A lower rim triazole linked calix[4]arene conjugate as a fluorescence switch on sensor for Zn2+ in blood serum milieu

Article abstract of DOI:10.1039/c0cc00219d

A fluorescence turn-on receptor based on triazole linked calix[4]arene (L) for selective recognition of Zn²⁺ in aqueous-methanolic HEPES buffer has been developed and showed its utility for sensing Zn²⁺ in blood serum milieu.

Full text of DOI:10.1039/c0cc00219d

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A lower rim triazole linked calix[4]arene conjugate as a fluorescence
switch on sensor for Zn2+ in blood serum milieuw
Rakesh Kumar Pathak,a Amol G. Dikundwar,b Tayur N. Guru Rowb and
Chebrolu Pulla Rao*a
Received 4th March 2010, Accepted 16th April 2010
First published as an Advance Article on the web 12th May 2010
DOI: 10.1039/c0cc00219d
A fluorescence turn-on receptor based on triazole linked calix[4]-
arene (L) for selective recognition of Zn2+ in aqueous-methanolic
HEPES buffer has been developed and showed its utility for
sensing Zn2+ in blood serum milieu.
The receptor L exhibits very weak fluorescence emission
owing to photoelectron transfer at B450 nm when excited at
380 nm in 1 : 4 water–methanol mixture at pH = 7.4 to have
an effective HEPES buffer concentration of 10 mM. Titration
of this by Zn2+, results in the enhancement of fluorescence
intensity as a function of added Zn2+ concentration (Fig. 1).
A plot of fluorescence intensity as a function of added [Zn2+]/[L]
mole ratio shows a stoichiometry of 1 : 1 between L and Zn2+
and exhibits saturation in the intensity at Z 1 equivalent and
yields an association constant of (1.49 Æ 0.03) Â 105 MÀ1 as
derived using Benesi–Hildebrand equation. Under UV light of
365 nm, the {L + Zn2+} is fluorescent while L is not.
Zn2+ is indispensable for human growth and development.1
Human serum contains B19 mM of this ion.2 Imbalanced
homeostasis of Zn2+ causes infection and diseases, such as,3
Alzheimer, hyperalgesia, diabetes and cancer, though the
reasons are little understood. The levels of Zn2+ were found
to be low in the serum of HIV infected patients.4a Cell
permeable Zn2+ chelators can enhance apoptosis in virally
infected cells.4a,b Therefore, the sensing and quantification of
the spectroscopically silent Zn2+ is a challenging task. This
may be circumvented by using a molecular receptor system
that selectively responds through its emission and/or absorption
properties rapidly with high sensitivity.5 Though, there are a
number of fluorescent sensors for Zn2+ based on quinoline,6a
fluorescein,6b,c NBD,6d BODIPY,6e coumarine,6f napthalimide,6g,h
DIPCY6i and benzimidazole6j in the literature, their utility
towards blood serum6k were little explored. Therefore, the
development of molecular receptors suitable for the blood
serum milieu measurements is still an important task. Having
realized the sensitive and selective recognition of metal ions by
the supramolecular lower rim 1,3-di-derivatives of calix[4]arenes,7
herein, for the first time we report a lower rim 1,3-di-conjugate
of calix[4]arene wherein the arms were built through a triazole-
linker and the resulting binding core responds to the Zn2+
even in the blood serum milieu.
The formation 1 : 1 complex has been further shown from
the m/z = 1403.8 {100%, [L + Zn + K–H+]}, 1388.9
{85–90%, [L + Zn + Na]}, 1365.9 {35–45%, [L + Zn]}
peaks observed in ESI MS, wherein the isotopic peak pattern
provides the signature of zinc in each of these peaks (ESI, S8w).
