A 1,3-Capped Calix[4] Conjugate Possessing an Amine Moiety as an Anion Receptor: Reversible Anion Sensing Detected by Spectroscopy and Characterization of the Supramolecular Features by Microscopy

Article abstract of DOI:10.1002/chem.201600609

A phenylenediamine-capped conjugate of calix[4]arene (L_amino) was synthesized by reducing its precursor, L_imino, with sodium borohydride in methanol. The L_aminosample binds to anions due to the more flexible and bent conformation of the capped aminophenolic binding core, compared to the precursor L_imino. The L_aminosample showed selectivity towards H₂PO₄^?by exhibiting a ratiometric increase in emission by about 11-fold with a detection limit of (1.2±0.2) μm ((116±20) ppb) over 15 anions studied, including other phosphates, such as P₂O₇^4?, adenosine monophosphate (AMP^2?), adenosine diphosphate (ADP^2?), and adenosine triphosphate (ATP^2?). The L_aminosample shows an increase in the absorbance at λ=315 nm in the presence of H₂PO₄^?, CO₃^2?, HCO₃^?, CH₃CO₂^?, and F^?. The¹H NMR spectroscopic titration of L_aminowith H₂PO₄^?, F^?, and CH₃CO₂^?showed major changes in the phenylene-capped and salicyl moieties, and thereby, confirming the aminophenolic region as the binding core. However, the binding strength of these anions followed the trend H₂PO₄^?>F^??CH₃CO₂^?>HSO₄^?. The heat changes observed by isothermal titration calorimetry support this trend. The L_aminosample showed reversible sensing towards H₂PO₄^?and F^?in the presence of Mg²⁺and Ca²⁺, respectively. NOESY studies of L_amino, in comparison with its anionic complexes, revealed that major conformational changes occurred in the capping region to facilitate the binding of anion. ESI-MS and the Job's method revealed 1:1 stoichiometry between L_aminoand H₂PO₄^?or F^?. In the SEM micrographs of L_amino, the spherical particles are converted into spherical aggregates and further form large agglomerates and even branched sheets in the presence of anions, depending upon their binding strength.

Full text of DOI:10.1002/chem.201600609

DOI: 10.1002/chem.201600609
Full Paper
&
Sensors
A 1,3-Capped Calix[4] Conjugate Possessing an Amine Moiety as
an Anion Receptor: Reversible Anion Sensing Detected by
Spectroscopy and Characterization of the Supramolecular
Features by Microscopy
Anita Nehra, Deepthi S. Yarramala, and Chebrolu Pulla Rao*[a]
Abstract: A phenylenediamine-capped conjugate of calix[4]-
arene (Lamino) was synthesized by reducing its precursor,
moieties, and thereby, confirming the aminophenolic region
as the binding core. However, the binding strength of these
À
L
imino, with sodium borohydride in methanol. The Lamino
anions followed the trend H2PO4À >FÀ @CH3CO2À >HSO4
.
sample binds to anions due to the more flexible and bent
conformation of the capped aminophenolic binding core,
compared to the precursor Limino. The Lamino sample showed
The heat changes observed by isothermal titration calorime-
try support this trend. The Lamino sample showed reversible
À
sensing towards H2PO4 and FÀ in the presence of Mg2+ and
À
selectivity towards H2PO4 by exhibiting a ratiometric in-
Ca2+, respectively. NOESY studies of Lamino, in comparison
with its anionic complexes, revealed that major conforma-
tional changes occurred in the capping region to facilitate
the binding of anion. ESI-MS and the Job’s method revealed
crease in emission by about 11-fold with a detection limit of
(1.2Æ0.2) mm ((116Æ20) ppb) over 15 anions studied, includ-
ing other phosphates, such as P2O74À, adenosine monophos-
phate (AMP2À), adenosine diphosphate (ADP2À), and adeno-
sine triphosphate (ATP2À). The Lamino sample shows an in-
crease in the absorbance at l=315 nm in the presence of
À
1:1 stoichiometry between Lamino and H2PO4 or FÀ. In the
SEM micrographs of Lamino, the spherical particles are con-
verted into spherical aggregates and further form large ag-
glomerates and even branched sheets in the presence of
anions, depending upon their binding strength.
1
H2PO4À, CO32À, HCO3À, CH3CO2À, and FÀ. The H NMR spec-
À
troscopic titration of Lamino with H2PO4À, FÀ, and CH3CO2
showed major changes in the phenylene-capped and salicyl
electrostatic when a quaternized ammonium center[30] was
present. In addition, the metal-ion complexes of calix conju-
gates have been developed as anion sensors.[31,32] However,
calix[4]arene conjugates that possess a reduced imine moiety
(ÀH2CÀNHÀ) and exhibit selective recognition for phosphates
and such systems are rather scarce in the literature.
