Lower rim 1,3-diderivative of calix[4]arene-appended salicylidene imine (H2L): Experimental and computational studies of the selective recognition of H2L toward Zn2+ and sensing phosphate and amino acid by [ZnL]

Article abstract of DOI:10.1021/jo1004247

A new 1,3-diderivative of calix[4]arene appended with hydroxymethyl salicylyl imine has been synthesized and its ion recognition toward biologically relevant Mⁿ⁺ ions studied. The receptor H₂L showed selectivity toward Zn²⁺

Full text of DOI:10.1021/jo1004247

pubs.acs.org/joc
Lower Rim 1,3-Diderivative of Calix[4]arene-Appended
Salicylidene Imine (H2L): Experimental and Computational Studies of the
Selective Recognition of H2L toward Zn2þ and Sensing Phosphate and
Amino Acid by [ZnL]
Roymon Joseph, Jugun Prakash Chinta, and Chebrolu P. Rao*
Bioinorganic Laboratory, Department of Chemistry, Indian Institute of Technology Bombay,
Mumbai 400 076, India
cprao@iitb.ac.in
Received March 10, 2010
A new 1,3-diderivative of calix[4]arene appended with hydroxymethyl salicylyl imine has been synthesized and its ion
recognition toward biologically relevant Mnþ ions studied. The receptor H2L showed selectivity toward Zn2þ by switch-
on fluorescence among the 12 metal ions studied with a detection limit of 192 ppb. The interaction of Zn2þ with H2Lhas
been further supported by absorption studies, and the stoichiometry of the complex formed (1:1) has been established on
the basis of absorption and ESI MS. Competitive ion titrations carried out reveal that the Zn2þ can be detected even in
the presence of other metal ions of bioimportance. The mode of interaction of Zn2þ with conjugate has been established
by a fleet of computational calculations carried out in a cascade manner, either on the ligand or on the complex, wherein
the final optimizations were carried out by the density functional theory (DFT) and found that the Zn2þ and Cd2þ
indeed bind differently. In situ prepared [ZnL] complex responds to both inorganic phosphate as well as AMP, ADP,
and ATP with a minimum detection limit of 426 ppb wherein the Zn2þ from the complex is detached and recomplexed
by the added phosphate moiety. It has been possible to build an INHIBIT logic gate for the conjugate using Zn2þ and
HPO42- as inputs by monitoring the fluorescence emission band at 444 nm as output. The amino acid sensing abilities of
[ZnL] have been explored by fluorescence and absorbance spectroscopy where it showed selectivity toward Cys, Asp,
and His through the formation of the Zn2þ complex of these amino acids by chelating through their side chain moieties.
Thus, while H2L is selective for Zn2þ among a number of cations, the [ZnL] is selective toward phosphate among a
number of anions and also toward Asp, Cys, and His among the naturally occurring amino acids.
DOI: 10.1021/jo1004247
r
Published on Web 04/14/2010
J. Org. Chem. 2010, 75, 3387–3395 3387
2010 American Chemical Society

Joseph et al.
JOCArticle
Introduction
functionalization.5 Though some conjugates of calix[4]arene
were demonstrated to have selectivity toward Zn2þ 6 or
phosphate7 or amino acids,8 to our knowledge there has
been no single calixarene-based receptor that recognizes all
three of these components. Our research group has been
involved in the synthesis of various calix[4]arene-appended
amide- and imine-based conjugates for the selective sensing
of ions and molecules.6d,f,9 Recently, we have also explored
the selective detection of amino acids using calix[4]arene
derivatives bearing transition-metal ions.8u,v It is of interest
to develop a calixarene-based molecular system that would
recognize both the ions and molecules. Thus, in this paper,
we discuss the synthesis and characterization of a calixarene
conjugate (H2L) that selectively recognizes Zn2þ and further
involvement of the corresponding Zn2þ complex in the
recognition of phosphate and amino acids. All the details
of spectroscopy studies relevant to these aspects as well as the
modeling the complexes of Zn2þ and Cd2þ are discussed in
this paper.
Zinc is one of the essential trace elements and second most
abundant transition-metal ion present in human body. The
zinc-containing proteins are abundant and act as both
structural and functional proteins by exhibiting a wide range
of biochemical activities.1 The deficiency of Zn2þ ion in brain
and pancreas may result in various disorders such as Par-
kinson’s disease, epilepsy, and certain cancers.2 In the pro-
teins, Zn2þ is generally coordinated by selected amino acid
side chains such as histidine, aspartic/glutamic acid, and
cysteine while exhibiting high affinity toward phosphate.1c,e
Thus, besides exhibiting a pivotal role in biology, the Zn2þ
ions are associated with amino acids in their free or peptide
form as well as anions including phosphate selectively. Both
the amino acids and phosphates are indispensable in bio-
logy,3 and the deficiency of these result in several side effects
in humans.4 Therefore, the selective recognition of the
species such as Zn2þ, phosphate and amino acids are im-
portant in biology. It is always desirable to have one single
molecular system that can provide features for recognition of
all these kinds of species. Therefore, in the search for
molecular systems which provide selectivity, conjugates of
calixarenes are worthy of note, since these contain both a
hydrophobic cavity and a hydrophilic rim and can also
provide an ideal platform for the development of receptors
toward ions or molecular species depending upon their
Results and Discussion
The receptor molecule (H2L) has been synthesized starting
from p-tert-butylcalix[4]arene (4) followed by dinitrile (5)
and then the diamine (6) derivative as shown in Scheme 1, as
already reported by us as well by others in the literature.6d,10
The receptor molecule has been synthesized by reacting the
diamine derivative 6 with 5-tert-butyl-2-hydroxy-3-(hydro-
xymethyl)benzaldehyde (3) in methanol. In turn, 3 has been
synthesized from p-tert-butylphenol (1) via 2,6-bis(hydro-
xymethyl)-4-tert-butylphenol (2).11 The precursors and final
products were characterized by analytical and spectral
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SCHEME 1. Synthesis of the Precursor Molecule 3 and Receptor Molecule H2La
aSynthesis of the precursor molecule, 3 (top row): (i) NaOH, CH2O, 7 d, rt, (ii) MnO2, CHCl3, 8 h, rt. Synthesis of the receptor molecule H2L (bottom
row): (a) K2CO3, ClCH2CN, acetone, reflux for 7 h; (b) LiAlH4, diethyl ether, reflux for 5 h; (c) 3, CH3OH, reflux. R = tert-butyl.
