Triazole-linked-thiophene conjugate of calix[4]arene: Its selective recognition of Zn2+ and as biomimetic model in supporting the events of the metal detoxification and oxidative stress involving metallothionein

Article abstract of DOI:10.1021/jo201865x

Supramolecular calix[4]arene conjugate (L) has been developed as a sensitive and selective sensor for Zn²⁺ in HEPES buffer among the 12 metal ion by using fluorescence, absorption and ESI MS and also by visual fluorescent color. The structural, electronic, and emission properties of the calix[4]arene conjugates L and its zinc complex, [ZnL], have been demonstrated using ab initio density functional theory (DFT) combined with time-dependent density functional theory (TDDFT) calculations. The TDDFT calculations reveal the switch on fluorescence behavior of L is mainly due to the utilization of the lone pair of electrons on imine moiety by the Zn²⁺. The resultant fluorescent complex, [ZnL], has been used as a secondary sensing chemo-ensemble for the detection of -SH containing molecules by removing Zn²⁺ from [ZnL] and forming {Cys/DTT·Zn} adducts as equivalent to those present in metallothioneins. The displacement followed by the release of the coordinated zinc from its Cys/DTT complex by heavy metal ion (viz. Cd²⁺ and Hg²⁺), as in the metal detoxification process or by ROS (such as H₂O₂) as in the oxidative stress, has been well demonstrated using the conjugate L through the fluorescence intensity retrieval wherein the fluorescence intensity is the same as that observed with [ZnL], which in turn mimics the zinc sensing element (MTF) in biology (Figure presented).

Full text of DOI:10.1021/jo201865x

Article
pubs.acs.org/joc
Triazole-Linked-Thiophene Conjugate of Calix[4]arene: Its Selective
Recognition of Zn2+ and as Biomimetic Model in Supporting the
Events of the Metal Detoxification and Oxidative Stress Involving
Metallothionein
Rakesh Kumar Pathak,† Vijaya Kumar Hinge,‡ Milon Mondal,† 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: Supramolecular calix[4]arene conjugate (L) has
been developed as a sensitive and selective sensor for Zn2+ in
HEPES buffer among the 12 metal ion by using fluorescence,
absorption and ESI MS and also by visual fluorescent color. The
structural, electronic, and emission properties of the calix[4]arene
conjugates L and its zinc complex, [ZnL], have been demonstrated
using ab initio density functional theory (DFT) combined with
time-dependent density functional theory (TDDFT) calculations.
The TDDFT calculations reveal the switch on fluorescence
behavior of L is mainly due to the utilization of the lone pair of
electrons on imine moiety by the Zn2+. The resultant fluorescent
complex, [ZnL], has been used as a secondary sensing chemo-
ensemble for the detection of −SH containing molecules by
removing Zn2+ from [ZnL] and forming {Cys/DTT·Zn} adducts as equivalent to those present in metallothioneins. The
displacement followed by the release of the coordinated zinc from its Cys/DTT complex by heavy metal ion (viz. Cd2+ and
Hg2+), as in the metal detoxification process or by ROS (such as H2O2) as in the oxidative stress, has been well demonstrated
using the conjugate L through the fluorescence intensity retrieval wherein the fluorescence intensity is the same as that observed
with [ZnL], which in turn mimics the zinc sensing element (MTF) in biology.
recent times, supramolecular calixarene conjugates have been
evolving as mimics for metalloproteins and metalloenzymes.8,9
Upon appropriate derivatization, the conjugates of calix[4]-
arene can be tuned to be sensitive and selective toward certain
metal ion.11 Therefore, the synthetic conjugates of calix[4]-
arene which senses Zn2+ selectively can be of great
advantage10−12 in this, while sulfhydryl bearing small organic
molecules, such as cysteine (Cys) and dithiothreotol (DTT),
can act as MT mimics.13 Hence, in the present paper we report
the synthesis, characterization of thiophene appended triazole-
linked calix[4]arene conjugate, L, and its selective recognition
behavior toward Zn2+. The structural, electronic, and emission
properties of the calix[4]arene conjugate L and its zinc
complex, [ZnL], have been demonstrated using ab initio
density functional theory (DFT) along with the calculations of
the time-dependent density functional theory (TDDFT).
Furthermore, this conjugate has been demonstrated as a
biomimetic model system to support the events involved in the
oxidative stress and metal detoxification pathways through the
sensitive and selective recognition of Zn2+.