Introduction
The synthesis of a novel receptor possessing an inherent capa-
bility to modify its photophysical properties upon interaction
with anions is of great interest to chemists because such a re-
ceptor can act as a sensor.[1–13] Anion sensing by typical supra-
molecular scaffolds, such as calixpyrrole,[14–17] azacyclo-
phanes,[18] calixarenes,[19–21] and cucurbiturils,[22] has been docu-
mented in the recent literature. Among these systems, calixar-
enes were preferential owing to the ease of functionalization
on both the upper and lower rims, and the presence of a preor-
ganized core, which is required to improve ion selectivity.[23–26]
Calix[n]arenes possessing urea/thiourea[27] and amide[28,29] moi-
eties were developed as anion sensors in which the interac-
tions were mainly manifested by hydrogen bonding and were
À
Phosphates play crucial roles in biology wherein H2PO4 is
involved in a variety of biological processes.[33–39] Therefore, the
À
detection of H2PO4 selectively among other biologically rele-
vant phosphates, such as adenosine monophosphate (AMP2À),
adenosine diphosphate (ADP2À), and adenosine triphosphate
(ATP2À), is very important.[40–53] Recently, we reported an anthra-
cenyl-appended triazole-linked receptor that was selective to
Co2+ [54]
When this receptor was quarternized at the triazole ni-
.
trogen atoms, effective binding to biologically relevant triphos-
phates was obtained.[55] Therefore, herein, we report a phenyle-
nediamine-capped conjugate of calix[4]arene, 5,11,17,23-tert-
butyl-25,27-bis-{[phenylenebis(azanediyl)]-bis(methylene)-bis-
(tert-butyl-2-methyl)phenoltriazolylmethoxy}-26,28-dihydroxy-
calix[4]arene (Lamino), that results in the loss of Mg2+ cation
sensing[56] and further transforms into a molecular system that
[a] Dr. A. Nehra, D. S. Yarramala, Prof. C. P. Rao
Bioinorganic Laboratory, Department of Chemistry
Indian Institute of Technology Bombay, Powai
Mumbai 400076 (India)
Fax: (+91)22-2572-3480
E-mail: cprao@iitb.ac.in
À
selectively senses anionic H2PO4 through a ratiometric fluores-
cence response. The binding strengths of anions were assessed
by isothermal titration calorimetry (ITC). Such anion sensing
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201600609.
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6.82 (s, 4H; Ar-OH), 6.68 (s, 2H; Ar-H), 5.5 (s, 4H; Sal-CH2), 5.1 (s,
4H; Ar-OCH2), 4.27 (s, 4H; Ar-CH2-Ar), 4.13 (d, 4H; Ar-CH2-Ar), 3.1
(d, 4H; Ar-CH2-Ar), 1.29 & 1.27 (d, 18H, -C(CH3)3), 0.90 ppm (s, 18H;
Ar-(CH3)3); 13C NMR (CDCl3): d=153.6, 151.2, 150.8, 147.8, 144.6,
143.8, 142.5, 137.5, 132.9, 128.8, 127.7, 127.6, 126.3, 125.7, 125.1,
123.9, 122.3, 115.8, 70.0, 50.1, 48.9, 34.3, 34.07, 34.04, 31.9, 31.8,
31.7, 31.6, 31.1 ppm; HRMS: m/z calcd for C80H95O6N8 [M+1]+:
1267.7688; found: 1267.7738; elemental analysis calcd (%) for
C82H106N8O8 (L+2CH3OH): C 73.95, H 8.02, N 8.41; found: C 73.62,
H 6.74, N 8.19.
can also be identified based on changes that occur in supra-
molecular aggregations of the receptor, as observed by SEM.
Experimental Section
Absorption and fluorescence studies
Bulk solutions of Lamino and the salts (possessing the requisite
anion) were prepared in acetonitrile at a concentration of 6À”
104À M. Bu4N+ salts were used in case of FÀ, ClÀ, BrÀ, IÀ, H2PO4
,
CO32À, ClO4À, HSO4À, and CH3CO2À; Na+ salts were used in case of
HCO3À, NO3À, SO42À, P2O74À, AMP2À, ADP2À, and ATP2À anions. All
fluorescence titrations were carried out at lex =290 nm. Excitation
and emission slit widths used were 5 nm and a scan speed of
200 nmminÀ1 was used. Absorption studies were carried out on
a JASCO V-570 spectrometer. All fluorescence and absorption titra-
tions were carried out in 1 cm quartz cells by maintaining a final
[Lamino] of 5 mm in a total volume of 3 mL; this was achieved by di-
luting with acetonitrile.