FIGURE 1. Fluorescence data for the titration of H2L by Zn2þ and Cd2þ in methanol: (a) spectral traces obtained during the titration of H2L
with Zn2þ, (b) plot of relative fluorescence intensity (I/I0) versus the mole ratio of [M2þ]/[H2L] added, and (c) plot of change in the wavelength
versus the mole ratio of [M2þ]/[H2L] added. b = Zn2þ; O = Cd2þ
.
techniques (see the Experimental Section and Supporting
Information).
The receptor molecule H2L has been characterized satis-
complex, at saturation the fluorescence enhancement is ∼30
fold. During the titration with Zn2þ, the metal ion is chelated
through phenolic oxygen and the imine nitrogen, resulting in
the utilization of lone pair of the nitrogen to block the PET
and thereby the fluorescence enhancement. However, a
similar titration carried out between H2L and Cd2þ resulted
in the fluorescence enhancement of only 10-fold even after
addition of 10 equiv of this ion, and hence, Cd2þ is at least
3-fold less effective as compared to that of Zn2þ. There is
about 12-15 nm shift toward blue as a function of addition
of Zn2þ or Cd2þ, suggesting the complex formation between
the conjugate and these ions. The sigmoidal plots obtained
in these cases exhibit a midpoint ratio of 1:1 for L2- to
Zn2þ and 4:1 for L2- to Cd2þ, in addition to a three-times
higher enhancement observed with Zn2þ (Figure 1b). Thus,
the selectivity difference observed between Zn2þ and Cd2þ
is ∼9-10 fold. The binding affinities of Zn2þ and Cd2þ
toward L2- have been calculated from the Benesi-
Hildebrand equation and found to have association con-
stants of 2.7 ( 0.1 ꢀ 104 and 3.6 ( 0.6 ꢀ 103 M-1, res-
pectively, suggesting an ∼8 times higher magnitude of
association of Zn2þ with L2- as compared to the Cd2þ. All
this seems to suggest that the selectivity is proportional to the
binding strength. A minimum concentration of ∼192 ppb of
Zn2þ has been detected by fluorescence studies carried out by
keeping H2L and Zn2þ at 1:1 and bringing appropriate
dilutions (Supporting Information).
1
factorily by H and 13C NMR, IR, ESI MS, and elemental
1
analysis. H2L exhibits a H NMR peak at 8.61 ppm char-
acteristic of the CHdN proton. The protons of the bridged
methylene were observed at 3.27 and 4.23 ppm owing to the
diastereotopic coupling constant (J = 13.4 Hz) indicating
the cone conformation.
A. Cation Recognition Studies. The binding, recognition,
and selectivity of H2L toward cations have been studied by
absorption and fluorescence spectral titrations.
Fluorescence Titration. The receptor H2L was excited at
320 nm, and its fluorescence emission was studied in the
range 330-600 nm. All of the studies were carried out in
methanol by maintaining the ligand concentration of 10 μM
throughout the experiment and varying the mole ratio of the
added metal ion. The metal ions subjected to the recognition
studies were Mn2þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cd2þ
,
Hg2þ, Ca2þ, Mg2þ, Naþ, and Kþ as their perchlorate salts.
H2L is a weak emitter as its fluorescence is quenched due to
the photoelectron transfer (PET) process from the lone pair
of nitrogen to the salicylidene moiety. During the titration
with Zn2þ, the fluorescence intensity of H2L increases as a
function of increase in the mole ratio of [Zn2þ]/[H2L]
(Figure 1a) and exhibits a sigmoidal behavior, wherein the
intensity is saturated at >3 equiv (Figure 1b). During the
titration, the emission band maximum shifts progressively
to exhibit a blue shift of ∼15 nm at saturation (Figure 1c),
and the titration exhibits a stoichiometric behavior. While
the midpoint of this sigmoidal plot corresponds to a 1:1
Titration by various Mnþ ions, viz., Mn2þ, Fe2þ, Co2þ
,
Ni2þ, Cu2þ, Hg2þ, Naþ, Kþ, Ca2þ, and Mg2þ, resulted in no
or minimal change in the fluorescence intensity (Figure 2),
suggesting that these ions are not recognized by H2L.
J. Org. Chem. Vol. 75, No. 10, 2010 3389

Joseph et al.
JOCArticle
FIGURE 2. Histogram representing the fluorescence response of
H2L with different Mnþ ions in methanol.
FIGURE 3. Plot of relative fluorescence intensity of H2L with Zn2þ
in the presence of 5 equiv of Mn2þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Cd2þ
,
Therefore, both the shift observed in the emission maximum as
well as the enhancement observed in the fluorescence intensity
during the titration will allow one to use H2L in the selective
and Hg2þ and 30 equiv of Naþ, Kþ, Ca2þ, and Mg2þ in methanol.
recognition of Zn2þ
.