INTRODUCTION
■
Metallothioneins (MTs) are low-molecular-weight proteins
having high cysteine content and thereby exhibit strong ability
to complex metal ions with greater affinity being imparted
toward softer ones.1 Two most important functions of MTs are
the heavy metal detoxification and antioxidant property.2 These
are controlled by the free and loosely bound zinc ions.2,3 The
reactive oxygen species (ROS) induced oxidation of thiols
triggers the formation of the oxidized protein thionine (Tox)
with the concomitant release of Zn2+.4 In the presence of toxic
heavy metal ions, Cd2+ and Hg2+ bind strongly with the MTred
by replacing Zn2+ and therefore hinder the process of oxidative
stress and other enzymatic pathways.5 At this stage, the
concentration of Zn2+ increases in the cytosol and the elevated
levels of free zinc binds to the metal-regulatory transcription
factor (MTF), thus inducing the expression of thionine, and the
expressed MTred state is stabilized by the Zn2+ through
binding.3,6 A molecular mechanistic based knowledge of the
release of Zn2+ from the Cys rich binding domains of MTred is
expected to demonstrate the bioinorganic pathways of the these
events. Synthetic fluorescent conjugates which selectively
recognize Zn2+ can be used to demonstrate the events involved
in the metal detoxification and the oxidative stress.7 In the
Received: September 10, 2011
Published: November 4, 2011
© 2011 American Chemical Society
10039
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
a
Scheme 1. Synthesis of the Precursor Molecule, L2, and the Final Receptor Molecule, L
a
Synthesis of receptor L: (a) propargyl bromide, K2CO3, acetone, reflux, 24 h, 82%; (b) 5-tert-butyl-3-(azidomethyl)-2-hydroxybenzaldehyde (L2),
CuSO4·5H2O and sodium ascorbate in dichloromethane: water (1:1) rt, 12 h, 89.9%; (c) thiophen-2-ylmethanamine, methanol, rt, 8 h, 93%; (d)
SnCl4, Bu3N, (CH2O)n, dry toluene, reflux, 63%; (e) 37% formaldehyde, concentrated HCl, rt, 24 h, 82%; (f) NaN3, DMF, rt, 12 h, 89%. R = tert-
butyl.
Figure 1. (a) Fluorescence spectra obtained during the titration of L with Zn2+ in aqueous ethanol (1:2 v/v) HEPES buffer (pH = 7.4), λex = 390
nm, [L] = 10 μM. (b) Relative fluorescence intensity (I/I0) as a function of [Zn2+]/[L] mole ratio.
mixture at pH = 7.4. Titration of L with Zn2+ results in the
RESULTS AND DISCUSSION
■
enhancement of the fluorescence intensity as a function of the
The calix[4]arene conjugate, L, was synthesized by going
added Zn2+ concentration (Figure 1a) and the intensity
through three consecutive steps11h starting from p-tert-
saturates at ≥1 equiv. The switch on fluorescence emission is
expected to the reversal of photoelectron transfer (PET) upon
butylcalix[4]arene followed by the preparation of its
dipropargyl ether and triazole linked conjugate as shown in
utilization of the lone pair of electrons on imine moiety by the
Scheme 1. The copper(I)-catalyzed 1,3-dipolar cycloaddition
Zn2+. A plot of the fluorescence intensity vs [Zn2+]/[L] mole
reaction was used for the introduction of L2 onto the calixarene
scaffold. The precursor azide derivative L2 has been synthesized
ratio (Figure 1b) fits well with the formation of a 1:1
stoichiometric complex between L and Zn2+, and the binding
constant derived from the Benesi−Hildebrand equation was
found to be (9.2 ± 0.2) × 10−4 M−1. The observed high Ka
value clearly indicates the strong affinity of Zn2+ to L. In order
to check whether the L is sensitive to only Zn2+ or even to the
other ions, similar fluorescence titrations were carried out in the
same medium with 11 other different metal ions, viz. Na+, K+,
Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Cd2+, and Hg2+, and
found no significant fluorescence enhancement in the presence
of any of these ions (Figure 2).
in three steps,11h,14 starting from the p-tert-butylphenol
(Scheme 1). The final receptor molecule has been synthesized
by the condensation of the L3 with thiophene methylamine as
shown in Schemes 1. All the precursor and receptor molecules
were characterized by various analytical and spectral techniques,
and the calix[4]arene conjugates were found to be in cone
conformation as per the data given in the Experimental Section
as well as in the Supporting Information, Figures S1−S4.