Synthesis and characterization of C
Compound 1 (0.1 g, 0.08 mmol) and benzyl azide (2; 0.15 g,
1.18 mmol) were dissolved in a mixture of dichloromethane, tert-
butanol, and water (20 mL, 1:1:2). CuSO4·5H2O (0.03 g, 1.15 mmol)
and sodium ascorbate (0.05 g, 0.23 mmol) were then added. The
resulting solution was stirred for 12 h at room temperature. Upon
completion of the reaction (based on TLC), the organic layer was
separated and the aqueous layer was extracted with dichlorome-
thane (2”50 mL). The combined organic layer was washed with
brine (2”50 mL), dried over Na2SO4, the solvent was removed
under reduced pressure, and hexane was added to give C as
1H NMR spectroscopy studies
1H NMR spectroscopy studies were carried out in CDCl3 at 258C at
400 MHz on a Brucker NMR spectrometer. Bu4N+ salts were used
for all anions used in this study. Solutions (40 mm) of the corre-
sponding anion were gradually added to a 4 mm solution of Lamino
(0.5 mL) during the titrations. 2D NMR studies were performed in
CDCl3; [Lamino]=5 mm.
1
a pure white product (0.09 g, 64%). H NMR (CDCl3, 400 MHz): d=
0.96 (s, 18H; C(CH3)3), 1.27 (s, 18H; C(CH3)3), 3.19 (d, J=15.08 Hz,
4H; Ar-CH2eq-Ar), 4.12 (d, J=15.24 Hz, 4H; Ar-CH2ax-Ar), 5.1 (s, 4H;
Ar-CH2), 5.54 (s, 4H; OCH2), 6.7 (s, 4H; Ar-H), 6.9 (s, 4H; Ar-H), 7.2 (s,
2H; Ar-OH), 7.26 (m, 4H; Ar-H), 7.34 (m, 6H; Ar-H), 7.82 ppm (s,
2H; triazole-H); 13C NMR (CDCl3, 100 MHz): d=31.2, (Ar-CH2-Ar),
31.9, 32.0, 34.0, 34.2, 54.4, 66.7 (-O-CH2), 123.8, 125.3, 125.9, 127.9,
128.3, 128.8, 129.2, 132.9, 135.2, 141.9, 144.6, 147.5, 149.7,
150.6 ppm; ESI-MS: m/z: 991 [M+1].
Isothermal titration calorimetry (ITC)
The calorimetric titrations were performed at 208C with a MicroCal
ITC 200 isothermal titration calorimeter from MicroCal (Northamp-
ton, MA, USA). The corresponding anion, as a 0.5 mm solution
(40 mL) in a syringe, was added to a 0.1 mm solution of ligand
Lamino in the cell by means of 20 injections. The first addition was
0.2 mL and successive additions of 2 mL were given by maintaining
a time spacing of 150 s between each injection. The ITC data were
fitted with the origin software package provided by MicroCal by
using one set of site curve-fitting models. Each time a control was
carried out without Lamino and the corresponding data were sub-
tracted from the main titration data; the resultant data was sub-
jected to the curve-fitting procedure.
Results and Discussion
The phenylenediamine-appended, triazole-linked calix[4]arene
Lamino was synthesized in a single step from Limino by the reduc-
tion of the imine group with NaBH4, as described in the Experi-
mental Section (Scheme 1). The Lamino sample was character-
1
ized by H and 13C NMR, COSY, and NOESY spectroscopy, ESI-
MS, and elemental analysis (SI 01 in the Supporting Informa-
tion).
The conjugate Lamino exists in a cone conformation in solu-
tion, as evident from the H NMR spectrum, which exhibits two
Sample preparation for SEM
1
For SEM studies, aliquots (40 mL) of Lamino (5 mm) and Lamino +anions
in acetonitrile were sonicated for 15–20 min and the samples were
drop-cast on an aluminum surface and dried under an IR lamp.