Competitive Ion Titration. In order to understand the
practical applicability of H2L in recognizing Zn2þ ions,
competitive metal ion titrations were carried out in the
presence of other Mnþ ions. During the titration, the
mole ratios of metal ions, viz., Naþ, Kþ, Ca2þ, and Mg2þ
,
were kept constant at a 1:30 ratio with respect to H2L, and
the mole ratios of the added Zn2þ were varied. In case of
other metal ions, viz., Mn2þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Cd2þ
and Hg2þ, the ligand to metal ion ratio was kept at 1:5 and
titrated against different equivalents of Zn2þ. It has been
found that the added Zn2þ is able to replace all the other ions
except Fe2þ, Cu2þ, and Hg2þ during the titration (Figure 3)
and hence its use in the presence of other ions.
Computational Modeling of the Complexes of Zn2þ and
Cd2þ by L2-. Having observed from the fluorescence studies
that the Zn2þ is about an order of magnitude more selective
toward H2L as compared to Cd2þ, computational calcula-
tions12 were carried out as discussed in this paper. The initial
model for the receptor molecule, H2L was prepared from the
published data of copper complex13 by bringing the follow-
ing modifications: (a) removing the copper center, (b) repla-
cing tert-butyl moiety by hydrogen, (c) protonating the
phenolate moieties, and (d) introducing a -CH2OH moiety
ortho to the phenolic OH of the Schiff’s base part of each of
the arm. This has been optimized by going through a cascade
process starting from PM3 f HF/STO-3G f HF/3-21G f
HF/6-31G f B3LYP/3-21G f B3LYP/6-31G. The output
FIGURE 4. B3LYP/6-31G-optimized structure of H2L stabilized
through both the intra- and inter-arm hydrogen bonds. Data for
these hydrogen bonds: (i) O6-H30 N2 hydrogen bond: N2
3 3 3
3 3 3
˚
˚
H30 = 1.479 A, O6 N2 = 2.467 A, O6-H30-N2 = 150.0°; (ii)
3 3 3
˚
O8-H46 O6 hydrogen bond: O6 H46 = 1.828 A, O8 O6 =
3 3 3 3 3 3 3 3 3
˚
2.641 A, O8-H46-O6 = 137.3°; (iii) O7-H41 O8 hydrogen
3 3 3
˚
˚
bond: O8 H41 = 1.759 A, O7 O8 = 2.738 A, O7-H41-O8 =
3 3 3 3 3 3
˚
167.5°; (iv) O4-H13 N1 hydrogen bond: N1 H13 = 1.618 A,
3 3 3 3 3 3
˚
O4 N1 = 2.545 A, O4-H13-N1 = 147.7°.
3 3 3
obtained at every stage has been given as input for the next
higher level of calculations. The optimized structure ob-
served at B3LYP/6-31G level of calculation shows well
ordered arms in an extended fashion wherein each arm is
(12) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.;
Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci,
B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada,
M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian,
H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain,
M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski,
J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin,
R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.;
Gonzalez, C.; Pople, J. A. Gaussian 03, Revision C.02; Gaussian, Inc.,
Wallingford CT, 2004.
stabilized through phenolic O-H N hydrogen bond in-
3 3 3
teraction (intra-arm). In addition, one of the arms extend a
weak hydrogen bond, viz., alcoholic O-H O(phenolic).
3 3 3
Further, the two arms are connected through a hydrogen
bond formed between their alocoholic-OH groups (inter-
arm), as can be seen from Figure 4. The hydrogen-bond
interactions observed at the phenolic lower rim indicate the
cone conformation; the inter-arm interactions also support
that conformation.
In order to make the Zn2þ or Cd2þ complex, the structure
of H2L obtained at B3LYP/6-31G level has been taken and
was deprotonated at its both the phenolic centers of the
Schiff’s base of the arms to form L2-. The metal ion has been
placed well above the arms of L2- and was allowed to
(13) Dey, M.; Rao, C. P.; Guionneau, P. Inorg. Chem. Commun. 2005,
8, 998.
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Joseph et al.
JOCArticle
TABLE 1. Conformational Angles (deg) of the Arms Obtained upon
Optimization for H2L and Its Zn2þ and Cd2þ Complexes
H2L
[ZnL]
[CdL]
arm-1 arm-2 arm-1 arm-2 arm-1 arm-2
C1-O1-C8-C12
O1-C8-C12-N1
C8-C12-N1-C21
C12-N1-C21-C26
N1-C21-C26-C29
C21-C26-C29-O4
-156
-74
23
179
-0.8
2
-169 -178 -178 -170
177
-49
-77
47
-64
-73
-64
-57
-73 -101 -125
176 -179 -179
177
-7
1
177
-5
1
-3
4
3
1
3
1
FIGURE 5. Space-filling models: (a) B3LYP/6-31G-optimized
structure of H2L, (b) B3LYP/LanL2DZ-optimized structure of
[ZnL], and (c) B3LYP/LanL2DZ-optimized structure of [CdL].
FIGURE 7. Absorption spectra obtained during the titration of
H2L with (a) Zn2þ and (b) Cd2þ in methanol.
FIGURE 6. Coordination cores of [ZnL] and [CdL]. The unfilled
circle “X” represents the vacant site. Metric data for [ZnL]: N1-
distances were found to be in the range 2.229-2.445 and
˚
2.323-2.377 A, respectively, the distances which were com-
˚
Zn = 2.071, N2-Zn = 2.072, O6-Zn = 1.979, O4-Zn = 1.979 A;
monly found in the literature.14 The coordination angles
observed with the Cd2þ core provide a better fit if it were to
be considered as a seven-coordinated capped octahedral
structure with one vacant site. The [ZnL] exhibits at least
40 kcal/mol higher stabilization energy as compared to that
of the [CdL] complex at the same level of DFT calculations.
On going from H2L f [ZnL] f [CdL], the conformations of
the arms vary progressively to accommodate the correspond-
ing metal ion by providing appropriate coordination. The list
of conformational angles given in Table 1 clearly indicate
that a major conformational change was observed with the
C-N bond (Supporting Information).