A. Metal Ion Recognition Studies. In order to under-
stand the selective and sensitive metal ion binding behavior of
L, fluorescence and absorption titrations were carried out.
Fluorescence Studies. The receptor L exhibits weak
fluorescence emission at ∼454 nm owing to the photoelectron
transfer when excited at 390 nm in 1:2 HEPES buffer:ethanol
Therefore, L is selective to Zn2+ among the 12 ions studied.
Visual fluorescent color change experiments have been carried
out to look at the behavior of L in the presence of various metal
ions. Under UV light, the solution of L is nonfluorescent,
10040
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Figure 2. Histogram showing the relative fluorescence response of L in
the presence of different metal ions, [L] = 10 μM. The inset shows the
visual fluorescent color change of L in presence of these ions under
365 nm incident light.
Figure 3. Histogram showing the fluorescence response of L with Zn2+
in the presence of various metal ion in aqueous ethanol (1:2 v/v)
HEPES buffer (pH = 7.4) solution. [L] = 10 μM, 2 equiv of Zn2+, and
5 equiv of other metal ions have been used.
whereas in the presence of Zn2+, it shows an intense blue
fluorescent color which is otherwise not present in case of the
other metal ions studied (Figure 2 inset). Therefore, Zn2+ can
easily be differentiated by visual color change among the other
metal ions.
NMR Titrations of L with Zn2+. In order to support the
1
formation of the complex between L and Zn2+, H NMR
titrations were carried out. During the titration, the
concentration of L was kept constant and the added [Zn2+]
mole ratio was varied, viz. 0, 0.5, 1, 1.5, 2.0, and 3.0 equiv
(Figure 6). Upon the interaction of L with Zn2+, the chemical
shifts of the protons of imine, thiophene, salicyl, and bridged
−CH2 showed marginal to considerable changes and that of the
salicylic−phenolic-OH disappears within 1 equiv. The absence
of any significant changes in the chemical shift of the triazole
proton rules out the interaction between this moiety and the
Zn2+. The considerable shift observed in the thiophene-CH2,
salicyl-H, and thiophene-H supports the role of salicylimine−
thiophene core in the Zn2+ binding. The 13C NMR spectra of L
and [L + 2 equiv Zn2+] have been compared and found to have
significant spectral differences supporting the complex
formation (Supporting Information, Figure S8).
Structural Features of [ZnL] by Computational Methods.
As no structure could be established for the zinc complex due
to its noncrystallinity, though the precursor L was crystalline
(Supporting Information, Figure S9), DFT-based calculations
were performed to understand the structural and coordination
features of [ZnL] complex. Therefore, both L and [ZnL] were
optimized by DFT using Gaussian 03 package15 as explained in
the Experimental Section, and the corresponding ground state
structures obtained from DFT calculations are shown in Figure
7. In [ZnL], Zn2+ shows highly distorted tetrahedral geometry,
where it is bonded through two salicyl oxygens, imine
nitrogens. One of the sulfur of thiophene showed weaker
coordination with zinc by a distance of 2.78 Å, which is higher
than the normal thiolate/thoethers-Zn bond distance. There-
fore, the interaction between Zn···S is of van der Waal’s type.
When this Zn···S interaction is being included in the
coordination sphere, the zinc exhibits a distorted square-
pyramidal geometry. However, the second thiophene moiety
does not participate in binding as the Zn to S distance is 5.13 Å
(Supporting Information, Figures SI-10−SI-14). The natural
bonding orbital (NBO) charge analysis shows that a more
positive charge on the zinc center and negative charges on the
coordinated O and N atoms of [ZnL] {Zn = 1.26, N1 = −0.66,
N2 = −0.66, O1 = −0.76, O2 = −0.75}. The charge on the zinc
center is >1 and indicates more of an interaction of the ionic
nature between the metal center and the ligands.
The sensitivity of L for Zn2+ has been further evaluated by
measuring the lowest concentration that can be determined.
The fluorescence titration carried out between L and [Zn2+] by
maintaining a 1:1 ratio gives a value of 47 ± 8 ppb (718 ± 122
nM), suggesting its applicability to detect Zn2+ ions in aqueous
medium at physiological conditions. To our knowledge, this is
one of those few calix[4]arene conjugates which possess high
sensitivity toward Zn2+ in the literature that can quantify Zn2+
even below 100 ppb level (Supporting Information, Figure S5).
