Imino-phenolic-pyridyl conjugates of calix[4]arene (L1 and L2) as primary fluorescence switch-on sensors for Zn2+ in solution and in HeLa cells and the recognition of pyrophosphate and ATP by [ZnL2]

Article abstract of DOI:10.1021/ic202426v

Pyridyl-based triazole-linked calix[4]arene conjugates, viz. L₁ and L₂, were synthesized and characterized. These two conjugates were shown to be selective and sensitive for Zn²⁺ among the 12 metal ions studied in HEPES buffer medium by fluorescence, absorption, and visual color change with the detection limit of ～31 and ～112 ppb, respectively, by L₁ and L₂. Moreover, the utility of the conjugates L ₁ and L₂ in showing the zinc recognition in live cells has also been demonstrated using HeLa cells as monitored by fluorescence imaging. The zinc complexes of L₁ and L₂ were isolated, and the structure of [ZnL₁] has been established by single-crystal XRD and that of [ZnL₂] by DFT calculations. TDDFT calculations were performed in order to demonstrate the electronic properties of receptors and their zinc complexes. The isolated zinc complexes, viz. [ZnL₁] and [ZnL ₂], have been used as molecular tools for the recognition of anions on the basis of their binding affinities toward Zn²⁺. [ZnL ₂] was found to be sensitive and selective toward phosphate-bearing ions and molecules and in particular to pyrophosphate (PPi) and ATP among the other 18 anions studied; however, [ZnL₁] was not sensitive toward any of the anions studied. The selectivity has been shown on the basis of the changes observed in the emission and absorption spectral studies through the removal of Zn²⁺ from [ZnL₂] by PPi. Thus, [ZnL ₂] has been shown to detect PPi up to 278 ± 10 ppb at pH 7.4 in aqueous methanolic (1/2 v/v) HEPES buffer.

Full text of DOI:10.1021/ic202426v

Article
pubs.acs.org/IC
Imino−Phenolic−Pyridyl Conjugates of Calix[4]arene (L1 and L2) as
Primary Fluorescence Switch-on Sensors for Zn2+ in Solution and in
HeLa Cells and the Recognition of Pyrophosphate and ATP by [ZnL2]
Rakesh Kumar Pathak,† Vijaya Kumar Hinge,‡ Ankit Rai,‡ Dulal Panda,‡ and Chebrolu Pulla Rao*,†,‡
‡
†Bioinorganic Laboratory & Department of Chemistry and Department of Biosciences & Bioengineering, Indian Institute of
Technology Bombay, Powai, Mumbai 400 076, India
S
* Supporting Information
ABSTRACT: Pyridyl-based triazole-linked calix[4]arene conjugates, viz. L1 and L2,
were synthesized and characterized. These two conjugates were shown to be selective
and sensitive for Zn2+ among the 12 metal ions studied in HEPES buffer medium by
fluorescence, absorption, and visual color change with the detection limit of ∼31 and
∼112 ppb, respectively, by L1 and L2. Moreover, the utility of the conjugates L1 and L2
in showing the zinc recognition in live cells has also been demonstrated using HeLa
cells as monitored by fluorescence imaging. The zinc complexes of L1 and L2 were
isolated, and the structure of [ZnL1] has been established by single-crystal XRD and
that of [ZnL2] by DFT calculations. TDDFT calculations were performed in order to
demonstrate the electronic properties of receptors and their zinc complexes. The
isolated zinc complexes, viz. [ZnL1] and [ZnL2], have been used as molecular tools for
the recognition of anions on the basis of their binding affinities toward Zn2+. [ZnL2]
was found to be sensitive and selective toward phosphate-bearing ions and molecules
and in particular to pyrophosphate (PPi) and ATP among the other 18 anions studied;
however, [ZnL1] was not sensitive toward any of the anions studied. The selectivity has been shown on the basis of the changes
observed in the emission and absorption spectral studies through the removal of Zn2+ from [ZnL2] by PPi. Thus, [ZnL2] has
been shown to detect PPi up to 278 10 ppb at pH 7.4 in aqueous methanolic (1/2 v/v) HEPES buffer.
systems such as calix[4]arene have emerged as molecular
scaffolds for building appropriate binding cores suitable for ion
and molecular recognition owing to their wide topological
features.10−13 Our research group has been involved in the
synthesis of various amide- and imine-based calix[4]arene
conjugates for the selective recognition of ions and molecules.14
We recently reported the selective detection of amino acids and
anions using calix[4]arene conjugates bearing transition-metal
ions.14 Therefore, it is of prime interest to develop calix[4]-
arene-based molecular systems to provide better sensitivity and
selectivity toward ions and molecules. Thus, in the present
paper, the synthesis and characterization of imino−phenolic−
pyridyl conjugates of calix[4]arene, L1 and L2, and their
selective recognition behavior toward Zn2+ have been reported.
These conjugates were also demonstrated as potential live-cell
fluorescence imaging agents upon treatment with Zn2+. The
differential recognition properties of the zinc complexes of
these receptors, viz., [ZnL1] and [ZnL2], have been explored
extensively using experimental and computational methods,
particularly to sense PPi and ATP selectively among common
biological phosphates.
INTRODUCTION
■
Zn2+ is one of the most abundant transition-metal ions present
in living cells, owing to its rich coordination chemistry.1 Pools
of labile Zn2+ are found in the cells of mammalian organs,
including brain,2 pancreas, and prostate.3 Metabolic disorders
of Zn2+ are responsible for various infections and diseases.2,4,5
Zn2+ is also associated with various anions, especially with
phosphates, by showing strong affinity.6 Phosphate-based
inorganic as well as organic molecules play fundamental roles
in a wide range of chemical and biological processes of human
life.6 Among the phosphates, adenosine triphosphate (ATP)
and inorganic pyrophosphate (PPi) have been of particular
interest because of their role in certain chemical and biological
processes such as energy storage, signal transduction, and DNA
sequencing. In the DNA polymerase chain reaction, PPi is
released; its detection is done as a real-time DNA sequencing
method.7 Biological fluids such as blood serum contain ∼0.80−
1.45 mM phosphates, and the higher phosphate levels are
responsible for cardiovascular disease and acute renal failure.8
Recently, metal ion complexes have been used as receptors for
phosphates, and this emerged as one of the most successful
strategies, since it provides specific metal ion−anion
interactions.9 Among the metal complex based receptors, the
ones that exhibit fluorescence enhancement in the presence of
Zn2+ have attracted considerable attention because of the
strong affinity of Zn2+ toward phosphates.10 Macrocyclic
Received: November 11, 2011
Published: April 20, 2012
© 2012 American Chemical Society
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Article
optimized with the B3LYP/6-31G(d,p) level and used for the single-
point energy calculations (Supporting Information, Figure S3).
Cellular Imaging Methodology. Human cervical carcinoma
(HeLa) cells were cultured in Eagle’s Minimal Essential Medium
(MEM) supplemented with 10% fetal bovine serum, 2.2 g/L sodium
bicarbonate, and 1% antibiotic-antimycotic solution containing
streptomycin, amphotericin B, and penicillin. Cells were maintained
under a humidified atmosphere of 5% CO2 and 95% air in a 37 °C
incubator. Stock solutions (10 mM) of the conjugates of calix[4]arene
were prepared in DMSO. HeLa cells were seeded at a density of 5 ×
104 cells/mL in polylysine-coated glass-bottom cell culture dishes.