N2-Zn-N1 =161.7, N2-Zn-O6 = 91.3, N2-Zn-O4 = 94.8,
N1-Zn-O6 = 94.8, N1-Zn-O4 = 91.3, O6-Zn-O4 = 141.5°.
The metric data for [CdL]: N1-Cd = 2.377, N2-Cdd2.323,
O2-Cd = 2.445, O4-Cd = 2.325, O5-Cdd2.441 and O6-Cd =
˚
2.229 A; N1-Cd-O5 = 147.4, N1-Cd-O2 = 98.5, N1-Cd-
O4 = 76.8, N1-Cd-N2 = 129.2, N1-Cd-O6 = 84.6, O2-Cd-
O5 = 82.7, N2-Cd-O2 = 125.5, N2-Cd-O4 = 91.0, N2-Cd-
O5 = 70.6, N2-Cd-O6 = 79.1, O2-Cd-O6 = 81.2, O2-Cd-
O4 = 128.9, O4-Cd-O5 = 77.3, O4-Cd-O6 = 146.2, O5-Cd-
O6 = 127.4°.
minimize. The minimization in the presence of metal ion was
carried out in a cascade fashion starting from PM3 f HF/
STO-3G
f HF/3-21G f HF/6-31G f B3LYP/
Absorption Titration. Absorption titrations were also car-
ried out to support the binding of Zn2þ with H2L. During the
titration, the concentration of H2L was kept constant at
20 μM and the mole ratio of Zn2þ was varied. Addition of
Zn2þ to a solution of H2L in methanol brought changes in its
absorption spectra with six absorption bands observed at
375, 320, 277, 262, 247, and 435 nm (Figure 7a). When H2L
was titrated against Zn2þ, a marginal increase was observed
in the absorbance of the bands at 375, 277, and 247 nm and a
significant decrease in the bands at 320, 262, and 435 nm.
Five isosbestic points were observed at 412, 345, 295, 269,
and 255 nm, and all these clearly indicate the complex
formation of H2L with Zn2þ. Absorption bands of the
conjugate obtained at 261 and 330 nm have been shifted to
277 and 370 nm, respectively, in the presence of Zn2þ. A plot
of absorbance versus mole ratio, [Zn2þ]/[H2L], has been
given in Figure 8a,b.
3-21G f B3LYP/6-31G f B3LYP/LanL2DZ in the case
of zinc and HF/STO-3G f HF/3-21G f B3LYP/3-21G f
B3LYP/LanL2DZ in the case of cadmium. Such minimiza-
tions resulted in the formation of Zn2þ or Cd2þ complex by
breaking the hydrogen-bonding interactions present bet-
ween the two arms. The Zn2þ interacts primarily at the
Schiff’s base core, and the Cd2þ primarily interacts at the
lower rim phenolic core (Figure 5).
The Zn2þ showed a distorted four-coordination geometry
bonded through two imine nitrogens and two Schiff’s base
phenolic oxygens. The Cd2þ showed a distorted six coordi-
nation geometry bonded through two imine nitrogens, two
Schiff’s base phenolic oxygens, and two lower rim phenolic
groups (Figure 6). The Zn-O and Zn-N were found to have
˚
distances of 1.979 and 2.072 A, respectively, distance which
were commonly found in the literature. The coordination
angles observed with the Zn2þ core provides a better fit if it
were to be considered as a trigonal bipyramidal with one
trigonal center being vacant, where the trans-angle, viz.,
N1-Zn-N2, is found to be 162°. The Cd-O and Cd-N
When similar titrations were carried out with Cd2þ, no
isosbestic point was observed, except for a slight increase in
the absorbance at 250 and 365 nm; however, this increase is
much less than that observed in the case of Zn2þ (Figures 7b
and 8c). In the case of the 263 nm band, the two ions exhibit
opposite trends in their absorbance. Such absorption spec-
tral changes observed with Cd2þ seem to indicate a different
(14) Xu, X.-Y.; Chen, J.-L.; Luo, Q.-H.; Shen, M.-C.; Haung, X.-Y.; Wu,
Q.-J. Polyhedron 1997, 16, 223.
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Joseph et al.
JOCArticle
FIGURE 8. Absorption spectral data for the titration of H2L with Zn2þ and Cd2þ in methanol: (a) and (b) are the plots of absorbance vs. mole
ratio of ([Zn2þ]/[H2L]) and (c) similar plots in case of Cd2þ. Symbols in figure (a) and (b) corresponds to 0 = 375, O = 320, b = 277, right-tilted
solid triangle = 262, 4 = 247, 9 = 435 nm and the symbols in figure (c) corresponds to 3 = 365, right-tilted solid triangle = 263, 4 = 250 nm.
FIGURE 9. Job’s plot obtained from the absorption titration of
H2L with Zn2þ in methanol, where A is the absorbance and nm and
n
H2L are the mole fractions of Zn2þ and H2L, respectively.
FIGURE 10. Mass spectral peak observed for the 1:1 complex of
H2L and Zn2þ: (a) experimental; (b) calculated.
mode of binding of L2- with Cd2þ as compared to that
observed for Zn2þ. Even the absorption spectral studies
resulted in association constants of 3.3 ( 0.1 ꢀ 104 and
4.4 ( 0.4 ꢀ 103 M-1, respectively, for Zn2þ and Cd2þ and are
in agreement with those derived from the fluorescence data.
Even the DFT studies showed higher stabilization energy of
the formation of the complex in the case Zn2þ as compared to
that with Cd2þ. Thus, both the absorption and fluorescence
studies clearly supported strong affinity of Zn2þ toward H2L
as compared to that of Cd2þ. The stoichiometry of the
complex formed was found to be 1:1 as derived from the
Job’s plot given in Figure 9. This has been further supported
by ESI MS as reported in this paper.