Therefore, L can be used as a sensitive and selective in vitro
receptor for Zn2+.
Competitive Metal Ion Titration. In order to show the
practical utility of L to detect Zn2+ selectively even in the
presence of other metal ions, competitive metal ion titrations
were carried out. These studies reveal that the Zn2+-induced
fluorescence enhancement was unaffected in the presence of
biologically relevant alkali (Na+, K+), alkaline earth (Mg2+ and
Ca2+), first row transition (Mn2+, Fe2+, Co2+, Ni2+), and the
toxic metal ions (Cd2+ and Hg2+). However, this is completely
quenched in the presence of Cu2+. Thus, it is notable that L can
be used as a selective Zn2+ fluorescent sensor in the presence of
most of the metal ions (Figure 3), except Cu2+.
Absorption Titrations with Zn2+. Absorption titrations were
carried out in aqueous ethanol (1:2 v/v) HEPES buffer (pH =
7.4) to support the binding of Zn2+ with L. The absorption
spectra of free L (10 μM) exhibits bands at ∼325 and ∼435
nm. Upon the addition of Zn2+, the new band observed at ∼380
nm shows increased absorbance, while that of the other two
bands at ∼325 and ∼435 nm show decrease. The isosbestic
points observed at 345 and 415 nm were indicative of the
transition between the unbound L and its Zn2+ complexed
species (Figure 4). The stoichiometry of the complex formed
between L and Zn2+ has been further shown to be 1:1 by the
Job’s plot (Supporting Information, Figure S6)
Electrospray Mass Spectrometry. ESI MS spectrum
obtained for the titration of L with Zn2+ results in a molecular
ion peak at m/z = 1443.62 that corresponds to [L + Zn + H]+.
The isotopic peak pattern observed is characteristic for the
presence of zinc supporting the complex formation (Supporting
Information, Figure S7) of L with Zn2+ (Figure 5).
10041
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Figure 4. (a) Absorption spectra obtained during the titration L (10 μM) with Zn2+ in aqueous ethanol (1:2 v/v) HEPES buffer (pH = 7.4). (b) Plot
of absorbance vs. [Zn2+]/[L] mole ratio for different absorption bands.
Figure 5. ESI MS spectrum obtained during the titration of L with Zn2+ for the 1:1 complex along with its isotopic peak pattern: (a) observed and
(b) calculated.
Figure 6. 1H NMR spectra measured during the titration of L with different mole ratio of Zn2+ in DMSO-d6: (a) 0, (b) 0.5, (c) 1.0, (d) 1.5, (e) 2.0,
and (f) 3.0: (●) imine-H; (⬡, ▲, △) thiophene-H; (★, ○, ■) bridged CH2 protons.
Calculations based on single-excitation time-dependent
density functional theory (TDDFT) were performed to explain
the electronic structural properties of the ground and excited
states behavior of L and [ZnL].16 The vertical transitions
calculated by TDDFT (Table 1) were compared (Figure 8) and
were found to be in good agreement with the experimental
data.
The TDDFT calculations were performed at the B3LYP/6-
31G(d,p) level and found to support the absorption bands
observed in the region of λmax = 270−450 nm. These studies
suggest that the band observed at ∼377 nm corresponds to
λmax of UV experimental spectra of [ZnL] and ∼321 nm
corresponds to L. In case of [ZnL], the observed band at ∼377
nm is assigned to the transition B (Table 1) that results from a
π → π* transition within the salicylimine fragment, which
mainly corresponds to two−one electron excitations from 318/
HOMO-2 → 321/LUMO and 320/HOMO → 322/LUMO+1.
The corresponding (molecular orbital’s) MO’s are shown in
Figure 9 (Table 1). In Figure 9A, the MO’s are majorly
localized on salicylimine part of [ZnL], and the transitions are
10042
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Figure 7. DFT optimized structure of (a) L and (b) [ZnL]. Hydrogen’s were omitted for the clarity. (c) Coordination sphere around the zinc ion,
bond angles (deg) in the coordination sphere are N1−Zn−O1 = 93.5, N1−Zn−O2 = 111.3, N1−Zn−N2 = 116.5, N1−Zn−S1 = 106.4, O1−Zn−O2 =
92.5, O1−Zn−N2 = 144.3, O1−Zn−S1 = 78.3, O2−Zn−N2 = 93.4, O2−Zn−S1 = 141.5, N2−Zn−S1 = 75.1.