The complexation has also been confirmed by comparing the
1H NMR spectrum of {L + Zn2+} with that of L, where some
resonances were shifted to down field (protons at 14 & 17) and
some to up field (protons at 7a, 7b, 13 & 16) as can be seen
from ESI, S9.w The absorption titration carried out between L
and Zn2+ in the same medium (ESI, S8w) exhibited three
isosbestic points at 290, 335 and 405 nm indicating a transition
between the unbound L and that of Zn2+ bound L. The
spectra also exhibited an increase in absorbance in the
B375 nm band and a decrease in absorbance in the case of
B320 and B420 nm bands. The stoichiometry of the complex
formed between L and Zn2+ has been derived to be 1 : 1 based
on Job’s plot (ESI, S10w).
In order to incorporate binding motifs in conjunction with
fluorophore onto the calix[4]arene platform, triazole has been
used as a linker. An aldehyde precursor, viz., L3, has been
synthesized as shown in Scheme 1.8a,b Final receptor molecule
(L) has been synthesized from L3 in quantitative yield
(Scheme 1). In order to address the role of the calix[4]arene
platform, a control molecule (L4) that is devoid of such platform
has also been synthesized by the same sequence of reactions.
All these were characterized satisfactorily (ESI, S2 to S7w).
In order to check whether L is sensitive to only Zn2+ or
even to other ions, similar fluorescence titrations were carried
out in the same medium with 16 different metal ions, viz., Li+,
a Bioinorganic Laboratory, Department of Chemistry,
Indian Institute of Technology Bombay, Mumbai 400 076, India.
E-mail: cprao@iitb.ac.in
b Solid State and Structural Chemistry Unit,
Indian Institute of Science, Bangalore 560012, India
w Electronic supplementary information (ESI) available: Synthesis,
characterization, absorption, mass spectra, NMR data of all
compounds. CCDC 768214. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c0cc00219d
Scheme 1 Synthesis of L and its control molecules: (a) 5-tert-butyl-3-
(azidomethyl)-2-hydroxybenzaldehyde(L2), CuSO4Á5H2O and sodium
ascorbate in dichloromethane: water (1 : 1), rt, 12 h; (b) n-butylamine,
methanol, rt, 4 h. R = tert-butyl.
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Fig. 2 Histograms showing the fluorescence response of L (10 mM)
with Zn2+ (30 mM) in the presence of proteins or serum in
aqueous–methanolic HEPES buffer at pH = 7.4: The [L + Zn2+
]
was titrated with varying mL of either the protein (HSA or BSA or LA)
(a = 0 mL; b = 20 mL; c = 40 mL; d = 60 mL; e = 80 mL; f = 100 mL;
g = 150 mL; h = 200 mL and i = 300 mL) or serum (a = 0 mL;
b = 10 mL; c = 20 mL; d = 30 mL; e = 40 mL; f = 50 mL; g = 60 mL;
h = 80 mL and i = 100 mL).
Fig. 1 Fluorescence spectra obtained during the titration of L
(cuvette concentration of 10 mM) with Zn2+ in aqueous methanolic
(1 : 4 v/v) HEPES buffer at pH = 7.4 (lex = 380 nm). The inset shows
the relative fluorescence intensity (I/I0) as a function of [Zn2+]/[L]
mole ratio and visual color change under UV light.
serum milieu. The minimum detection limit of Zn2+ was
found to be 332 ppb in blood serum (ESI, S12w).
Na+, K+, Cs+, Mg2+, Ca2+ Sr2+, Ba2+, Mn2+, Fe2+
,
Co2+, Ni2+, Cu2+, Cd2+, Ag+ and Hg2+, and found no
significant fluorescence enhancement in the presence of these
ions (ESI, S11w). L exhibited a minimum detection limit of
36 ppb for Zn2+ under these conditions (ESI, S12w). To the
best of our knowledge, this is the first known Zn2+ sensor
based on a calix[4]arene derivative in the literature that can
quantify Zn2+ even below 100 ppb. When titrations were
carried out between L and Zn2+ in the same medium, varying
the pH from 6 to 9 resulted in no variation in the fluorescence
intensity (ESI, S13w). While the quantum yield of L is only
F = 0.028, that of the Zn2+ bound L enhances to F = 0.32.