Formation of [ZnL] by ESI MS. Mass spectral peak
corresponding to the 1:1 complex of the conjugate and
Zn2þ has been observed as isotopic multiplet centered about
m/z = 1178.9 and is spread in the m/z values of 1177.8-
1184.8. The presence of zinc ion in the complex is evident
from the observed isotopic peak pattern (Figure 10).
B. Anion Recognition Studies. Since the receptor molecule
detects Zn2þ selectively, the in situ prepared [ZnL] complex
has been used for its anion recognition studies.
FIGURE 11. (a) Plot of relative fluorescence intensity (I/I0) of [ZnL]
with added anions and (b) similar plots with nucleotides, viz., AMP,
ADP, and ATP in methanol. 9=F-, b=Cl-, 2=Br-, 1=I-, left-
tilted solid triangle = ClO4-, right-tilted solid triangle = BF4-, ( =
-
AcO-, 3 = SCN-, left-tilted open triange = CO32-, f = HCO3
,
4 = NO3-, / = SO42-, O= H2PO4-, 0 = HPO42-, solid pentagon =
PO43-, ) = AMP, right-tilted open triangle = ADP, and g = ATP.
to 1:1 (Figure 11a). Thus, out of the 15 anions studied,
3-
only H2PO4-, HPO42-, and PO4 exhibited changes in the
fluorescence intensity and all other ions exhibited no significant
change. Since the Zn2þ complex of H2L was found to be sensi-
tive toward phosphate ions, fluorescence titrations were ex-
tended to biorelevant molecules possessing phosphate moiety,
viz., adenosine monophosphate (AMP), adenosine diphos-
phate (ADP), and adenosine triphosphate (ATP). The titra-
tions carried out with these nucleotides exhibited changes in the
fluorescence intensity similar to that shown by simple inorganic
phosphates (Figure 11b). The fluorescence quenching observed
in the titration of the complex with phosphate is attributable to
the removal of Zn2þ ions from the complex by the phosphate
Fluorescence Titration. The anion recognition studies
have been carried out by titrating an in situ generated
complex, [ZnL], by different anions, viz., F-, Cl-, Br-, I-,
2-
ClO4-, BF4-, AcO-, SCN-, CO32-, HCO3-, NO3-, SO4
,
H2PO4-, HPO42-, and PO43-. All the anion titrations
exhibited no significant change in the fluorescence inten-
sity except those based on phosphate. During the titra-
tion with phosphate, the initially observed fluorescence
intensity of [ZnL] has been found to be quenched gradually
to reach an intensity that is similar to that of H2L. The
corresponding fluorescence behavior can very well be fitted
to a sigmoidal type, and the inflection point corresponds
2-
ions. The minimum concentration of HPO4 that can be
detected by [ZnL] has been found to be 426 ppb.
3392 J. Org. Chem. Vol. 75, No. 10, 2010

Joseph et al.
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FIGURE 12. Absorption spectral data for the titration of [ZnL] with HPO42- in methanol. (a) Spectra obtained during the titration; inset
shows the absorption spectral traces corresponds to H2L (black), [ZnL] (red) and [ZnL] with HPO42- (blue); (b, c) Plots of absorbance vs mole
ratio of HPO42- added. 4 = 250, 3 = 263, g = 276, O = 325, 0 = 360, 9 = 435 nm.
FIGURE 13. (a) INH logic gate represented using a conventional
gate notation; an active output signal is obtained when Zn2þ = 1
and HPO42- = 0. (b) Truth table for the INH logic gate; Zn2þ and
2-
HPO4 are inputs to the system; I444 is the output signal of H2L
at 444 nm.
FIGURE 14. (a) Plot of relative fluorescence intensity (I/I0) of
[ZnL] versus the mole ratio of added Cys and Asp. (b) Histogram
Absorption Titration. In order to understand the removal
of Zn2þ by phosphate ions, absorption titrations were car-
representing the fluorescence quenching fold caused by the titration
of amino acids with [ZnL] (2:1) in methanol. b = Asp and 4= Cys.
2-
ried out. During the titration of the [ZnL] by HPO4
(Figure 12), the absorption bands observed at 358 and 275 nm
arising from the precursor complex have been progressively
shifted to 330 and 264 nm, respectively, and the shifted bands
correspond to that of free L. All this suggests the disruption of the
fluorescence quenching to different extents depending upon
the chelation ability of the added amino acid toward Zn2þ
(Figure 14). [ZnL] showed a maximum fluorescence quench-
ing with some amino acids, and this follows a trend, Cys >
Asp . His. In fact, these are the three amino acids present
to the maximum extent in the coordination sphere by bind-
ing through their side chains, viz., thiolato, carboxylate,
and imidazolate, in zinc enzymes. A quick search of the 94
catalytic centers present in different zinc enzymes suggests
that 50% of the coordination sphere is occupied by His, 20%
by the carboxylate of Asp and Glu, and ∼13-15% by Cys.
Fluorescence quenching observed in the presence of these
amino acids can be attributed mainly to the protonation of
the Zn2þ coordination sphere followed by the formation
of a complex of Zn2þ with the amino acid. This suggests
that both the pKa as well as the ability to chelate Zn2þ using
the side chain are the major factors responsible for the
observations.8u The dechelation of Zn2þ from [ZnL] by
amino acids has been further confirmed by the absorption
titration.
Absorption Titration. Upon titration of [ZnL] with Cys,
the initial absorption bands of [ZnL] exhibited a red shift in
the absorption maximum, and the spectra obtained after
2 equiv addition of Cys are similar to that of the simple H2L
(Figure 15a). Therefore, the changes observed during the
titration of phosphate and amino acids are similar to each
other, and [ZnL] senses both these species through a dis-
placement mechanism. Titrations carried out with Asp and
His also resulted in the release of H2L (Figure 15b).