Table 1. Main Singlet Vertical Electron Transition Energies (ΔE), Wavelengths (λ), Oscillator Strengths (F), and Configuration
Interaction Coefficients (CIcoeff) for [ZnL], Calculated at the TDDFT Level
transition
ΔE [eV] (λnm
)
F
key excited state (CIcoeff
)
contribution (%)
assignment
A
B
3.2047 (386)
3.2885 (377)
3.4914 (355)
3.9349 (315)
0.0852
0.1534
0.0041
0.0048
HOMO-2 → LUMO (0.62332)
HOMO-2 → LUMO (0.13094)
HOMO-2 → LUMO+1 (0.6988)
HOMO → LUMO+2 (0.70082)
78
78
98
98
salicylimine → salicylimine (π−π*)
salicylimine→ salicylimine (π−π*)
salicylimine 1 → salicylimine 2 (π−π*)
LMCT
C
D
Figure 8. Normalized TDDFT singlet monoelectronic vertical transitions and normalized diffuse reflectance spectrum: (a) L and (b) [ZnL].
Figure 9. Pictorial representation of the molecular orbital’s (MOs) calculated for the singlet vertical electron transitions A to D (given in Table 1)
using TDDFT for [ZnL].
10043
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Figure 10. (a) Fluorescence spectra obtained during the titration of {L + Zn2+} with Cys in aqueous ethanolic (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 [Cys]/[ZnL] mole ratio. (b) Histogram showing relative
fluorescence response of various biological thiols with [ZnL]. The inset shows the visual fluorescent color change of [ZnL] in the presence of
different −SH-containing molecules, along with controls, under 365 nm incident light.
similar to those observed in B but with a lesser oscillator
strength when compared to that in Figure 9B (Table 1). Part C
mainly corresponds to the transition from the π MO’s of one
salicylimine to the π* MO of the other salicylimine of [ZnL]
and corresponds to the pure 318/HOMO-2 → 322/LUMO+1
excitation. In case of part D, the ligand-to-metal charge transfer
(LMCT) was observed from one of the salicylimine moiety to
zinc ion, and the corresponding 320/HOMO and 323/LUMO-
2 MO’s are given in Figure 9. The TDDFT calculations have
been carried out for L and the corresponding electronic
transitions details included in the Supporting Information,
Figures S11 and S15.
fluorescence and forming a 1:1 complex. Therefore, the
resulting fluorescent chemo-ensemble zinc complex [ZnL]
has been used for the study of the recognition of thiol (−SH)
bearing molecules, since the ligating centers in metallothionein
mainly arise from the side chain −SH function of the Cys
residues.
Fluorescence Titrations. The chemosensing ensemble used
in these titrations was prepared by in situ mixing of L with Zn2+.
The in situ complex [ZnL] shows strong fluorescence emission
at ∼454 nm when excited at 390 nm. The fluorescence
titrations carried out with cysteine (Cys), dithiothreitol (DTT),
glutathione reduced (GSH), mercaptopropionic acid (MPA),
homocysteine (Hcy), mercaptoethanol (ME), and cysteamine
(Cyst) all exhibited fluorescence quenching; however, a
considerably high quenching was observed mainly in the case
of Cys, DTT, and GSH in the order Cys > DTT > GSH
(Figure 10), while the others exhibited only minimal changes
(Figure 10b), suggesting that the chelatable −SH-containing
molecules are effective and not the others (Supporting
Information, Figure S14). In fact, the Cys and DTT are good
mimics of the Zn2+ binding regions of MTred during oxidative
stress and heavy metal ion detoxification process. The
fluorescence quenching observed in the presence of Cys and
DTT may be attributed initially to their interaction with the
[ZnL], followed by the removal of Zn2+ from this complex, and
the chelation of the freed Zn2+ by Cys and DTT. All these
suggests that the chelating ability of the −SH molecules is
The origin of fluorescence behavior of L in presence of Zn2+
can be explained based on the fluoroscence enhancement by
orbital control (FEOC) phenomena.17 In the present case the
fluorescence behavior of L and [ZnL] has been demonstrated
on the basis of the involvement of imine-nitrogen lone pair
toward Zn2+ binding. In L, the major contribution to the
excitation is from the nitrogen lone pair, viz. HOMO-3 to
LUMO (77%) and HOMO-5 to LUMO (57%), and therefore
contributes for the fluorescence quenching (Supprting
information, Figures S12 and S13 and Table S4). However,
in case of [ZnL], the nitrogen lone pair orbitals are involved in
the binding of Zn2+ and therefore not available for the
excitation transitions which otherwise correspond to the
fluorescence quenching. Hence, upon complexation, Zn2+
blocks the involvement of nitrogen lone pair in the excitation
and thereby enhances the fluorescence of [ZnL].
