4,4-[bis-(N,N-dimethylamino)phenyl]squaraine dye (Φf ) 0.70 in
chloroform) as standard at an excitation wavelength of 580 nm.29
Fluorescence lifetimes were measured using a Tsunami Spectra Physics
single photon counting system. The stability constant Ks was determined
from absorption spectral changes using eq 1,
reaction mixture was then refluxed for 20 h. The hot reaction mixture
was filtered, and the solid was washed with 2-propanol until the filtrate
was almost colorless. Column chromatography (chloroform/methanol,
9/1) of the crude product over silica gel (100-200 mesh) gave the
pure product. The monochromophore 2 was prepared by the reaction
of 7 and 8 as described above.
1a. Yield: 22%. mp 261-263 °C. FT-IR (KBr): ν ) 2362, 1585,
1
1487, 1363, 1171, 1117, 933, 836, 791 cm-1. H NMR (300 MHz,
CDCl3, TMS): δ ) 3.17 (s, 18H, -NCH3), 3.55-3.68 (m, 20H,
-NCH2 and -OCH2), 6.73-6.78 (dd, J ) 1.38, 9.37 Hz, 8H, aromatic),
8.32-8.37 (dd, J ) 2.74, 9.32 Hz, 8H, aromatic). 13C NMR (75 MHz,
CDCl3): δ ) 39.6, 40.34, 52.35, 68.5, 70.58, 70.63, 70.89, 112.35,
112.53, 119.86, 120.2, 133.52, 154.46, 154.95, 183.32. Anal. Calcd
for C48H54N4O8: C, 70.74; H, 6.68; N, 6.87. Found: C, 70.36; H, 6.78;
N, 6.96. HRMS-FAB: [M + H]+ calcd for C48H54N4O8, 815.9761;
found 815.975.
A0
ꢀL
1
)
+ 1
(
)
A0 - A ꢀL - ꢀML
Ks[M]
where ꢀL and ꢀML are the molar extinction coefficients of the ligand
and the complex, respectively. The quantity A0/(A0 - A) is plotted versus
[M]-1, and the stability constant is then given by the ratio of intercept/
slope.
1b. Yield: 24%. mp 225-227 °C. FT-IR (KBr): ν ) 2361, 1593,
1
1401, 1367, 1182, 1122, 937, 830, 784.5 cm-1. H NMR (300 MHz,
CDCl3, TMS): δ ) 3.18 (s, 18H, -NCH3), 3.59-3.69 (m, 24H,
-NCH2 and -OCH2), 6.74-6.79 (dd, J ) 1.2, 9.2 Hz, 8H, aromatic),
8.33-8.38 (dd, J ) 2.8, 9.1 Hz, 8H, aromatic). 13C NMR (75 MHz,
CDCl3): δ ) 39.5, 40.64, 52.75, 68.7, 70.48, 70.33, 70.99, 112.15,
112.33, 119.75, 120.6, 133.12, 154.28, 154.98, 183.92. Anal. Calcd
for C50H58N4O9: C, 69.91; H, 6.81; N, 6.52. Found: C, 69.61; H, 6.41;
N, 6.65. HRMS-FAB: [M + H]+ calcd for C50H58N4O9, 859.4282;
found 859.4272.
General Procedure for the Metal Ion Binding Titrations. Metal
perchlorate solutions were prepared in spectroscopic grade acetonitrile.
Bichromophores were completely dissolved in acetonitrile with soni-
cation and slight warming in a water bath. Metal ion titrations were
carried out by adding small volumes (1-5 µL) of the metal solutions
(10-6 M in CH3CN, 4 mL) in quartz cuvette. After the addition of
metal salt solution to the cuvette, using a microliter syringe, the solution
was shaken well and kept for 1 min before recording the absorption
and emission spectra of the metal complexed dye.
1c. Yield: 20%. mp 235-237 °C. FT-IR (KBr): ν ) 2355, 1588,
1
1480, 1366, 1174, 1118, 831, 791 cm-1. H NMR (300 MHz, CDCl3,
TMS): δ ) 3.18 (s, 18H, -NCH3), 3.59-3.69 (m, 28H, -NCH2 and
-OCH2), 6.73-6.79 (dd, J ) 1.28, 9.5 Hz, 8H, aromatic), 8.34-8.39
(dd, J ) 2.43, 9.2 Hz, 8H, aromatic). 13C NMR (75 MHz, CDCl3): δ
) 39.3, 40.48, 52.53, 68.62, 70.42, 70.54, 70.78, 112.2, 112.53, 119.86,
120.2, 133.52, 154.46, 154.95, 183.32. Anal. Calcd for C52H62N4O10:
C, 69.16; H, 6.92; N, 6.2. Found: C, 68.97; H, 6.78; N, 6.35. HRMS-
FAB: [M + H]+ calcd for C52H62N4O10, 904.0653; found 904.0647.