2-
[ZnL] complex followed by the removal of Zn2þ by the HPO4
ions to result in free conjugate. It has been noted from the mole
ratio versus absorbance plot (Figure 12b,c) that the changes
observed for different bands during the titration between the
[ZnL] and HPO42- is exactly reverse to that observed during the
titration of H2L with Zn2þ (Figure 8a,b). This suggests a
displacement mechanism during the titration of HPO42-. Similar
observations were noticed even with H2PO4-, PO43-, AMP,
ADP, and ATP (Supporting Information).
INHIBIT Logic Gate. Logic gate properties of receptor
2-
molecule have also been explored by using Zn2þ and HPO4
as input signals and the fluorescence emission response of
H2L at 444 nm monitored as output. In the absence of two
inputs, the output signal showed a fluorescence quench for
the emission band at 444 nm. H2L did not exhibit any
response in the presence of simple HPO42- without addition
of Zn2þ. The fluorescence intensity of H2L has been in-
creased upon the addition of 2 equiv of Zn2þ and resulted in
an output signal at 444 nm. There was no output signal
observed in the presence of equimolar solutions of both Zn2þ
and HPO42- in methanol. Hence, these studies suggests that
H2L can be used as an INHIBIT logic gate toward Zn2þ in
2-
the absence of HPO4
by monitoring the fluorescence
enhancement at 444 nm (Figure 13).
C. Amino Acid Recognition Studies. Since Zn2þ has the
ability to form complexes with amino acids, in situ prepared
[ZnL] has been used for the recognition of amino acids by
fluorescence and absorption titrations.
Conclusions and Correlations
Fluorescence Titration. The complex [ZnL] was titrated
with the 20 naturally occurring amino acids which resulted in
The salicylyl imine conjugate of calix[4]arene (H2L) re-
ported in this paper possesses an additional CH2OH group
J. Org. Chem. Vol. 75, No. 10, 2010 3393

Joseph et al.
JOCArticle
FIGURE 15. (a) Absorption spectra of L during the titration of
[ZnL] with Cys. (b) Absorption spectra of H2L (black), [ZnL] (red),
{[ZnL] þ Cys} (blue), {[ZnL] þ Asp} (green), and {[ZnL] þ His}
(purple). The mole ratio of H2L, Zn2þ, and amino acid was 1:2:2,
and the solvent used was methanol.
when compared to a simple naphthyl derivative.6d This
conjugate shows better selectivity toward Zn2þ among the
12 metal ions studied by fluorescence spectroscopy. Metal
ion binding of the receptor molecule with Zn2þ has been
further supported by absorption titration, and the new
species of formation was demonstrated from the observed
isosbestic points. There is a blue shift of ∼15 nm observed in
FIGURE 16. Cartoon representation of ion binding by H2L and
the reactivity of [ZnL] toward HPO42- and amino acids.
exhibits INHIBIT logic gate properties when the inputs are
2-
Zn2þ and HPO4
emission intensity observed at 444 nm.
and the out put is the fluorescence
the emission band of the ligand when titrated with Zn2þ
.
Competitive titrations suggest the feasibility of sensing Zn2þ
by H2L even in the presence other biologically relevant metal
ions, except Fe2þ, Cu2þ, and Hg2þ. ESI MS spectroscopy
and the Job’s plot suggest the formation of a 1:1 complex
between L2- and Zn2þ, and the fluorescence studies reveal a
lowest detection limit of 192 ppb of Zn2þ. Thus, H2L can act
Interaction of [ZnL] with naturally occurring amino acids
has been demonstrated by spectroscopic techniques that
resulted in dechelation of Zn2þ from the complex of [ZnL]
and the formation of the amino acid complex of zinc, as
reflected from the emission and the absorption spectral
studies. The fluorescence spectra obtained during the titration
indicates that the rate of dechelation directly depends on the
nature of the amino acid. Therefore, in this paper we demon-
strate the ability of H2L as a sensor for Zn2þ and further the
use of [ZnL] as a sensor for phosphate. The [ZnL] also exhibits
as fluorescence switch on sensor to Zn2þ
.
The experimentally observed higher selectivity of H2L
toward Zn2þ over Cd2þ has been addressed by DFT calcula-
tions and found to have a different binding site in H2L for
each of these ions. While Zn2þ binds at the Schiff’s base
region by exhibiting a N2O2 core to result in trigonal bipy-
ramidal geometry with one vacancy, the Cd2þ binds primar-
ily at the lower rim by exhibiting a N2O4 core to result in
capped octahedral geometry with one vacancy. In order to
accommodate either Zn2þ or Cd2þ, the arms of H2L needs to
undergo appropriate conformational changes. In the case of
H2L, the two salicylyl moieties on the arms of calixarene are
oriented parallel to each other and also parallel to the central
axis of calix[4]arene scaffold. Upon complexation with Zn2þ
or Cd2þ, the phenyl moieties orient themselves almost per-
pendicular to the central axis of the calixarene platform as a
result of the major conformational change brought about the
C-N of the arm. As the extent of conformational changes
occurred differ between the Zn2þ and Cd2þ cases, their
binding regions in H2L also differ.
sensitivity primarily toward Cys and Asp which chelatesZn2þ
All these events have been pictorially depicted in Figure 16.
.