doublets at d=3.10 and 4.13 ppm that correspond to the
bridge CH2 groups of the calixarene rim. As the electronic envi-
ronment in the capped arm region changes upon reduction,
the chemical shifts of protons e and f were affected considera-
bly due to the conversion of ÀHC=NÀ (Limino) into ÀH2CÀNHÀ
(Lamino). As a result of the conversion of imine to amine, the
Dde,f value changes from 0.34 to 0.01 ppm; the environments
of e and f turn out to be identical. Signals a and b were identi-
fied at d=7.29 and 7.21 ppm, respectively, by COSY due to
cross correlations (Figure 1a). Conformational changes that oc-
curred in the capped arms upon reduction of the imine moiety
were supported by NOESY results (Figure 1b). The set of pro-
tons a and g show strong correlations in the NOESY spectrum,
which suggest that the capped region is bent to a large extent
due to the presence of ÀH2CÀNHÀ (Lamino), thereby introducing
greater flexibility than that of ÀHC=NÀ present in Limino. This is
Synthesis and characterization of Lamino
Synthesis and characterization of all precursors were the same as
that reported recently.[56] The Limino derivative (0.1 g, 0.08 mmol)
was taken in dry methanol (5 mL) before NaBH4 (0.06 g,
0.0016 mol) was added at 08C and the mixture was stirred for 1 h
at room temperature. The solvent was evaporated under vacuum,
water (10 mL) was added to the residue, and the product was ex-
tracted into dichloromethane (3”10 mL). The collected organic
layer was washed with water (2”10 mL), followed by brine, and
was dried over anhydrous Na2SO4. Removal of the solvent under
reduced pressure afforded Lamino as a pure product (0.05 g, 50%).
1H NMR (CDCl3): d=8.14 (s, 2H; triazole-H), 7.29 (d; Ar-H), 7.10 (d;
Ar-H), 6.98 (s, 2H; calix-Ar-H), 6.89 (s, 2H; Ar-H), 6.87 (s, 2H; Ar-H),
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Scheme 1. Synthesis of Lamino and C: a) 3-(azidomethyl)-5-tert-butyl-2-hydroxybenzaldehyde, CuSO4·5H2O, sodium ascorbate, dichloromethane/water (1:1), RT,
12 h (ꢀ90%); b) o-phenylenediamine, CH3OH, RT, 12 h (74%); c) NaBH4, CH3OH, RT, 1 h; d) NaN3, DMSO, RT, 12 h; e) 2, CuSO4·5H2O, sodium ascorbate, tBuOH/
water (1:1), RT, 12 h.
1
Figure 1. Two-dimensional H NMR spectra of Lamino: a) COSY, b) NOESY.
further supported by a weak correlation observed between
b and e and f. The calix[4]arene conformation is still a cone in
Absorption studies
the case of Lamino
.
The binding of anions to Lamino has been evaluated by carrying
The Lamino sample has been subjected to anion-binding stud-
ies by H NMR, absorption, and fluorescence spectroscopy, and
out absorption titrations with different anions, such as halides
1
(FÀ, ClÀ, BrÀ, IÀ), phosphates (H2PO4À, P2O74À, AMP2À, ADP2À,
À
the masses of the species were established by ESI-MS. The con-
trol, C, was synthesized from 2 and dipropargyl (1) through
a click reaction and was characterized (SI 02 in the Supporting
Information). Control C was used to affirm the necessity of the
capped aminophenolic binding core for receiving the anion.
and ATP2À), and other oxoanions (CO32À, NO3À, SO42À, ClO4
,
HSO4À, HCO3À, CH3CO2À), in acetonitrile. The Lamino sample ex-
hibited an absorption band centered at l=290 nm that corre-
sponded to p–p* transitions (Figure 2a). Upon titration, some
of these anions exhibited a new band at l=315–320 nm (Fig-
ure 2a), the absorbance of which increased with increasing
concentration of the added anion. This new band is attributa-
ble to intermolecular charge transfer (ICT) between the anion
and NH of Lamino, through which the interaction leads to hydro-
gen bonding. Considerable changes of this type were ob-
served with FÀ, among the halides; with H2PO4À, among the
phosphates; and with HCO3À, CO32À, and CH3CO2À, among the
Response towards anions
The interactions of Lamino with anions have been studied by
1
ESI-MS and H NMR, fluorescence, and absorption spectrosco-
py.
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Figure 2. Absorption spectra obtained during the titration of Lamino with anions: a) H2PO4À, b) FÀ, c) CH3CO2À, d) CO32À, e) HCO3À, and f) HSO4À. g) Absorbance
versus molar ratio plots for all anion titrations. h) Histogram showing changes in the absorbance of Lamino at l=315 nm in the presence of anions.