After 24 h of seeding, MEM media was removed and the dishes were
washed twice carefully with PBS. Cells were incubated in the presence
of 10 μM (diluted in PBS) L1 and L2 separately for 20 min. Then, the
PBS containing some traces of ligand was removed and the dishes
were washed three times with PBS. At this stage, cells were imaged for
the intrinsic fluorescence of the conjugates using an Eclipse TE 2000
U microscope (Nikon, Tokyo, Japan) at 40× magnification. Further,
the cells were incubated with 10 μM ZnSO4/pyrithione diluted in PBS
buffer for 20 min. After this incubation, the PBS containing Zn2+ was
removed and the cells were washed three times with PBS.
Subsequently, different dishes were further incubated with 10 μM
(diluted in PBS) of L1 and L2 separately for 20 min. After the
incubation, the dishes were washed three times with PBS and the cells
were imaged using an Eclipse TE 2000U microscope at 40×
magnification. The exposure time was kept the same for all the
cases. The images were processed using Image-Pro Plus software
(Media Cybernetics, Silver Spring, MD).
EXPERIMENTAL SECTION
■
General Information and Materials. H and 13C NMR spectra
were measured on a Varian Mercury NMR spectrometer working at
400 MHz. The mass spectra were recorded on a Q-TOF Micromass
instrument (YA-105) using electrospray ionization methods. Steady-
state fluorescence spectra were measured on a Perkin-Elmer LS55
instrument. The absorption spectra were measured on a Varian Cary
100 Bio instrument. The diffraction data were collected on an Oxford
Diffraction XCALIBUR-S diffractometer. All the perchlorate salts, viz.,
NaClO4·H2O, KClO4, Ca(ClO4)2·4H2O, Mg(ClO4)2·6H2O, Mn-
(ClO4)2·6H2O, Fe(ClO4)2·xH2O, Co(ClO4)2·6H2O, Ni-
(ClO4)2·6H2O, Cu(ClO4)2.·H2O, Zn(ClO4)2·6H2O, Cd(ClO4)2·H2O,
Hg(ClO4)2·xH2O, and Zn(OAc)2·2H2O, were procured from Sigma
Aldrich Chemical Co. (USA). Salts of different anions, viz., Bu4NF,
Bu4NCl, Bu4NBr, Bu4NI, Bu4NClO4, Bu4NH2PO4, Bu4NHSO4,
Na4P2O7, NaSCN, Na2SO4, Na2CO3, NaNO3, NaNO2, NaN3, AMP,
ADP, and ATP, were purchased from Sisco Research Laboratories Pvt
Ltd. (India).
1
Bulk solutions of L1 and L2 and the metal salts were made up in
MeOH, at 6 × 10−4 M concentration. The fluorescence studies
performed in aqueous methanolic (1/2 v/v) HEPES buffer, pH 7.4
solution, always used a 50 μL amount of the bulk solution of L1 or L2
prepared at 6 × 10−4 M concentration in methanol. A 50 mM HEPES
buffer stock solution was prepared with deionized water, and 1000 μL
of this bulk solution was used for each titration of 3 mL of solution.
During the metal ion titrations, the concentrations of metal
perchlorates were varied accordingly to result in requisite mole ratios
of metal ion to receptors, and the total volume of the solution was
maintained at 3 mL in each case by the addition of CH3OH and
HEPES buffer. For anion titrations, the [ZnL1] and [ZnL2] were
initially dissolved in the minimum amount of CHCl3 (100 μL in 2 mL
of bulk) and the final volumes were made up to 2 mL by adding
methanol in order to get a final concentration of 6 × 10−4 M. Stock
solutions of AMP, ADP, ATP, and PPi at 6 × 10−4 M concentration
were made up in the HEPES buffer medium. During the anion
titrations, the concentrations of anions were varied accordingly to
result in requisite mole ratios of anions to zinc complexes, and the
total volume of the solution was maintained at 3 mL in each case by
the addition of appropriate volumes of CH3OH and HEPES buffer
solution. For absorption studies, the final concentrations of L1 and L2
were kept constant at 20 μM, and the procedure used for the titrations
was the same as that used for fluorescence titrations. All the solvents
used were procured from local sources and were dried and distilled by
the usual procedures immediately before use. Distilled and deionized
water was used for the studies. Quantum yields for the calix[4]arene
conjugates, viz., L1 and L2, were measured in the absence and in the
presence of Zn2+ using quinine sulfate as standard. Fluorescence and
absorption titrations have also been carried out between these
calix[4]arene conjugates, viz., L1 and L2 and zinc acetate (Supporting
Information, Figures S1 and S2).
Synthesis and Characterization of 1−3. Detailed information
about the synthesis and characterization of compounds 1−3 is given in
Supporting Information.
Synthesis and Characterization of 4.14e A mixture of potassium
carbonate (5.10 g, 36.72 mmol) and p-tert-butylcalix[4]arene (10.0 g,
15.43 mmol) in dry acetone (200 mL) was stirred at room
temperature for 1 h. A solution of propargyl bromide (6.49 g, 30.80
mmol) in dry acetone (50 mL) was added dropwise into this stirred
mixture over a period of 30 min. The reaction mixture was refluxed for
24 h and was then cooled to room temperature. The reaction mixture
was filtered over Celite to remove insoluble particles, and the filtrate
was concentrated by a rotary evaporator. A 100 mL portion of 2 M
HCl was added to the concentrated reaction mixture, and the product
was extracted with dichloromethane (3 × 100 mL). The combined
organic extracts were successively washed with water and brine (100
mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness
in vacuo. The crude product was recrystallized from CH2Cl2/CH3OH
1
to afford 4 as a white solid (9.10 g, 82% yield). H NMR (400 MHz,
CDCl3): δ (ppm) 7.07 (s, 4H, Ar-H), 6.73 (s, 4H, Ar-H), 6.50 (s, 2H,
OH), 4.74 (d, J = 2.4 Hz, 4H, OCH2), 4.37 (d, J = 13.4 Hz, 4H,
ArCH2Ar), 3.33 (d, J = 13.4 Hz, 4H, ArCH2Ar), 2.54 (t, J = 2.4 Hz,
2H, C≡CH), 1.30 (s, 18H, (CH3)3), 0.90 (s, 18H, (CH3)3). 13C NMR
(CDCl3, 100 MHz): δ (ppm) 150.51, 149.62, 147.40, 141.77, 132.74,
128.16, 125.70, 125.19, 78.93, 63.45, 34.05, 34.02, 32.19, 31.88, 31.11.
HRMS: m/z calcd for C50H60O4 (M + H) 725.4570, found 725.4576.