Experimental Section
Materials. All the perchlorate salts, viz., Mn(ClO4)2 6H2O,
3
Fe(ClO4)2 xH2O, Co(ClO4)2 6H2O, Ni(ClO4)2 6H2O, Cu-
(ClO4)2 6H2O, Zn(ClO4)2 6H2O, Cd(ClO4)2 H2O, Hg(ClO4)2
3
3
3
3
3
3
3
xH2O, Na(ClO4)2 H2O, K(ClO4)2, Ca(ClO4)2 4H2O, and Mg-
(ClO4)2 6H2O, were procured from a US commercial sup-
3
3
3
plier. Salts possessing different anions, viz., Bu4NF, Me4NCl,
Bu4NBr, Bu4NI, Bu4NClO4, Bu4NH2PO4, Na2HPO4, Na3PO4,
NaSCN, NaOAc, Na2SO4, Na2CO3, NaHCO3, NaNO3, and
NaBF4 were procured from a commercial supplier in India. All
the amino acids and nucleotides were purchased from the same
source in India. Materials used for the synthesis of p-tert-
butylcalix[4]arene and its derivatives, viz., p-tert-butylphenol
and formaldehyde, were procured from a US commercial suppli-
er, and the other chemicals, viz., NaOH, LiAlH4, MnO2, K2CO3,
and the solvents, viz., acetone and diethyl ether, were purchased
from a commercial supplier in India.
The in situ prepared [ZnL] complex can sense phosphate
among the 15 anions studied by fluorescence and absorption
spectroscopy. The response of nucleotides to the [ZnL] has
also been established by spectroscopic studies resulting in the
fluorescence quenching that is same as that of inorganic
phosphates. During the anion titration, only phosphate
could dechelate Zn2þ from [ZnL] and release the free H2L,
and the Zn2þ ion in turn is complexed by the phosphate.
Hence, the present study unambiguously proves the use of
H2L as a sensor for Zn2þ and the corresponding [ZnL] as a
selective phosphate sensor. The detection limit observed for
Solvents. All the solvents used were dried and distilled by
usual procedures immediately before use. For the purification of
methanol, 50-100 mL of analytical (AR)-grade methanol was
first treated with 10 g of magnesium turnings and 1.0 g of iodine
in a flask until the iodine disappeared and all of the magnesium
was converted to the methoxide. An additional 2 L of methanol
was added and the mixture refluxed for 2-3 h followed by
distillation under nitrogen atmosphere immediately before use.
Chloroform was also purified before use by treating AR-grade
2-
HPO4 with [ZnL] has been found to be 426 ppb. H2L
3394 J. Org. Chem. Vol. 75, No. 10, 2010

Joseph et al.
JOCArticle
chloroform with CaCl2 followed by distillation under nitrogen
atmosphere.
washed the Celite with dichloromethane to obtain light brown
clear solution. This was concentrated to give a brown solid,
which was recrystallized from chloroform/methanol to give a
Details of Solution Studies. Bulk solutions of H2L and the
metal salts were made in CHCl3 and MeOH, respectively, at
6 ꢀ 10-4 M. All the fluorescence titrations were carried out in
1 cm quartz cells by using 50 μL of H2L, and the total volume in
each measurement was made to 3 mL to give a final concentra-
tion of the ligand as 10 μM (i.e., the 3 mL solution contains
2.950 mL of CH3OH and 0.050 mL of CHCl3). During the
titration, the concentration of metal perchlorate was varied
accordingly to result in requisite mole ratios of metal ion to
H2L, and the total volume of the solution was maintained at
3 mL in each case by addition of CH3OH. For absorption
studies, the final concentration of [H2L] was kept constant at
20 μM, and the procedure used for the titrations was the same as
that used for fluorescence titrations. The bulk solution of
anions, amino acids, and nucleotides were made by dissolving
the corresponding compound initially in ∼500 μL of deionized
water and was diluted by using methanol to reach a final volume
of 10 mL. During the titration, the in situ prepared H2L (50 μL)
and Zn2þ (100 μL) of 6 ꢀ 10-4 M were titrated against different
mole ratios of anions, amino acids, and nucleotides in methanol
by maintaining a total volume of 3 mL through out the experi-
ment. A 100 μL portion of H2L and Zn2þ of 6 ꢀ 10-3 M has been
used for ESI MS titration studies after dilution by at least 1000-
fold or higher. 1H NMR titrations were carried out by dissolving
5 mg of H2L in 400 μL of CDCl3.
white crystalline solid: yield (900 mg) 80%; FTIR (KBr, cm-1
)
1482 (νCN), 3515 (νOH); 1H NMR (CDCl3, δ ppm) 0.88, (s, 18H,
C(CH3)3), 1.32 (s, 18H, C(CH3)3),3.45(d,J= 13.55 Hz, 4H, Ar-CH2-
Ar), 4.22 (d, J = 13.55 Hz, 4H, Ar-CH2-Ar), 4.81 (s, 4H, OCH2), 6.73
(s, 4H, Ar-H), 7.12 (s, 4H, Ar-H). Anal. Calcd for C48H58N2O4: C,
79.30; H, 8.04; N, 3.85. Found: C, 79.49; H, 8.30; N, 3.92.
Synthesis and Characterization of 6. To a vigorously stirred
solution of 5 (0.58 g, 0.81 mmol) in 32 mL of diethyl ether was
added LiAlH4 (0.25 g, 7.13 mmol) , and the reaction mixture was
refluxed for 5 h. After that, the reaction flask was immersed into
an ice-water bath, and excess LiAlH4 was destroyed by the
addition of wet benzene into the reaction mixture. The clear
organic layer was decanted, and the inorganic salts were rinsed
with benzene. The combined organic layers were evaporated to
dryness to yield diamine as a light yellow solid: yield (470 mg)
1
85%; FTIR (KBr, cm-1) 3362 (νOH/NH); H NMR (CDCl3, δ
ppm) 1.10 (s, 18H, C(CH3)3), 1.24 (s, 18H, C(CH3)3), 3.29 (t, J =
4.76 Hz, 4H, NCH2), 3.37 (d, J = 12.82 Hz, 4H, Ar-CH2-Ar),
4.07 (t, J = 4.76 Hz, 4H, OCH2), 4.32 (d, J = 12.82 Hz, 4H,
Ar-CH2-Ar), 6.97 (s, 4H, Ar-H), 7.04 (s, 4H, Ar-H); 13C NMR
(CDCl3, 100 MHz, δ ppm) 31.3, 37.8 (C(CH3)3), 34.0, 34.3
(C(CH3)3), 32.3 (Ar-CH2-Ar), 42.7 (CH2N), 78.7 (OCH2),
125.6, 126.0, 127.8, 133.3, 142.2, 147.7, 149.3, 150.4 (Ar-C);
ES MS m/z 735.26 (Mþ, 100). Anal. Calcd for C48H66N2O4: C,
78.43; H, 9.05; N, 3.81. Found: C, 78.29; H, 8.87; N, 3.97.