other oxoanions studied (Figure 2b–f and SI 03 in the Support-
ing Information). The changes observed in absorbance in the
new band were plotted as a function of the molar ratio of the
anion added for all five anions (Figure 2g). An increase in the
absorbance of lꢀ255–260 nm was observed for all five anions
(Figure 2a–e). The other 11 anions studied showed no consid-
erable change to the absorption spectra; Figure 2 f shows the
spectra for one such case: HSO4À. The highest absorbance ob-
served in the new band is shown as a histogram in Figure 2h,
from which the five strongly interacting and binding ions can
be easily identified. To understand the relative interaction abili-
ties of these five anions with Lamino, further studies were carried
(SI 06 in the Supporting Information). The binding constant de-
rived based on this data by using the Benesi–Hildebrand equa-
À
tion resulted in Ka =6.18”104 mÀ1 for the binding of H2PO4 by
Lamino (SI 07 in the Supporting Information). The minimum de-
tection limit observed is (1.2Æ0.2) mm ((116Æ20) ppb; SI 08 in
the Supporting Information). To investigate the effect of H2O,
a similar titration was performed in a 5% aqueous solution of
CH3CN (v/v), in which Lamino exhibited a 3- to 4-fold increase in
À
fluorescence intensity at l=425 nm when H2PO4 was added
(SI 04 in the Supporting Information). No other phosphate ion,
namely, P2O74À, AMP2À, ADP2À, and ATP2À, studied showed any
change in the fluorescence emission of Lamino
.
1
out by fluorescence and H NMR spectroscopy.
Similar fluorescence studies carried out with the halides re-
sulted in a ratiometric change only in case of FÀ and not with
other halides; however, the extent to which the lꢀ430 nm
Fluorescence studies
band increased was much lower in case of FÀ than that of
2À
The Lamino sample exhibited an emission band at l=315 nm
upon excitation at l=290 nm in acetonitrile, and was titrated
with each of those anions listed for the absorption studies (SI
04 in the Supporting Information). When Lamino is titrated
against H2PO4À, it shows a decrease in the fluorescence intensi-
ty of the band at l=315 nm and a concomitant increase in
a new emission band at l=425 nm, with a clear isoemissive
point at l=372 nm (Figure 3a and b). Thus, the Lamino sample
exhibited a ratiometric response towards the incremental addi-
tion of H2PO4À, which implied the interaction of the conjugate
with the anion. Emission is attributed to the presence of hy-
drogen-bonding interactions and to the aggregational behav-
ior of the complexes with anions. Such blue emission has been
reported in the literature.[57] The quantum yield of Lamino is
0.0024, which is enhanced by about 10-fold in the presence of
H2PO4À, and is 0.026 with respect to 2-aminopyridine (SI 05 in
the Supporting Information). The 1:1 stoichiometry between
H2PO4À. Of all other oxoanions studied, only CH3CO2À, CO3
,
À
and HCO3 exhibited similar ratiometric changes, although the
increase in intensity observed for the lꢀ430 nm band was
much lower and comparable to that observed with FÀ. Based
on the ratiometric enhancement of I425/I315, the anion interact-
ing ability follows the order H2PO4À @FÀ ꢀCO32À ꢀCH3CO2À ꢁ
À
HCO3 (Figure 3b–f). This enhancement is at least four- to six-
fold higher for H2PO4À than that of any other anion, which sug-
gests that the interaction with this ion is stronger and may
result in greater changes to the conformation of the arms. The
relative fluorescence intensity pertaining to the l=420 nm
band is shown in Figure 3g as a histogram for all anions. No
other anions showed significant changes in the fluorescence
À
spectra of Lamino and were the same as that of HSO4 given in
Figure 3h.
À
Lamino and H2PO4 was established based on the Job method
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Figure 3. Fluorescence spectra obtained during the titration of Lamino with anions: a) H2PO4À, c) FÀ, d) CH3CO2À, e) HCO3À, f) CO32À, and h) HSO4À. b) The titra-
À
tion of Lamino with H2PO4 is shown as molar ratio versus intensity (squares: l=315 nm, triangles: l=425 nm); the blue line indicates the molar ratio versus
ratiometric response. Inset: photographs of vials under a UV lamp. g) Histogram showing the relative intensity for the band at l=425 nm with 10 equivalents
of anions.
À
for H2PO4À, FÀ, CH3CO2À, and HSO4 (Figure 4 f). All of these
1D and 2D NMR spectroscopy studies with anions
1H NMR spectra reveal that the binding strengths of these
anions follow the order H2PO4À >FÀ @CH3CO2À >HSO4À; this
À
Having observed that H2PO4À, FÀ, and CH3CO2 ions showed
considerable changes in their absorption and emission spectra,
trend is consistent with the absorption and emission results
1
À
we carried out H NMR spectral studies with these ions (SI 09
given herein. All of these results provide evidence that H2PO4
in the Supporting Information). In addition, another ion that
did not show any change in absorption and emission, namely,
interacts with Lamino through conformational changes to the
capped region, but without forming any interactions with the
triazole proton or without affecting the cone conformation of
the calixarene platform. On the other hand, FÀ interacts with
Lamino through the triazole moiety without resulting in signifi-
cant conformational changes to the capped region or affecting
the cone conformation of the calix[4]arene platform.