Synthesis and Characterization of 5. Compound 4 (4.0 g, 5.52
mmol) was added to a solution of 3 (2.83 g, 12.70 mmol) in 50 mL of
a dichloromethane and water (50/50) mixture. To this solution were
added CuSO4·5H2O (165.38 g, 0.662 mmol) and sodium ascorbate
(437.4 mg, 22.08 mmol). The resulting solution was stirred for 12 h at
room temperature. Upon completion of the reaction, as checked on
the basis of TLC, the organic layer was separated and the aqueous
layer was extracted with dichloromethane (2 × 50 mL). The combined
organic layer was washed with water and then with brine (2 × 50 mL)
and dried over anhydrous Na2SO4, and the solvent was removed in
vacuo. The crude product was purified by triturating with hexane
followed by filtering the precipitate. Yield: 81%. 1H NMR (CDCl3, 400
MHz): δ (ppm) 11.30 (s, 2H, Sal-OH), 9.83 (s, 2H, sal-CHO), 8.08
(s, 2H, triazole-H), 7.62 (s, 2H, Sal-H) 7.49 (d, 2H, Sal-H), 7.15 (s,
2H, Ar-OH), 6.98 (s, 4H, Ar-H), 6.77 (s, 4H, Ar-H), 5.56 (s, 4H, Sal-
CH2), 5.18 (s, 4H, Ar-O-CH2), 4.14 (d, J = 13.0 Hz, 4H, ArCH2Ar),
3.17 (d, J = 13.0 Hz, 4H, ArCH2Ar), 1.27 (s, 18H, Ar-(CH3)3), 1.26 (s,
Computational Methodology. In the DFT calculations,15 the
initial model for L2 was prepared from the crystal structure of the
complex [ZnL1] upon using the following modifications: (a) removal
of Zn2+, (b) protonation of the salicyl OH moieties, and (c)
introduction of one more −CH2 group to the pyridyl moiety in each of
the arms. This has been optimized by going through a cascade process
starting from PM3 → HF/STO-3G → HF/3-21G → HF/6-31G →
B3LYP/3-21G → B3LYP/6-31G. The output obtained at every stage
has been given as the input for the next higher level of calculations. In
order to create the neutral complex with Zn2+, the salicyl OH groups
were deprotonated and the resulting L22− was used for further studies.
2−
The complex of L2 with Zn2+ was made by simply placing the Zn2+
2−
far away from the binding arms of the DFT optimized L2 in such a
way that the salicyl O and imine N atoms are pointed toward Zn2+. To
understand the electronic properties of L1, L2, and their Zn2+
complexes, TDDFT studies were carried out.16 Single-point energies
of the complexes formed were calculated using the formula ΔEs =
E[Zn (L)] − (EL + EZn2+). In addition, the complex of PPi with Zn2+ was
2+
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Article
a
Scheme 1. Synthesis of the Precursors and the Final Receptor Molecules, L1 and L2
a
Synthesis of L1 and L2: (a) propargyl bromide, K2CO3, acetone, reflux, 24 h; (b) 5-tert-butyl-3-(azidomethyl)-2-hydroxybenzaldehyde (3),
CuSO4·5H2O, and sodium ascorbate in dichloromethane/water (1/1), room temperature, 12 h; (c) pyridin-2-ylmethanamine or 2-(pyridin-2-
yl)ethanamine, methanol, room temperature, 8 h; (d) SnCl4, Bu3N, OH(CH2O)nH, dry toluene, reflux; (e) 37% formaldehyde, concentrated HCl,
room temperature, 48 h; (f) NaN3, DMF, room temperature, 12 h.
18H, Ar-(CH3)3), 0.96 (s, 18H, Sal-(CH3)3). 13C NMR (CDCl3, 100
MHz): δ (ppm) 196.6, 157.1, 150.4, 149.6, 147.2, 144.2, 143.2, 141.5,
135.3, 132.6, 130.7, 127.8, 125.6, 125.0, 124.2, 123.1, 120.2, 69.8, 48.2,
34.2, 33.9, 33.8, 31.7, 31.2, 31.1, 31.02. IR: ν 3463, 2959, 1656, 1483
cm−1. HRMS (ESI): m/z calcd for C74H90N6O8 [M + H]+ 1191.6898,
found 1191.6898.
solution of Zn(CH3COO)2·2H2O (0.026 g, 0.120 mmol, 5 mL), and
the resulting reaction mixture was refluxed for 5 h. After the solution
was concentrated, a light yellow precipitate started to form and the
solid that formed was then filtered, washed with cold methanol, and
1
dried in vacuo to give the desired product, [ZnL1]. Yield: 92%, H
NMR (CDCl3, 400 MHz): δ (ppm) 9.23 (s, 2H, imine-H), 8.04 (s,
2H, triazole-H), 7.9 (d, 2H, Py-H), 7.70 (s, 2H, Ar-OH), 7.61 (broad
d, 2H, Sal-H), 7.36 (t, 2H, Py-H), 6.83−6.9 (m, 10H, Ar-H, Py-H),
6.60 (dd, 4H, Sal-H, Py-H), 5.6 (s, 4H, Ar-OCH2), 5.12 (d, 2H, Sal-
CH2), 5.02 (d, 2H, Py-CH2), 4.33 (d, 2H, Sal-CH2), 4.15 (d, 2H, Py-
CH2), 3.55 (d, 2H, Ar-CH2-Ar), 3.44 (d, 2H, Ar-CH2-Ar), 2.78 (d, 2H,
Ar-CH2-Ar), 2.31(d, 2H, Ar-CH2-Ar), 1.36 (s, 18H, Ar-C(CH3)3),
1.19 (S, 18H, Sal-(CH3)3), 1.02 (s, 18H, Ar-C(CH3)3). 13C NMR
(CDCl3, 100 MHz): δ (ppm) 170.95, 167.84, 156.05, 150.45, 148.46,
148.40, 147.31, 143.08, 141.32, 137.42, 134.96, 133.72, 133.36, 133.23,
132.70, 127.92, 127.65, 127.47, 126.91, 126.19, 125.48, 125.07, 124.67,
122.73, 121.90, 117.51, 69.65, 61.76, 51.98, 34.10, 33.86, 33.81, 31.88,
31.71, 31.54, 31.17. HRMS: m/z calcd for C86H100N10O6Zn
1433.7197, found 1433.7190. Crystal data for [ZnL1]: empirical
formula C88H96N10O9Zn; formula weight 1503.12; temperature
150(2) K; radiation Mo Kα; wavelength/Å 0.710 73; crystal system
Synthesis and Characterization of L1. A mixture of 5 (1.0 g,
0.839 mmol) and pyridin-2-ylmethanamine (0.182 g, 1.68 mmol) in
methanol was stirred for 8 h, which gives rise to a yellow precipitate.
The precipitate was filtered under vacuum to get a yellow solid, which
was further recrystallized using methanol to get pure solid product L1.