The observed ten fold increase in the quantum yield of L in the
presence of Zn2+, the presence of water and buffer in the
medium of measurements and the observed low detection limit
seemed to qualify L for sensing as well as quantifying Zn2+ by
switching on fluorescence at biological pH. Since biological
systems possesses large concentrations of alkali and alkaline
earth ions, the selectivity of Zn2+ has been studied by carrying
out appropriate competitive metal ion titrations and found no
significant change in the fluorescence enhancement of L with
Zn2+ (ESI, S11w). As cadmium and mercury belong to the
same family, the selectivity of Zn2+ towards L has been
checked by carrying out the corresponding competitive ion
titrations and found no interference of these ions in the
detection of Zn2+ (ESI, S11w).
The necessity of the Schiff’s base portion as well as the
calix[4]arene platform in L for sensing Zn2+ were addressed
by selecting L3 (a precursor that possesses an aldehyde but not
imine) and L4 (a single stranded version of L) as control
molecular systems (Scheme 1). Fluorescence titration results
of L3 and L4 towards Zn2+ were compared with that of L and
found that L is certainly highly sensitive towards Zn2+, while
its control molecular partners were not (Fig. 3). In both L3 and
L4, the fluorescence enhancement (ESI, S14w) was found to be
very low even at Z 30 equivalents.
The isolated complex (ESI, S15w) of [ZnL] resulted in the
single crystals of diffraction quality when grown from a 1 : 1
methanol–acetonitrile mixture.10 The structure clearly exhibited a
distorted tetrahedral Zn2+ center where both the arms of L act
as bidentate through their imine nitrogen and phenolate-
oxygen to give a N2O2 core wherein the total complex is
neutral (Fig. 4).
L has been demonstrated to be sensitive and selective
towards Zn2+ by switch on fluorescence spectroscopy.
Further, its selectivity has been demonstrated, (a) in aqueous
methanolic HEPES buffer, (b) in the pH range of 6–9, (c) in
blood serum milieu, (d) in the presence of albumins, (e) in the
Biological applicability of L to sense Zn2+ has been
addressed by carrying out fluorescence titrations9 using blood
serum as well as related albumin proteins, viz., human serum
albumin (HSA), bovine serum albumin (BSA) and even an
a-lactalbumin (LA), capable of capturing Zn2+.8 Fluorescence
experiments were carried out by taking an in situ generated
Zn2+ complex of L and titrating this by varying the concen-
trations of blood serum or the related proteins (HSA, BSA,
LA). Almost no change was observed in the fluorescence
intensity of the B450 nm band of L either in the presence of
these proteins individually or in the presence of the serum
(Fig. 2). Thus L can selectively sense Zn2+ even in the blood
Fig. 3 Fluorescence intensity as a function of {[Zn2+]/[Ligand]} mole
ratio. Symbols corresponds to # = L, y = L4, $ = L3.
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.
CPR acknowledges the financial support from DST, CSIR
and DAE-BRNS. RKP and AGD acknowledge CSIR for their
fellowships.
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9 Serum samples were obtained from a healthy volunteer after
fasting. The blood sample was allowed to clot and serum was
obtained via centrifugation. The serum samples were kept at
À20 1C for storage. 100 mL of serum was dissolved in 3 mL of
HEPES buffer and used as stock solution. The bulk solution for
proteins was made by using 1 mg mLÀ1. [P.S.: Experiments could
not be carried out beyond this concentration of either the serum or
the proteins, owing the problem of precipitation].
10 Crystal data for [ZnL]. Empirical formula: C82H106N8O6Zn; F.wt.:
ꢀ
1365.16; Crystal system: Triclinic, P1; Unit cell Dimension ((A)):
15.5813(6), 17.3524(5), 18.4381(6), 79.959(3), 65.028(4), 76.432(3);
V = 4351.7(1) (A3); Z = 2; Dc = 1.15 (g mlÀ1); Unique reflections:
28611, R_obs: 0.068, wR2_obs: 0.205.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 4345–4347 | 4347