B. Secondary Sensing of Thiols by L. Thus, L recognizes
Zn2+ with high selectivity and sensitivity by switching on the
Figure 11. (a) Absorption spectra obtained during the titration of [ZnL] with DTT. The inset shows a plot of absorbance vs [DTT]/[ZnL] for
different bands. ESI MS for {DTT·Zn} complex with its isotopic peak pattern: (b) observed and (c) calculated.
10044
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Figure 12. (a) Fluorescence spectra obtained during the titration of {[DTT + Zn2+] + L} with H2O2 in aqueous ethanol (1:2 v/v) containing
HEPES buffer (pH = 7.4), λex = 390 nm. The inset shows the relative fluorescence intensity (I/I0) as a function of [H2O2]/{[DTT + Zn2+] + L}
mole ratio. Note: the 30% H2O2 {1 mL in 2 mL buffer} has been diluted twice with buffer and used for the titrations. (b) Comparison of the
fluorescence spectra for [L + Zn2+], {[L + Zn2+] + DTT}, and {[DTT + Zn2+] + L + H2O2}.
important for the removal process. This indeed results in the
fluorescence quenching, and the final fluorescence is similar to
that exhibited by L, since the Zn2+ complex of Cys or DTT do
not contribute to the fluorescence.
to understand the biochemical mechanism of zinc release from
MT during oxidative stress and metal detoxification process, it
is necessary to model this process by using in vitro biomimetic
molecular systems. The sulfhydryl-containing organic molecule
DTT has been used as a model in the present study which can
be a better mimic of MT, and the conjugate L has been used as
fluorescent probe for Zn2+.
Under the UV light, the solutions of [ZnL] were found to be
nonfluorescent in the case of Cys and DTT, weakly fluorescent
in the case of GSH, and highly fluorescent {like that of the
[ZnL]} in the presence of the other −SH-containing molecules
(Figure 10b, inset). The fluorescent or nonfluorescent behavior
observed in the presence of these −SH-containing molecules
clearly support the release of Zn2+ and L from the [ZnL]
mainly in the case of Cys and DTT, partly in the case of GSH,
and not at all with the other −SH molecules. Therefore, this
clearly distinguishes those capable molecules which can remove
Zn2+ from [ZnL] and release L, viz. Cys and DTT, from the
rest of the −SH molecules as can be noted from eq 1.
Absorption Titrations. The release of L from [ZnL] by Cys
or DTT has been supported even from the absorption titration
studies. The absorbance of the ∼380 nm band decreases and
the new bands observed in the presence of Cys or DTT at
∼325 and ∼430 nm, corresponding to the free receptor L
increases (Figure 11a; for others see Supporting Information,
Figure S15) as a function of the concentration of the added Cys
or DTT. However, the other −SH-containing molecules do not
show significant change in the absorbance of the 380 nm band
and 325 and 430 nm bands. Therefore, the changes observed in
case of Cys or DTT are attributable to the displacement of Zn2+
from [ZnL] and the complexation of the released Zn2+ by Cys
or DTT (Figure 11a).
Fluorescence and Absorption Titrations of {[ZnL] + DTT}
with H2O2. The titration of [ZnL] with DTT results in the
formation of the zinc−DTT complex and releases L, and hence
the corresponding mixture is denoted as {[DTT + Zn2+] + L}.