Yield: 83%, 1H NMR (CDCl3, 400 MHz): δ (ppm) 13.6 (broad s, 2H,
Sal-OH), 8.54 (m, 2H, Py-H, J = 4.88 Hz), 8.5 (s, 2H, imine-H), 8.0
(s, 2H, triazole-H), 7.60 (t, 2H, Py-H, J = 7.9 Hz), 7.42 (t, 2H, Py-H),
7.25−7.27 (m, 4H, Sal-H), 7.16 (t, 2H, Py-H), 7.16 (s, 2H, Ar-OH),
6.95 (s, 4H, Ar-H), 6.7 (s, 4H, Ar-H), 5.6 (s, 4H, Ar-OCH2), 5.1 (s,
4H, Sal-CH2), 4.85(s, 4H, Py-CH2), 4.12 (d, 4H, Ar-CH2-Ar, J = 13.1
Hz), 3.12 (d, 4H, Ar-CH2-Ar, J = 13.1 Hz), 1.26 (s, 18H, Ar-
C(CH3)3), 1.24 (s, 18H, Sal-(CH3)3), 0.92 (s, 18H, Ar-C(CH3)3). 13
C
NMR (CDCl3, 100 MHz): δ (ppm) 166.93, 157.75, 157.13, 150.5,
149.6, 149.4, 147.0, 144.0, 141.62, 141.44, 137.0, 132.60, 130.82,
128.98, 127.83, 125.60, 125.0, 124.23, 122.44, 122.26, 122.0, 118.23,
69.73, 64.80, 48.90, 34.04, 33.91, 33.83, 31.73, 31.38, 31.00. ES/MS:
m/z 1371.5 ([M]+, 100%). HRMS: m/z calcd for C86H102N10O6
1371.8062, found 1371.8115.
triclinic, P1; unit cell dimensions a = 11.0065(3) Å, b = 16.4816(7) Å,
̅
and c = 24.5981(10) Å; α = 106.453(4)°, β = 90.525(3)°, γ =
93.464(3)°; V = 4270.1(3) Å3; Z = 2; Dcalc= 1.169 g/cm3; μ = 0.348
mm−1; F(000) = 1592; θ = 3.33, 25.0°(min, max); no. of unique
Synthesis and Characterization of L2. L2 was prepared by using
reflections 14 984; no. of parameters 991; Robs = 0.0873, wR2obs
=
the procedure given for L1, but with 2-(pyridin-2-yl)ethanamine. Yield:
1
0.2147; GOF = 1.005.
Synthesis and Characterization of [ZnL2]. This complex has
89%. H NMR (DMSO-d6, 400 MHz): δ (ppm) 13.9 (broad s, 2H,
Sal-OH), 8.53 (s, 2H, imine-H), 8.48 (d, 2H, Py-H, J = 4.8 Hz), 8.0 (s,
2H, triazole-H), 7.9 (s, 2H, Ar-OH), 7.7 (t, 2H, Py-H), 7.42 (d, 2H,
Sal-H), 7.37 (d, 2H, Sal-H), 7.23−7.17 (m, 4H, Py-H), 7.0 (s, 4H, Ar-
H), 6.9 (s, 4H, Ar-H), 5.60 (s, 4H, Ar-OCH2), 5.0 (s, 4H, Sal-CH2),
3.98 (d, 4H, Ar-CH2-Ar, J = 12.8 Hz), 3.90 (t, 4H, N-CH2-Py), 3.17
(d, 4H, Ar-CH2-Ar, J = 12.8 Hz), 3.04 (t, 4H, N-CH2-Py), 1.18 (s, 18
H, Ar-C(CH3)3), 1.14 (s, 18 H, Sal-(CH3)3), 1.05 (s, 18H, Ar-
C(CH3)3). 13C NMR (CDCl3, 100 MHz): δ (ppm) 165.40, 158.95,
157.47, 150.50, 149.63, 149.47, 147.00, 143.88, 141.43, 141.21, 136.49,
132.56, 130.51, 128.52, 127.84, 125.57, 125.00, 124.22, 123.78, 122.45,
121.56, 118.05, 69.77, 58.66, 48.92, 39.33, 33.99, 33.91, 33.81, 31.72,
31.37, 31.0. ES/MS: m/z 1400.13 ([M + 1]+, 100%). HRMS: m/z
calcd for C88H106N10O6 1399.8381, found 1399.8375.
been synthesized by adapting the same procedure that was used for
1
[ZnL1] but using L2 instead of L1. Yield: 86%. H NMR (DMSO-d6,
400 MHz): δ (ppm) 8.7 (s, 2H, imine-H), 8.4 (d, 2H, Py-H, J = 4.8
Hz), 8.23 (s, 2H, Ar-OH), 8.13 (s, 2H, triazole-H), 7.7 (t, 2H, Py-H),
7.35 (d, 2H, Sal-H), 7.16 (t, 2H, Py-H), 6.9−7.0 (m, 12H, PyH, Sal-H,
Ar-H), 5.66 (broad d, 4H, Ar-OCH2), 4.95 (s, 4H, Sal-CH2), 3.91
(broad d, 8H, Ar-CH2-Ar, Py-NCH2, J = 12.8 Hz), 3.67 (broad d, 8H,
Ar-CH2-Ar, Py-NCH2, J = 12.8 Hz), 1.20 (s, 18 H, Ar-C(CH3)3), 1.16
(s, 18 H, Sal-(CH3)3), 1.09 (s, 18H, Ar-C(CH3)3). 13C NMR (CDCl3,
100 MHz): δ (ppm) 171.45, 166.20, 158.10, 150.35, 149.33, 149.08,
147.10, 143.98, 141.52, 137.01, 136.42, 132.99, 132.87, 132.45, 132.27,
127.99, 127.60, 127.47, 125.63, 125.14, 124.99, 123.67, 121.87, 117.55,
69.79, 59.57, 50.44, 38.27, 33.95, 33.82, 33.70, 31.74, 31.40, 31.01.
HRMS: m/z calcd for C88H104N10O6Zn 1461.7498, found 1461.7510.
Synthesis and Characterization of [ZnL1]. To a solution of L1
(0.150 g, 0.109 mmol) in CHCl3 (6 mL) was added a methanolic
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Figure 1. Fluorescence titration of L1 and L2 by metal ions. (a) Fluorescence spectra obtained during the titration of L1 with Zn2+ in aqueous
methanolic (1/2 v/v) HEPES buffer (pH 7.4), λex 390 nm. The inset shows the relative fluorescence intensity (I/I0) as a function of [Zn2+]/[L1 or
L2] mole ratio. (b) Histogram showing the fluorescence response of various metal ions with L1 (gray bars) and L2 (bars with hash marks) and the
vials exhibiting visual color changes of L1 and L2 in the presence of these ions under 365 nm incident light.
connected calix[4]arene conjugates bind to Zn2+, these
RESULTS AND DISCUSSION
■
processes are inhibited to result in fluorescence enhancement.18
Imino−phenolic−pyridyl conjugates of calix[4]arene, L1 and
The binding of Zn2+ to these conjugates has also been
L2, were synthesized in three consecutive steps starting from p-
tert-butylcalix[4]arene followed by the preparation of its
dipropargyl ether and the triazole-linked conjugate, as shown
delineated from other spectral methods, as reported in this
paper. Even the chelation by Zn2+ to the imine N, phenolic O,
and pyridyl N brings rigidity to the conjugates and results in
chelation-enhanced fluorescence (CHEF). During the chelation
in Scheme 1. The precursor azide derivative 3 has been
synthesized in three steps14i,17 (Supporting Information)
process, the phenolic −OH from the salicyaldimine moiety is
starting from p-tert-butylphenol (Scheme 1). The receptor
deprotonated and forms a six-membered chelate ring. The
molecules L1 and L2 have been synthesized by the
condensation of 5 with pyridin-2-ylmethanamine and 2-
(pyridin-2-yl)ethanamine, respectively. The zinc complexes of
these conjugates have been isolated in quantitative yield by
reacting L1 or L2 with zinc acetate in methanol, as detailed in
the Experimental Section. All the precursor and receptor
molecules were characterized by various analytical and spectral
techniques. The conjugates of calix[4]arene were found to be in
binding affinities of Zn2+ toward L1 and L2 have been calculated
from the Benesi−Hildebrand equation and found to have
association constants of [9.3( 0.11)] × 104 and [7.2( 0.09)]
× 104 M−1, respectively. A 30% increase observed in the
association constant in the case of L1 as compared to L2 speaks
for the stronger binding of Zn2+ with L1, as explained on the
basis of the coordination cores later in this paper.