À
HSO4 was also studied by 1H NMR spectroscopy to further
prove that this ion indeed did not bind to Lamino (SI 09 in the
À
Supporting Information). When H2PO4 is added to Lamino incre-
mentally, signals corresponding to the phenylene motif (a, b)
and salicyl moiety (e, f, s) shift upfield, whereas the signal ob-
served at d=4.2 ppm (d) undergoes a downfield shift, which
In the pursuit of identifying the conformational changes
that occur in Lamino upon binding to an anion, 2D NMR spec-
troscopy studies were carried out (SI 10 in the Supporting In-
À
supports the hypothesis that H2PO4 interacts with Lamino in
this region. However, the signal of the triazole proton (i) and
calixarene platform signals (m, n, q, r) exhibit negligible
À
formation). In the presence of H2PO4 or FÀ, the triazole
À
changes upon the addition of H2PO4 (Figure 4).
proton i loses its correlation with protons k and j. However,
the cross correlation between protons a and g remains intact,
which indicates that the bent conformation of the capped
region persists to facilitate the interaction. Protons e and f
showed interactions with a in the presence of H2PO4À, whereas
this was not present for FÀ (Figure 5a). The connectivity be-
tween k and j is lost in the presence of FÀ (Figure 5b). Thus,
this experiment suggests that the major conformational
changes observed are primarily in the capped region for
H2PO4À, whereas in the presence of FÀ the triazole part of the
arms is also affected.
The following are some important comparisons observed
1
from the H NMR spectra upon titration with different anions.
1) The triazole proton i exhibits a large downfield shift by
about 0.22 ppm (Dd) only in presence of FÀ; the other anions
showed negligible changes (Figure 4b and c). 2) The ÀCH2 (d)
signal shifted downfield by about 0.12 ppm in the presence of
H2PO4À, whereas in the case of FÀ the signal shifted upfield by
À
À
about 0.1 ppm. However, CH3CO2 and HSO4 showed much
smaller downfield shifts of about 0.04 and 0.018 ppm, respec-
tively, which was, at most, one-third to one-fifth of that ob-
À
served for H2PO4 and FÀ (Figure 4d). 3) Although the protons
showed large upfield shifts of 0.27, 0.22, and 0.12 ppm for
H2PO4À, FÀ, and CH3CO2À, respectively, the shift was almost in-
Species detection by ESI-MS
À
significant in the presence of HSO4 (Dd=0.03 ppm; Fig-
To identify the complex(es) formed between Lamino and the
anion, mass spectrometry studies were carried out by using
(Bu4N)H2PO4 and (Bu4N)F salts. The mass spectra showed sig-
ure 4e). 4) The upfield shifts observed in the signal for tert-
butyl proton s are 0.1, 0.076, 0.048, and 0.02 ppm, respectively,
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Figure 4. 1H NMR spectroscopy titration of Lamino with H2PO4À (a) and FÀ (b) in CDCl3 at RT (the number given on the left side of each spectrum corresponds
to the number of equivalents of anion added). Proton labeling is given in Scheme 1. Plots of chemical shift versus molar ratio for protons c) i, d) d, e) a, and
À
À
À
: F , : CH3CO2À, and : HSO4
*
, .
~
!
&
f) s; : H2PO4
À
Figure 5. 2D NOESY spectra of a) Lamino +H2PO4 and b) Lamino +FÀ.
nals corresponding to the anion-bound species in both cases
shows the molecular ion signal at m/z 1268. When (Bu4N)H2PO4
was added to Lamino, an anion-bound species at m/z 1365
(SI 11 in the Supporting Information). The Lamino sample alone
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À
[Lamino +H2PO4 ] was observed. Even in the case of FÀ, upon
performed (SI 14 in the Supporting Information). The heat
changes obtained from one set of site fitting for all of these
anions for this interaction followed the order H2PO4À @FÀ >
the addition of (Bu4N)F, the spectra exhibited one or more fluo-
ride-bound species at m/z 1303 and 1327.
À
CH3CO2À >HSO4 (Figure 6c and SI 14 in the Supporting Infor-
mation). The data clearly show that the heat release is maximal
for H2PO4À, which supports the hypothesis that this anion in-
teracts more strongly with Lamino than any other anion studied.
Thus, this result is in accordance with that observed from spec-
tral studies.