All this has been studied by fluorescence, absorption, and ESI
MS. The fluorescence and absorption experiments were carried
out with the model system, viz. {[DTT + Zn2+] + L} in the
presence of the oxidizing agent, H2O2, where the L probes the
release of Zn2+ (through the changes observed in the
fluorescence intensity) from the DTT complex. Upon the
addition of H2O2, the weakly fluorescent mixture, viz. {[DTT +
Zn2+] + L}, gains its full fluorescence intensity corresponding
to the [ZnL] (Figure 12). This is attributed to the oxidation of
DTT and the consequent release of Zn2+ followed by its
capture by L to form [ZnL], which is highly fluorescent. These
observations have been further substantiated based on the
absorption study where the original band of the [ZnL]; i.e., 380
nm reappears upon the addition of H2O2 to a mixture of
{[DTT + Zn2+] + L} (Scheme 2). Also noted was the oxidized
Scheme 2. Involvement of L as Zinc Recognition Element
and DTT as Mimic to MT
ESI MS Titration of [ZnL] with Cys and DTT. The complex
species of Cys or DTT formed with the freed Zn2+ has been
shown by ESI MS through the observation of the peaks at
1382.29 for (L + H) and 338.75 for (2Cys + Zn + CH3OH +
H2O + 2Na+) and 371.90 for (2DTT + Zn + 2H) (Figure 11b,c
and Supporting Information, Figure S16). Therefore, the
changes observed in the titration of Cys or DTT are
attributable to the release of Zn2+ and L from the [ZnL]
followed by the complexation of Zn2+ by Cys or DTT. Both the
absorption and fluorescence spectral studies clearly demon-
strate the release of L from [ZnL] upon titration with Cys or
DTT.
C. Oxidative Stress and Heavy Metal Toxicity Events:
An Attempt To Mimic These by [ZnL]. Zn2+ is released
from the metallothionein during the oxidative stress and heavy
metal detoxification pathways. The elevated concentration of
Zn2+ is responsible for various infections and diseases. In order
product of DTT by H2O2 observed around ∼238 nm, which
has been further confirmed by carrying out a titration between
simple DTT and H2O2 (Supporting Information, Figure S17).
10045
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Effect of Toxic Metal Ions (Cd2+ and Hg2+) on Sensing of
Cys and DTT by [ZnL]. During the heavy metal ion toxicity,
the Zn2+ is freed from the metallothionein by sequestering Cd2+
or Hg2+ because these ions have high affinity of binding to the
−SH functions of MTred. Indeed, Cys or DTT can act as small
molecular mimic for MTred. Therefore, this pathway has been
modeled in the present case by carrying out the fluorescence
titrations of a mixture of {[Zn(Cys/DTT)] + L} by Cd2+/Hg2+
and found to regain the fluorescence enhancement upon the
addition of the heavy metal ions, which is equivalent to that of
the [ZnL] (Figure 13). The fluorescence data clearly fit well
between Cd2+/Hg2+ and DTT/Cys, and the release of Zn2+
from the [Zn(DTT/Cys)], ESI MS titrations were carried out.
The mass spectral data show the peaks at m/z 446.73 for (2Cys
+ Cd + CH3OH + H+), 440.89 for (2Cys + Hg + 2H+), and
419.90 for (2DTT + Cd + 3H + Na+), 434.92 for (DTT + Hg
+ 2CH3OH + 2H2O) (Supporting Information, Figures S23−
S26). All this clearly support the displacement of zinc from
[Zn(DTT/Cys)] by Cd2+ or Hg2+ as shown through eq 2.
CONCLUSIONS AND CORRELATIONS
■
Development of a synthetic organic molecular model system
that can provide us with some clues on the complex nature of
the oxidative stress and the heavy metal ion detoxification
process would indeed be a welcome feature of the bioinorganic
chemistry of metallothioneins. A suitably modified supra-
molecular calix[4]arene system (L) has been developed as the
sensitive and selective recognition tool for Zn2+. The high
sensitivity and selectivity of L toward Zn2+ has been
demonstrated on the basis of the fluorescence, absorption,
and ESI-MS and also by visual fluorescent color change. The
structural, electronic, and emission properties of the calix[4]-
arene conjugates L and its zinc complex, [ZnL], have been
demonstrated using DFT computational calculations. L can
detect Zn2+ up to 47 ± 8 ppb (718 ± 122 nM) by switch on
fluorescence, suggesting its applicability to detect Zn2+ ions in
aqueous medium at physiological conditions. Therefore, L can
be used as a sensitive and selective switch on fluorescence
receptor for Zn2+. The TDDFT calculations reveal that the
switch on fluorescence behavior of L is mainly due to the
utilization of the lone pair of electrons on imine moiety by the
Zn2+. The resultant fluorescent [ZnL] has been used as a
secondary chemosensor ensemble for the detection of −SH-
containing molecules.