In order to check whether L1 and L2 are sensitive to only
a cone conformation on the basis of NMR studies
(Experimental Section and Supporting Information, Figures
Zn2+ or even to the other ions, fluorescence titrations were
carried out in the same medium with 12 different metal ions,
S4−S9). The molecular structure of the zinc complex of
viz. Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Cd2+,
receptor L1 has been established on the basis of single-crystal
XRD data, and the structure of [ZnL2] has been optimized
and Hg2+, and no significant fluorescence enhancement was
found in the presence of these ions (Figure 1b). Minimum
detectable concentrations of ∼31 and ∼112 ppb of Zn2+ for L1
and L2, respectively, has been detected by the fluorescence
titrations carried out by keeping the receptor to Zn2+ mole ratio
as 1/1 (Supporting Information, Figure S10). Therefore, L1 and
L2 can be used for the selective recognition of Zn2+ among the
12 different metal ions studied. The titrations carried out at
different pHs of the medium exhibited no variation in the
fluorescence intensity in the pH range 6−10. However, at
highly acidic and basic pH, the fluorescence of these conjugates
and their Zn2+ complexes shows a decreas,e indicating the
instability of the complex under these conditions (Supporting
Information, Figure S11). The quantum yields of L1 and L2
were found to be 0.024 and 0.020, respectively, and those of
their Zn2+ complexes show higher ϕ values of 0.190 and 0.165,
respectively. The observed 8-fold increase in the quantum yield
of the Zn2+-bound species, the presence of water and buffer in
the medium of measurements, and the observed low detection
limits seem to qualify L1 and L2 for sensing as well as
quantifying Zn2+ by switch-on fluorescence at physiological pH.
Under UV light, the solutions of L1 and L2 are nonfluorescent,
whereas in the presence of Zn2+ an intense blue fluorescent
using DFT calculations.
Zn2+ Recognition. The sensitivity of L1 and L2 toward
different metal ions and their preferential selectivity toward
Zn2+ over the other ions has been studied by fluorescence and
absorption titrations.
Fluorescence Titrations of L1 and L2 by Metal Ions.
The receptors L1 and L2 exhibit very weak emission at ∼455
nm when excited at 390 nm in 1/2 water/methanol mixture at
pH 7.4 to have an effective HEPES buffer concentration of 10
mM. Titration of L1 or L2 with Zn2+ results in the enhancement
of fluorescence intensity as a function of the added Zn2+
concentration (Figure 1a). A plot of fluorescence intensity as
a function of added [Zn2+]/[L] mole ratio (Figure 1a, inset)
shows a stoichiometry of 1/1 between the receptor and Zn2+
and the intensity saturates at ≥1 equiv. The observed
fluorescence enhancement may be viewed as follows. The
calix[4]arene conjugates, viz., L1 and L2, exhibit low
fluorescence owing to the isomerization of the imine (CN
bond) as well as to the excited-state proton transfer (ESPT)
from salicyl −OH to the imine nitrogen in the excited state,
which has been well documented in the literature in the case of
Schiff base molecular systems. When such Schiff base
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color was observed (Figure 1b, bottom) and no such
fluorescent color was observed in case of the other metal
ions. Thus, Zn2+ can easily be differentiated by its fluorescent
color change from the other metal ions.
The selectivity of Zn2+ has been studied by carrying out
appropriate competitive metal ion titrations in the presence of
alkali and alkaline-earth ions (being major components of
biological fluids) and also Cd2+ and Hg2+ (being environ-
mentally toxic), and no significant change was found in the
fluorescence enhancement when Zn2+ interacted with L1 and L2
in the presence of different ions. The marginal fluorescence
quenching observed in the presence of Cd2+ and Hg2+ may be
considered as a heavy atom effect upon interaction with the
excited species. However, the fluorescence of these is
completely quenched in the presence of Cu2+. Thus, L can
be used for the selective recognition of Zn2+ in the presence of
most of the metal ions except Cu2+ (Supporting Information,
Figure S12).
Figure 3. Molecular ion peak observed in ESI MS with its isotopic
peak pattern: (a) [ZnL1]; (b) [ZnL2].
bridging and arm CH2 protons showed minimal to marginal
changes in their δ values. The absence of any changes observed
in the chemical shift of the triazole proton rules out interactions
between it and Zn2+. Considerable shifts observed in the Py
CH2 and Py H protons supports the binding of the pyridyl
moiety. All these changes are suggestive of the complex
formation of L1 and L2 with Zn2+ by utilizing both of its
chelating arms.
Structures of the Zinc Complexes. While the structure of
[ZnL1] could be established crystallographically owing to the
availability of nicely diffracting single crystals, the same had to
be established by DFT computational methods in the case of
[ZnL2], as no single crystals could be obtained.
Absorption Titration of L1 and L2 by Zn2+. The
absorption spectra of free L1 and L2 in aqueous methanolic
(1/2 v/v) HEPES buffer (pH 7.4) exhibited different bands
from 200 to 450 nm. Upon the addition of Zn2+, a new band
was observed at ∼385 nm in case of L1 and at 375 nm in case of
L2, and their absorbance increases. The absorbance of the other
two bands in the case of L1 (∼330 and ∼440 nm) and L2 (325
and 425 nm) decreases. The isosbestic points observed in the
cases of L1 (345 and 425 nm) and L2 (340 and 415 nm) were
indicative of the transition between the free and the complexed
species (Figure 2). The stoichiometry of the complex formed
Single-Crystal XRD Structure of [ZnL1]. Single crystals of
[ZnL1] suitable for X-ray diffraction were obtained by slow
diffusion of methanol into a solution of [ZnL1] in chloroform.
It crystallizes in the triclinic system with space group P1. The
̅
details of the data collection and the structure refinement are
given in the Experimental Section. The calix[4]arene unit
adopts a cone conformation by forming intramolecular O−
H···O hydrogen bonds at the lower rim. In the crystal structure
of [ZnL1], it is observed that one of the coordinating arms is
bent toward the calix[4]arene cavity and the other arm projects
from the top by forming an N3O2 core to coordinate Zn2+
(Figure 5). The structure shows that Zn2+ has a square-
pyramidal geometry formed by using two phenolic oxygens,
two imine nitrogens of both the arms, and one pyridyl nitrogen
from one of the arms. In the lattice of [ZnL1], the two
calix[4]arene units form a dimerlike structure at the lower rim
through weak hydrogen bonding of the types O−H···N and
O−H···O, mediated via two water molecules (Figure 6),
wherein the two Zn2+ centers are separated by 7.55 Å. Further,
these dimers were again stacked in its lattice and exhibit both
hydrophobic and hydrophilic columnar stacked patterns formed
by the tert-butyl part of the calix[4]arene and the lower rim of
the zinc binding portion, respectively (Supporting Information,
Figures S14 and S15, Tables S1 and S2).
Structure of [ZnL2] by Computational Modeling.