Requirement of the capped aminophenolic core for anion
recognition
To show that the binding core formed in Lamino is due to cap-
ping, the control molecule, C, devoid of the capped aminophe-
nolic core was studied for the absorption and emission spectra
in the presence of H2PO4À. No changes were found in the
spectra, which supported the necessity of the capped amino-
phenolic binding core for the binding and recognition of
H2PO4À. Furthermore, the importance of the flexible ÀH2CÀ
Supramolecular features of Lamino and its anion adducts
Calixarenes are known to show distinctive supramolecular fea-
tures in the presence of different cations, anions, or molecules
when placed on surfaces and studied by microscopy.[58–61] The
supramolecular characteristics of Lamino and its anion adducts
were studied by SEM. The Lamino sample shows discrete spheri-
cal particles of 200–500 nm (Figure 7a, b, o, and p), and the
features are the same, regardless of whether the molecule was
taken in acetonitrile or ethanol. Even in the presence of the
À
NHÀ core in the capped region for the recognition of H2PO4
was demonstrated when the titrations were carried out with
L
imino, which contained ÀHC=NÀ instead of the amine moiety
(ÀH2CÀNHÀ); no changes in the absorption and emission spec-
À
tra were observed upon addition of H2PO4 to Limino (SI 12 in
the Supporting Information). Thus, the capped aminophenolic
core is essential for the recognition of anions.
À
HSO4 anion, the observed SEM features are similar to that of
simple Lamino with regard to size and shape; this suggests that
no significant interactions are present between HSO4 and
Reversible anion sensing by Lamino
À
The fluorescence enhancement observed upon the addition of
Lamino which alter the supramolecular characteristics (Figure 7c
and d). However, in the case of CH3CO2À, the Lamino sample re-
veals discrete spherical particles with a tendency to form clus-
ters to a lesser extent, which suggests that the interaction of
À
H2PO4 to Lamino could be reversed with the addition of Mg2+
/Zn2+. Therefore, the reversible sensing of Lamino towards
À
H2PO4 has been demonstrated when H2PO4À, followed by
Mg2+/Zn2+, were added consecutively for five cycles, and a re-
producible fluorescence response was found for each cycle
(Figure 6a and SI 13 in the Supporting Information). Thus, the
À
CH3CO2 results in only minimal conformational changes to
Lamino (Figure 7e and f). The tendency to form such aggregates,
followed by their interconnection, leads to larger agglomerates
and supports the formation of chainlike structures in the pres-
ence of CO32À (Figure 7g and h). This indicates that the carbon-
ate ion indeed interacts more strongly with Lamino than that of
À
addition of H2PO4 to Lamino switches ON the fluorescence,
whereas the same is switched OFF when Mg2+ or Zn2+ is
added consecutively. Similar responses were observed in case
of the titration of Lamino with FÀ followed by Ca2+ (Figure 6b).
The repeatedly demonstrated ON/OFF fluorescence behavior
suggests that Lamino can be used as a reversible sensor for
the acetate ion. When FÀ (Figure 7i and j) interacts with Lamino
,
chainlike aggregation dominates and is more extensive than
À
that observed with carbonate ions. In the presence of H2PO4
,
À
H2PO4 and FÀ.
the Lamino sample exhibits extensively branched fibrils filled
with spherical particles when taken in acetonitrile (Figure 7k
and l). On the other hand, the same sample in ethanol exhibits
additionally aggregated spherical particles (Figure 7m and n);
thus solvent plays a role in the nature of the supramolecular
structures formed.
Anion binding to Lamino measured by ITC
To understand the heat changes that occurred upon binding
À
of H2PO4À, FÀ, CH3CO2À, and HSO4 to Lamino, ITC studies were
À
Figure 6. Reversible anion sensing detected by fluorescence spectroscopy: a) H2PO4 followed by Mg2+; b) FÀ followed by Ca2+. c) Histogram showing the
heat changes observed during the interaction of anions with Lamino, as monitored by ITC.
Chem. Eur. J. 2016, 22, 8980 – 8989
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Figure 7. SEM micrographs of Lamino (a,b), Lamino +HSO4À (c,d), Lamino +CH3CO2À (e,f), Lamino +CO32À (g,h), Lamino +FÀ (i,j), and Lamino +H2PO4À in acetonitrile (k,l)
and ethanol (m,n). All samples were prepared in acetonitrile, unless otherwise stated. Particle size distributions for Lamino (o) and {Lamino +HSO4À} (p).
Thus, there is a smooth and sequential change in the mor-
phological features of the particles of Lamino in the presence of
different anions, and these changes seem to reflect the
strength of their interactions with the calix conjugate upon
going from acetate to carbonate to fluoride to dihydrogen
phosphate, as observed by SEM. Indeed, the strength of inter-
actions derived from spectroscopy and thermodynamics is re-
flected in aggregation, and the trend follows the order
H2PO4À >FÀ >CO32À >CH3CO2À. This clearly demonstrates the
anion-induced aggregational features of the supramolecular
aspects of Lamino and the influence of solvent.
cursor, which suggested that Limino was not a receptor for
H2PO4À. On the other hand, the reduced form, Lamino, did not
show any spectral changes in the presence of cations, includ-
ing Mg2+ (SI 15 in the Supporting Information), but was sensi-
tive towards anions. The Lamino sample exhibited different inter-
action strengths with different anions; the most prominent
À
were H2PO4 and FÀ. These two ions are differentiated not
only based on their spectra, but also on the supramolecular
features revealed by SEM. Thus, these two conjugates, Limino
and Lamino, exhibit complementarity in their sensing behavior
towards ions (Scheme 2).