Synthesis and Characterization of H2L. A mixture of 6 (0.37 g,
0.53 mmol) and 3 (0.22 g, 1.06 mmol) was stirred in methanol
(75 mL) at room temperature for 12 h followed by refluxing for
6 h. A yellow solution thus formed was concentrated, and
the product was recrystallized in hexane: yield (49%, 0.29 g);
FTIR (KBr, cm-1) 1641 (νCdN), 3394 (νOH); 1H NMR:(CDCl3,
δ ppm) 0.92 (s, 18H, C(CH3)3), 1.25 (s, 18H, C(CH3)3), 1.26 (s,
18H, C(CH3)3), 3.27 (d, 4H, Ar-CH2-Ar, J = 13.44 Hz), 4.08 (t,
4H, OCH2, J = 5.20 Hz), 4.20-4.26 (m, 8H, Ar-CH2-Ar and
NCH2), 4.64 (s, 4H, CH2OH), 6.76 (s, 4H, Ar-H), 7.00 (s, 4H,
Ar-H), 7.12 (s, 2H, Ar-OH), 7.22 (d, 2H, Ar-H, J = 2.42 Hz),
7.29 (d, 2H, Ar-H, J = 2.35 Hz), 8.61 (s, 2H, CHdN), 13.64 (s,
2H, Ar-OH); 13C NMR (CDCl3, 100 MHz, δ ppm) 31.1, 31.5,
31.8 (C(CH3)3), 31.8 (Ar-CH2-Ar), 33.9, 34.0, 34.1 (C(CH3)3),
58.5 (NCH2), 62.4 (OCH2), 75.3 (CH2OH), 117.8, 125.2, 125.7,
127.5, 127.7, 128.2, 129.3, 132.5, 141.0, 141.6, 147.3, 149.6,
150.5, 157.7 (Ar-C), 168.18 (NCH); ES MS m/z 1115.77 (Mþ,
70). Anal. Calcd for C72H94N2O8.C2H5OH: C, 76.51; H, 8.68;
N, 2.41. Found: C, 76.98; H, 8.34; N, 2.34.
Synthesis and Characterization of 211. To a solution of NaOH
(5.0 g, 125 mmol) in H2O (150 mL) was added 4-tert-butylphe-
nol (19.0 g, 125 mmol), and a clear solution was obtained after
heating. A 37% formaldehyde (19 mL, 250 mmol) solution was
added dropwise under mechanical stirring after the reaction
mixture was cooled to 0 °C. The mixture was stirred at room
temperature for 7 days. The precipitate formed after the addi-
tion of NaCl (30 g) was filtered and suspended in 300 mL of
water. The suspension was cooled, and 5% aqueous HCl was
added. The resulting suspension was extracted with CH2Cl2
(150 mL ꢀ 3 times), and the combined organic phases were dried
over anhydrous sodium sulfate. The product was purified by
silica gel column chromatography using petroleum ether and
ethyl acetate as eluent (6:4): yield (16.0 g, 60%); 1H NMR
(CDCl3,δ ppm) 1.27 (s, 9H, C(CH3)3), 2.74 (s, 2H, CH2OH),
4.76 (s, 4H, CH2OH), 7.06 (s, 2H, Ar-H).
Synthesis and Characterization of 311. A mixture of 2 (5.0 g,
25 mmol) and MnO2 (22.0 g, 250 mmol) in CHCl3 (150 mL) was
stirred at room temperature for 8 h. After filtration, the product
was purified by silica gel column chromatography using petro-
1
leum ether and ethyl acetate as eluent: yield (1.5 g, 30%); H
NMR (CDCl3, δ ppm) 1.33 (s, 9H, C(CH3)3), 2.42 (t, 1H,
CH2OH, J = 6.5 Hz), 4.76 (d, 2H, CH2OH, J = 6.3 Hz), 7.47
(d, 1H, Ar-H, J = 2.4 Hz), 7.62 (d, 1H, Ar-H, J = 2.4 Hz), 9.9
(s, 1H, Ar-OH), 11.22 (s, 1H, Ar-CHO).
Acknowledgment. C.P.R. acknowledges financial support
by DST, CSIR, and BRNS-DAE. R.J. thanks UGC and J.P.
C. thanks CSIR for their research fellowships. We thank
SAIF, IIT Bombay, for ESI MS measurements.
Synthesis and Characterization of 510a. A mixture of p-tert-
butylcalix[4]arene, 4 (1.0 g, 1.55 mmol), K2CO3 (0.85 g, 6.20
mmol), NaI (0.92 g, 6.13 mmol), and chloroacetonitrile (0.4 mL,
5.33 mmol) in 50 mL of acetone was refluxed under nitrogen
atmosphere for 7 h. The reaction mixture was allowed to cool
down to room temperature and filtered through Celite and
Supporting Information Available: Spectral data of L,
fluorescence data, minimum detection limit, computational
data, absorption data, and pie chart. This material is available
free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 10, 2010 3395