Computational calculations were carried out using the Gaussian
03 package15 in order to establish the complexation features of
L2 by Zn2+. In the optimized structure of [ZnL2] (Figure 7 and
Supporting Information, Tables S3 and S4), the Zn2+ was found
in an N3O2 core as was observed in the case of [ZnL1], where
the second pyridyl nitrogen group is disposed at a distance of
5.65 Å, but with a greater distortion in the geometry. The
comparison of the metric data of the coordination spheres of
both complexes clearly supports this through an elongated
Figure 2. Absorption titration of receptors by Zn2+ in aqueous
methanol (1/2 v/v) HEPES buffer (pH 7.4): (a) spectra obtained for
L1; (b) spectra obtained for L2. The insets show the plot of absorbance
vs [Zn2+]/[L] for different absorption bands for both (a) L1 and (b)
L2.
between the receptors and Zn2+ has been derived to be 1/1 on
the basis of a Jobs plot (Supporting Information, Figure S13).
The binding affinities of Zn2+ toward L1 and L2 have also been
calculated from the Benesi−Hildebrand equation using
absorption data and found to have the association constants
of [6.8( 0.5)] × 104 and [3.7( 0.17)] × 104 M−1, respectively.
Electrospray Mass Spectrometry. ESI mass spectra
obtained for the isolated complexes of L1 and L2 with Zn2+
resulted in molecular ion peaks at m/z 1433.72 and 1461.75,
respectively (Supporting Information, Figures S8 and S9). The
isotopic peak patterns observed for these peaks are character-
istic of the presence of zinc, thus supporting the complex
formation of Zn2+ with L1 and L2 (Figure 3).
1
1H NMR Studies. H NMR spectral measurements of L1
and L2 and their Zn2+ complexes were carried out (Figure 4 and
Supporting Information, Figures S6−S9). Upon the interaction
of L1 or L2 with Zn2+, the imine H, phenolic OH, Py H, and
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1
1
●
■
▲
,
△
Figure 4. (a) H NMR spectra of L1 and [ZnL1]. (b) H NMR spectra of L2 and [ZnL2]. Legend: ( ) imine-H; (⬡,
) Py H; (★) CH2
⧫
○
connecting to triazole with aldimine; ( ) CH2 connecting to calix[4]arene with triazole; ( ) Py CH2; ( ) calix[4]arene bridging CH2; (☆) Calix
OH; (⬢) triazole H.
Figure 6. Dimer of [ZnL1] formed in the lattice via intermolecular
hydrogen bonding mediated through water molecules. W = water
oxygen.
its homologue [ZnL1], with a major change being reflected in
lengthening the PyN−Zn bond in the case of the former
(Figure 8 and Supporting Information, Tables S3−S7). This
has further resulted in the lowering of crowding by the pyridyl
moiety at the Zn2+ center in [ZnL2]. Only marginal differences
are observed in the dihedral angles (Supporting Information,
Figure S16 and Table S8) of the arms on going from [ZnL1] to
[ZnL2], while the arms move farther apart in comparison to
their free ligands in order to accommodate the Zn2+ in the
binding core formed by the two salicylimine, two phenolic, and
one pyridyl moiety, as was also observed in the crystal structure
of [ZnL1]. All of this can be reconciled by the higher Kassoc
value observed with [ZnL1] as compared to that of [ZnL2].
Computational Studies of [ZnL1] and [ZnL2] by Time-
Dependent Density Functional Theory. TDDFT calcu-
lations were performed to explain the electronic properties of
these complexes in their ground and excited states. The vertical
transitions calculated by TDDFT (Supporting Information,
Tables S9 and S10) were compared with the solid-state UV−vis
diffuse reflectance spectra of [ZnL1] and [ZnL2] and were
found to have good agreement with the experimental data.16
The TDDFT calculations performed at the B3LYP/6-31G(d,p)
level of theory revealed the absorption bands in the region of
Figure 5. ORTEP diagram of [ZnL1] drawn with 50% probability
ellipsoids. The inset shows the coordination sphere around the zinc
ion. Hydrogen atoms are omitted for clarity. Bond lengths (Å) and
bond angles (deg) in the coordination sphere: Zn−N6 = 2.130(5),
Zn−N7 = 2.071(5), Zn−N4 = 2.049(4), Zn−O5 = 1.989(3), Zn−O6 =
1.985(5); O5−Zn−O6 = 92.24(17), O5−Zn−N6 = 93.37(17), O5−
Zn−N7 = 158.50(16), O5−Zn−N4 = 94.06(16), O6−Zn−N6
=
157.52(17), O6−Zn−N7 = 89.44(19), O6−Zn−N4 = 101.44(19),
N6−Zn−N7 = 77.60(19), N6−Zn−N4 = 99.86(18), N7−Zn−N4
106.62(18).
=
PyN−Zn bond observed in the case of [ZnL2] as compared to
that in [ZnL1].
Binding Core Comparison and Conformational
Changes Observed in the Arms. A careful examination of
the binding cores of both the structures reveals that the Zn2+
center is more distorted in the case of [ZnL2] as compared to
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of the arms (Figure 9, Supporting Information, Table S9 and
S10). Corresponding molecular orbitals (MOs) of the two one-
electron excitations are shown in Figure 10 and the Supporting
Figure 7. B3LYP/6-31G(d,p) optimized structure of [ZnL2]. The
inset shows the coordination sphere around the zinc ion. Bond lengths
(Å) and bond angles (deg) in the coordination sphere: Zn−N1 = 2.35,
Zn−N2 = 2.07, Zn−N3 = 2.05, Zn−O1 = 2.00, Zn−O2 = 1.98; O1−
Zn−O2 = 87.21, O1−Zn−N1 = 141.60, O1−Zn−N2 = 88.29, O1−Zn−
N3 = 112.42, O2−Zn−N1 = 86.00, O2−Zn−N2 = 161.00, O2−Zn−N3
= 93.42, N1−Zn−N2 = 86.09, N1−Zn−N3 = 105.68, N2−Zn−N3 =
105.34.
Figure 10. Pictorial representation of the molecular orbitals (MOs)
calculated for the singlet vertical electronic transitions A−E (given in
the Supporting Information, Table S9) using TDDFT for [ZnL1].
Contributions of key transition MOs are given above the arrows as
percentages.
Figure 8. Comparison of the binding cores around zinc in [ZnL1] and
[ZnL2]: (a) overlap of the binding core of the structures of [ZnL1]
(gray) and [ZnL2] (pink); (b) schematic diagram of the binding core.
λmax 270−400 nm (Figure 9). The studies suggest that the
vertical transitions observed at ∼369 and ∼354 nm are
comparable with those from experimental data for [ZnL1]
and [ZnL2], respectively. In both cases, the highest observed
oscillator strengths (F) correspond to the experimental λmax (at
∼369 and ∼354 nm), which is assigned to the transition A that
results from π → π* of the salicylimine and pyridyl fragments
Information (Figure S17). These MOs are mostly localized on
the salicylimine and pyridyl parts of [ZnL1] and [ZnL2], and
the percentages of localization are given in the Supporting
Information (Table S9 and S10). Similar studies have also been
carried out for L1 and L2, and the corresponding electronic
transition data have been given in the Supporting Information
(Figures S18 and S19 and Tables S11 and S12).
Figure 9. Normalized TDDFT singlet monoelectronic vertical transitions and normalized diffuse reflectance spectra: (a) [ZnL1]; (b) [ZnL2].