À
The Lamino sample showed good selectivity towards H2PO4
over 15 anions studied, including other phosphates, such as
P2O74À, AMP2À, ADP2À, and ATP2À. The absorption studies of
Conclusion
À
2À
À
A phenylenediamine-capped conjugate of calix[4]arene (Lamino
)
Lamino with anions revealed that H2PO4 , CO3 , HCO3 ,
CH3CO2À, and FÀ showed an increase in the absorbance at l=
was synthesized by reducing the precursor, Limino, with sodium
borohydride in methanol. The Limino sample was recently
shown by us to act as a selective receptor to Mg2+ cations.[56]
315 nm among the phosphates, halides, and oxoanions
À
(HSO4À, NO3À, SO42À, and ClO4 ) studied. Upon the interaction
À
The anion H2PO4 did not affect the spectra of the Limino pre-
of Lamino with H2PO4À, the emission band at l=425 nm
Chem. Eur. J. 2016, 22, 8980 – 8989
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Scheme 2. Schematic representation of the complementarity of the sensing behavior of Limino versus Lamino
.
Figure 8. Relationship between binding strength and aggregation.
showed an enhancement of about 21-fold, whereas the ratio-
metric enhancement (I425/I315) was about ꢀ11-fold; thus, an in-
crease in the quantum yield by an order of magnitude in pres-
cal aggregates, large agglomerates, and finally to branched
sheets in the presence of the anions, depending upon the
binding strength (Figure 8).
À
ence of H2PO4 was observed. The present study, including ESI-
Thus, although Limino is sensitive to cations, in particular, to
Mg2+, the Lamino sample, obtained from the reduction of the
imino-capped derivative to its amino form, is sensitive to
anions, in particular, to H2PO4À. The conformational flexibility
of Lamino, particularly in the capped region, is responsible for
this transformation in the sensing behavior observed. Indeed,
this was reflected in features observed by both spectroscopy
and microscopy.
MS experiments, supported the formation of a 1:1 complex
with Ka =6.18”104 mÀ1 and provided a minimum detection
À
limit of (1.2Æ0.2) mm ((116Æ20) ppb) for H2PO4 with Lamino
.
,
Even other anions, such as FÀ, CO32À, HCO3À, and CH3CO2
À
also exhibited a ratiometric increase, but to a much lesser
extent.
À
1H NMR spectroscopy titration of Lamino with H2PO4 revealed
that the interaction led to changes in chemical shift mainly in
the capped region, and thus, provided evidence of the amino-
phenolic region as the binding core for H2PO4À. However,
other anions, such as FÀ, CH3CO2À, and HSO4À, exhibited much
smaller changes in chemical shifts of the protons present in
Acknowledgements
C.P.R. acknowledges financial support from the DST (SERB and
Nano mission), CSIR, and DAE-BRNS. C.P.R. acknowledges the
DST (SERB) for a J. C. Bose National Fellowship and IIT Bombay
for an Institute Chair Professorship. Fellowships from UGC for
A.N. and D.S.Y. are gratefully acknowledged. We thank the
FEGSEM central facility of IIT Bombay for providing the service.
the binding region, which suggested that the binding strength
À
of these anions followed the order H2PO4À >FÀ @CH3CO2
>
HSO4À. This trend is the same for the absorption and emission
data. The conformational changes induced in Lamino upon inter-
action with anion were detected by NOESY experiments, and
the data supported changes to the capped region upon bind-
ing H2PO4À; this also extended to the triazole part for FÀ. The
Lamino sample exhibited a reversible ON/OFF response in the
Keywords: anions · calixarenes · sensors · structure–activity
relationships · supramolecular chemistry
À
presence of H2PO4 followed by Mg2+ for five cycles, and simi-
lar results were obtained for the addition of FÀ followed by
Ca2+. The ITC experiments provided evidence that the changes
in heat observed upon interaction with an anion followed
a trend that paralleled the binding strengths monitored from
the chemical shifts observed in the spectra. In SEM micro-
graphs of Lamino, spherical particles are transformed into spheri-
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Received: February 9, 2016
Published online on May 24, 2016
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