General Procedure for the Preparation of the Bichromophores
1a-d and the Monochromophore 2. To a 100 mL round-bottomed
flask containing 50 mL of 2-propanol were added 0.22 mmol of 5a-d,
117 mg (0.54 mmol) of 3-(4-(N,N-Dimethylamino)phenyl)-4-hydroxy-
cyclobut-3-ene-1,2-dione (8), and 1 mL of tributyl orthoformate. The
(28) Bueschel, M.; Stadler, C.; Lambert, C.; Beck, M.; Daub, J. J. Electroanal.
Chem. 2000, 484, 24 and references therein.
(29) Cornelissen-Gude, C.; Rettig, W.; Lapouyade, R. J. Phys. Chem. A 1997,
101, 9673.
1d. Yield: 22%. mp 218-220 °C. FT-IR (KBr): ν ) 2359, 1595,
1372, 1183, 1120, 833, 784 cm-1. 1H NMR (300 MHz, CDCl3, TMS):
9
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A R T I C L E S
Arunkumar et al.
δ ) 3.18 (s, 18H, -NCH3), 3.59-3.7 (m, 32H, -NCH2 and -OCH2),
6.73-6.79 (dd, J ) 1.27, 9.28 Hz 8H, aromatic), 8.34-8.39 (dd, J )
2.46, 9.26 Hz, 8H, aromatic). 13C NMR (75 MHz, CDCl3): δ ) 39.71,
40.33, 52.32, 68.56, 70.58, 70.8, 112.3, 112.4, 119.8, 119.9, 154.4,
154.9, 183.4. Anal. Calcd for C54H66N4O11: C, 68.48; H, 7.02; N, 5.92.
Found: C, 68.25; H, 6.88; N, 5.63. HRMS-FAB: [M + H]+ calcd for
C54H66N4O11, 948.1733; found 948.1726.
2. Yield: 32%. mp 48-49 °C. FT-IR (KBr): ν ) 2874, 2355, 1588,
1370, 1339, 1179, 1096, 838, 784 cm-1. 1H NMR (300 MHz, CDCl3):
δ ) 3.2 (s, 3H, -NCH3), 3.21 (s, 6H, -NCH3), 3.37 (s, 3H, -OCH3),
3.55-3.72 (m, 24H, -NCH2 and -OCH2), 6.75-6.8 (m, 4H, aromatic),
8.36-8.41 (m, 4H, aromatic). 13C NMR (75 MHz, CDCl3): δ ) 39.7,
40.33, 52.32, 58.99, 68.56, 70.46, 70.51, 70.58, 70.61, 70.86, 71.88,
112.33, 112.45, 119.85, 119.93, 133.2, 154.43, 154.98, 183.38 ppm.
Anal. Calcd for C32H44N2O8: C, 65.73; H, 7.58, N, 4.79. Found: C,
65.57; H, 7.89; N, 4.99. HRMS-FAB: [M + H]+ calcd for C32H44N2O8,
585.8849; found 585.884.
SF/C6/99-2000) and DAAD, Germany, for the exchange visits
under the DST-DAAD-PP program. We are thankful to Dr. P.
Ramamurthy, National Centre for Ultrafast Processes, Chennai,
for permitting us to use the single photon counting facilities.
E.A. thanks CSIR, Government of India, for a research
fellowship. This is contribution number RRLT-PPD-195 from
the Regional Research Laboratory.
Supporting Information Available: Fluorescence lifetime
profiles of 1b. Changes in the absorption and emission properties
of 1b under aqueous buffer solution, with Mg(ClO4)2, Sr(ClO4)2,
Ba(ClO4)2, LiClO4, NaClO4, and KClO4 . Changes in the optical
properties of 1a, 1c, and 1d with Ca(ClO4)2. 1H NMR spectral
changes of 2 with the addition of Ca(ClO4)2. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank the Department of Science and
JA045760E
Technology, Government of India, for financial support (DST/
9
3164 J. AM. CHEM. SOC. VOL. 127, NO. 9, 2005