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Figure 11. Fluorescence images of L2 with Zn2+ in HeLa cells (excitation at ∼358 nm and emission at ∼461 nm) in PBS buffer: (a) differential
interference contrast microscopy (DIC) image of the HeLa cells treated with L2 (10 μM), PBS buffer pH 7.4; (b) fluorescence image of the same
case as (a); (c) merged image of (a) and (b); (d) DIC microscopy image of the HeLa cells treated with L2 followed by 10 μM of Zn2+/pyrithione
(1/1) solution; (e) fluorescence image of the same case as (d); (f) merged image of (d) and (e). Each scale bar is 10 μm.
Intracellular Zn2+ Sensing by Fluorescence Micros-
copy. In order to demonstrate the utility of these amphiphilic
calix[4]arene conjugates, viz., L1 and L2, in the recognition of
Zn2+ in biological systems, HeLa cells were used as models
(Experimental Section). HeLa cells were incubated in PBS
buffer (pH 7.4) containing 10 μM of the conjugate (L1 or L2)
for 20 min at 37 °C, followed by washing the cells with the
same buffer to remove the excess of the conjugates. At this
stage, the fluorescence microscopy image of HeLa cells
displayed weak intracellular fluorescence. However, upon the
addition of exogenous Zn2+ into the cells via incubation with
ZnSO4/pyrithione solution for 20 min at 37 °C, the cells
exhibited highly intense blue fluorescence (Figure 11 and
Supporting Information, Figure S20). Differential interference
contrast microscopy (DIC) measurements for each experiment
confirm that the cells are viable throughout the imaging
experiments and the merged images support that the
fluorescence is exhibited through the cells (Figure 11 and
Supporting Information, Figure S20). The control experiments
carried out with ZnSO4/pyrithione solution alone do not show
any fluorescence (Supporting Information, Figure S21).
Thus, HeLa cells incubated with L1 and L2 in the presence of
Zn2+ showed much greater fluorescence emission as compared
to the cells which were not incubated with this, suggesting that
Zn2+ is responsible for enhancing the fluorescence of L1 and L2
in the cells, as it has also been shown to exhibit fluorescence
enhancement in the solution medium (Figure 1). These results
clearly indicate that the imino−phenolic−pyridyl conjugates of
calix[4]arene, viz., L1 and L2, are effective intracellular Zn2+
imaging agents with cell permeability. To the best of our
knowledge, this is the first known calix[4]arene-based conjugate
in the literature which has been demonstrated to recognize
Zn2+ in live cells.
out by titrating them with different anions, viz., F−, Cl−, Br−, I−,
N3 , CO32−, NO2 , NO3 , SCN−, SO42−, ClO4 , HSO4 ,
−
−
−
−
−
−
−
4−
HCO3 , H2PO4 , and P2O7 (PPi). The [ZnL1], which is
deprived of one methylene moiety in the arm as compared to
[ZnL2] (CH2−Py vs CH2−CH2−Py), does not show any
significant fluorescence change (Figure 12 and Supporting
Figure 12. Fluorescence titration of [ZnL1] and [ZnL2] by different
anions: (a) fluorescence spectra obtained during the titration of
[ZnL2] with PPi in aqueous methanolic (1/2 v/v) HEPES buffer (pH
7.4), with [ZnL2] = 10 μM and λex 390 nm; (b) relative fluorescence
intensity (I0/I) obtained during the titration of [ZnL1] or [ZnL2] with
different anions (black bars are for [ZnL1], and the bars with hatch
lines are for [ZnL2]). The inset shows the relative fluorescence
intensity (I/I0) as a function of [anion]/[ZnL2] mole ratio.
Information, Figure S22) toward any of these anion; however,
[ZnL2] shows quenching of fluorescence intensity in the case of
almost all the phosphate ions. Thus, in the case of inorganic
pyrophosphate (PPi), it shows maximum quenching and the
sigmoidal plot exhibits saturation at ∼1−2 equiv, as compared
to all other inorganic phosphates studied, which show
saturation at much higher equivalents, suggesting the strong
chelating affinity of PPi toward Zn2+ (Figure 12). The fast and
selective response of [ZnL2] toward PPi as compared to many
anions, including other phosphates, allows it to be a selective
sensing molecular system for PPi.
As [ZnL2] has been found to recognize PPi among the 15
anions, the studies were extended to organic as well as
biorelevant molecules bearing phosphate moiety, viz., β-
naphthyl phosphate (NP) and adenosine mono-, di-, and
triphosphates (AMP, ADP, and ATP). Among these, the
Phosphate Recognition. Owing to the strong affinity of
Zn2+ toward the phosphate, the highly fluorescent Zn2+
complexes [ZnL1] and [ZnL2] have been studied for their
secondary sensing property toward phosphate-based anions.
Fluorescence Titration of [ZnL2] with Anions. Isolated
Zn2+ complexes of L1 and L2 show strong fluorescence emission
at ∼455 nm when excited at 390 nm. The secondary
recognition capabilities of these complexes have been carried
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fluorescence quenching follows the order ATP > ADP ≫ AMP
≈ β-naphthyl phosphate, suggesting that the quenching is
dependent both on the bulkiness of the organic moiety and on
the number of phosphate moieties present (Figure 13). In
of the 377 nm band corresponding to [ZnL2]. The absorption
spectra obtained from the titration do not conform to the
formation of free L2 and hence are suggestive of the presence of
intact [ZnL2] in the case of all the other anions (Figure 16).
1H NMR Titrations. 1H NMR titrations were carried out to
check the removal of Zn2+ from [ZnL2] by polyphosphates. For
this purpose ATP has been used instead of PPi because of the
solubility and the precipitation problem faced in case of the use
of Na4(PPi) when taken in DMSO-d6. During the titration of
[ZnL2] with ATP, the signals corresponding to the complex
start to disappear and the signals corresponding to free L2 start
to appear as the concentration of the added ATP increases and
the signals of the complex completely disappeared at higher
equivalents (Figure 17). and thus clearly supporting Zn2+
removal from its complex by releasing free L2.
Figure 13. Relative fluorescence intensity (I0/I) obtained during the
titration of [ZnL2] with different organic phosphates in aqueous
methanolic (1/2 v/v) HEPES buffer (pH 7.4), with [ZnL2] = 10 μM
and λex 390 nm. The inset shows a relative fluorescence intensity plot
(I/I0) for the titration of [ZnL2] with these pshosphates.
Reusability and Reversibility of the Receptor. The
reusability of the calix[4]arene conjugate L2 has been
demonstrated by carrying out four alternative cycles of the
titration of L2 with Zn2+ followed by PPi. Zn2+ shows
remarkable switch-on fluorescence changes through formation
of [ZnL2], and further the titration (Supporting Information,
Figure S24) of this fluorescent complex with PPi quenches the
same by removing the Zn2+ from [ZnL2]. The chemical
reversibility behavior of the receptor with Zn2+, followed by the
removal of Zn2+ by PPi, was studied in HEPES buffered water−
methanol solution. The switch-on and -off action of L2 could be
studied by monitoring the fluorescence changes as a function of
the addition of Zn2+ followed by PPi, for four consecutive
cycles, wherein remarkable reversal of the fluorescence intensity
was observed (Figure 18). Hence, L2 is a reversible and
reusable sensor for Zn2+ and its zinc complex [ZnL2] as a
secondary sensor for PPi.
order to assess the sensitivity of [ZnL2] for PPi sensing,
fluorescence titrations were carried out between [ZnL2] vs
[PPi] by keeping a 1/1 ratio. The results clearly demonstrate
that [ZnL2] can detect up to 278 10 ppb of PPi, suggesting
that the detection limit is low enough to have some biological
applications (Supporting Information, Figure S23).
As [ZnL2] shows strong visual fluorescent color under UV
light, experiments were carried out between [ZnL2] and
different anions, including phosphate-bearing nucleotides, by
keeping a 1/2 ([ZnL2]/anion) mole ratio. The visual color
changes from fluorescent to nonfluorescent in the presence of
ATP and PPi, where the color is reminiscent of L2. Thus,
[ZnL2] clearly distinguishes biologically important ATP and
PPi from a variety of other anions. The color changes thus
observed in the fluorescence is attributable to the interaction
with followed by the removal of Zn2+ from [ZnL2] by these
anions. However, the color of [ZnL2] remains fluorescent in
the presence of other anions, suggesting that Zn2+ is intact in
the complex in the presence of these anions (Figure 14).
Absorption Titration of [ZnL2] with Anions. In order to
support the results obtained from the fluorescence studies,
similar titrations were carried out with absorption spectroscopy.
Titration of [ZnL2] with PPi exhibits lowering of absorbance of
the band at ∼378 nm and further shows two new bands, one at
∼325 nm and the other at ∼420 nm, which correspond to the
free receptor, and the absorbance of these bands increases
(Figure 15). The absorbance vs mole ratio plot shows sigmoidal
behavior, with saturation being acquired within ∼1−2 equiv,
similar to that observed from the fluorescence study (Figure
12).
CONCLUSIONS AND CORRELATIONS
■
The imino−phenolic−pyridyl conjugates of calix[4]arene L1
and L2 have been synthesized and characterized. These were
found to be sensitive and selective receptors for Zn2+ in HEPES
buffer medium. Their selectivity and sensitivity were demon-
1
strated on the basis of fluorescence, absorption, and H NMR
spectroscopy, ESI mass spectrometry, and visual fluorescent
color changes. L1 and L2 can detect Zn2+ up to ∼31 and ∼112
ppb, respectively by switch-on fluorescence, suggesting its
applicability to detect Zn2+ ions in aqueous HEPES buffer
medium. TDDFT calculations were carried out to demonstrate
the electronic properties of L1 and L2 and their corresponding
zinc complexes. The structure of [ZnL1] has been established
by single-crystal XRD, and in this the zinc center shows a
square-pyramidal geometry by using an N3O2 core formed by
two phenolic oxygens, two imine nitrogens of both the arms,
and one pyridyl nitrogen from one of the arms of the
calix[4]arene unit. In the lattice, two water molecules were
found which connect the two neighboring calix[4]arene units
through hydrogen bonding, thereby resulting in the formation
The titrations carried out with other anions exhibited no
significant change except in the case of ATP. The ADP and
simple phosphates showed marginal changes in the absorption
Figure 14. Vials showing the color changes observed when different anions were added to a solution of [ZnL2] in 2/1 mol ratio under (near-UV)
365 nm light.
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Figure 15. (a) Absorption spectra obtained during the titration of [ZnL2] with PPi in aqueous methanolic (1/2 v/v) HEPES buffer (pH 7.4). (b)
Plot of absorbance vs [PPi]/[ZnL2] for different absorption bands.
Figure 16. (a) Absorption spectra obtained during the titration of [ZnL2] with different anions in aqueous methanolic (1/2 v/v) HEPES buffer (pH
7.4). (b) Histogram showing the absorbance of [ZnL2] at the 377 nm band, on titration with different anions.
Figure 17. 1H NMR spectral titration in a DMSO-d6/D2O mixture (2/1): (a) L2; (b) [ZnL2]; (c−e) [ZnL2] + n equivalents ATP (n = 1 (c), 2 (d),
●
▲ △ △
, ,
and 3 (e)). Legend to symbols: ( ) imine-H; (⬡,
with bar) Py-H; (☆) calix-OH; (⬢) triazole-H; (⬡ with bar) Sal-H.
of dimers. The structure of [ZnL2] was computationally
optimized using DFT calculations and found an almost similar
type of zinc geometry, except that the Py-N···Zn distance is
longer in the case of [ZnL2] as compared to that of [ZnL1]. L1
and L2 were demonstrated as potential live-cell fluorescence
imaging agents under microscopy using HeLa cells. Non-
fluorescent images were observed when HeLa cells were
incubated with these conjugates alone. However, strong
fluorescence was observed in HeLa cells in the presence of
Zn2+. Hence, these results clearly indicate that the imino−
phenolic−pyridyl conjugates of calix[4]arene, viz., L1 and L2,
are effective intracellular Zn2+ imaging agents with cell
permeability.
properties toward various anions. The fluorescence intensity of
[ZnL1] has been found to be unaltered in the presence of all 18
anions studied, thus indicating the nonavailable nature of Zn2+
in its complex. However, [ZnL2] was found to be selective
toward phosphate-bearing ions and molecules and is sensitive
as well as selective in particular to PPi and ATP among all the
anions studied, including phosphates. The selectivity has been
shown on the basis of fluorescence and absorption spectros-
copy studies. [ZnL2] was able to detect a minimum
concentration of PPi up to 278
10 ppb at pH 7.4 in
aqueous methanolic HEPES buffer. The unusual sensitivity and
reactivity of [ZnL2] over [ZnL1] toward phosphates is
attributable to a highly distorted geometry with long a Zn−
NPy bond found in the former.
These fluorescent zinc complexes, viz., [ZnL1] and [ZnL2],
have been subjected to studies of their secondary sensing
5003
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Article
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Figure 18. Fluorescence experiment to show the reversibility and
reusability of the receptor for sensing Zn2+ and PPi by alternate
addition of these two to L2: (bottom) relative fluorescence intensity
(I/I0) obtained during the titration of L2 with Zn2+ and PPi in aqueous
methanolic (1/2 v/v) HEPES buffer (pH 7.4), with [L2] = 10 μM and
λex 390 nm; (top) visual fluorescent color change after each addition.
Labels: (a) L2; (b) {(a) + Zn2+}; (c) {(b) + PPi}; (d) {(c) + Zn2+};
(e) {(d) + PPi}; (f) {(e) + Zn2+}; (g) {(f) + PPi}; (h) {(g) + Zn2+};
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ASSOCIATED CONTENT
* Supporting Information
■
S
Figures, text, tables, and a CIF file giving X-ray crystallographic
data for [ZnL1], synthesis and characterization details of
1
precursor molecules, H NMR, 13C NMR, and mass spectral
data for all calix[4]arene conjugates, XRD data of [ZnL1],
computational data for [ZnL2], TDDFT data, and fluorescence
and absorption spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 91 22 2576 7162. Fax: 91 22 2572 3480. E-mail: cprao@
iitb.ac.in.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.P.R. acknowledges financial support by the DST, CSIR, and
BRNS-DAE. D.P. acknowledges financial support by a DAE-
SRC fellowship, Department of Atomic Energy, Government of
India. R.K.P. and A.R. thank the CSIR for their Senior Research
Fellowships.
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