Figure 13. Relative fluorescence intensity plots obtained during the
titration of [ZnL + Cys/DTT] with Cd2+ or Hg2+ in aqueous ethanol
(1:2 v/v) HEPES buffer (pH =7.4), λex = 390 nm: (a) in the case of
{Zn(Cys) + L}; (b) in the case of {Zn(DTT) + L}.
with the fact that the [Zn(Cys/DTT)] complex present in the
mixture goes through a transmetalation by Cd2+/Hg2+, and thus
the released Zn2+ forms a complex with L to exhibit
fluorescence enhancement through the formation of [ZnL]
(Figure 13 and Supporting Information, Figure S18). These
results were further confirmed by carrying out an absorption
titration between the mixture, viz. {[Zn(DTT/Cys) + L} by
Cd2+/Hg2+, and the spectra supported the formation of [ZnL]
as well as the complex formed by Cd2+/Hg2+ with DTT/Cys
(Supporting Information, Figures S19−S22).
The observed fluorescence enhancement of the {Zn(Cys/
DTT) + L} when titrated with Cd2+ or Hg2+ suggests the
reformation of the [ZnL] followed by the transfer of Cd2+/Hg2+
to Cys/DTT. Therefore, the data reveal the importance of MTs
and free −SH residues in the biological medium for the
detoxification and sensing of heavy metal ions by using OFF
and ON fluorescence of L and [ZnL], respectively (Figure 14).
In the presence of Cys or DTT, the Zn2+ is being freed from
[ZnL] and is complexed by Cys or DTT, as demonstrated by a
combination of different techniques. The displacement
followed by the release of the coordinated zinc from its Cys/
DTT complex by heavy metal ion (viz. Cd2+ or Hg2+), as in the
metal detoxification pathway or by ROS (such as H2O2) as in
the oxidative stress (Scheme 3), has been well demonstrated by
the L. The L in turn mimics the zinc sensing element (MTF) in
biology. Therefore, to our knowledge, this is the first report of a
synthetic calix[4]arene conjugate that mimics some critical
steps of the metal detoxification and oxidative stress events
noticed in biology, in which metallothionein plays a central
role.
Figure 14. Histogram showing the relative fluorescence response of L
in aqueous ethanol (1:2 v/v) HEPES buffer (pH =7.4) solution in
different condition: (a) L; (b) [L + Zn2+]; (c) {[L + Zn2+] + Cys}; (d)
{[L + Zn2+] + DTT}; (e) {{[L + Zn2+] + Cys} + Cd2+}; (f) {{[L +
Zn2+] + Cys} + Hg2+}; (g) {[L + Zn2+] + DTT} + Cd2+; (b) {[L +
Zn2+] + DTT} + Hg2+.
EXPERIMENTAL SECTION
■
General Information and Materials. 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)2·6H2O, Hg(ClO4)2·4H2O, Zn-
(ClO4)2·6H2O, and Cd(ClO4)2·H2O, and different thiols, viz. Cys,
ESI MS Titration of {Zn(DTT) + L} with Cd2+ and Hg2+. To
provide further proof for the formation of the complexes
10046
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Scheme 3. Schematic Representation of the Events Involving the Heavy Metal Toxicity and Oxidative Stress Wherein the DTT
a
Mimics MTs and L Mimics the Zinc Recognition Element
a
Cd2+ and Hg2+ have been used as heavy metals and H2O2 as oxidant.
DTT, GSH, MPA, Hcy, ME, and Cyst, were purchased from the
commercial suppliers in India and the US. All the fluorescence
titrations were carried out in 1 cm quartz cells by using 50 μL of L, and
the total volume in each measurement was made to 3 mL to give a
final concentration of the ligand as 10 μM. The fluorescence studies
performed in aqueous ethanolic HEPES buffer, pH = 7.4 solution used
Synthesis and Characterization of 1.14 SnCl4 (1.07 g, 4.1
mmol) was added to a solution containing p-tert-butylphenol (6.01 g,
40 mmol) in dry toluene. Bu3N was added dropwise to the above
reaction mixture and stirred the solution for 20 min. To the reaction
mixture, paraformaldehyde (2.43 g, 81 mmol) was added and heated
to reflux for 8 h. After cooling the reaction mixture to room
temperature, the solution was poured into a separating funnel and then
extracted with diethyl ether (2 × 50 mL). The combined organic layer
was washed with saturated sodium chloride and dried over anhydrous
sodium sulfate. The product formed was purified by silica gel column
chromatography using petroleum ether and ethyl acetate. Yield (4.5 g,
always a 50 μL of bulk solution of L prepared at 6 × 10−4
M
concentration in ethanol. A 50 mM HEPES buffer stock solution was
prepared with deionized water, and 1000 μL of this bulk solution is
used for each titration of 3 mL solution. During the titration, the
concentration of the metal perchlorate has been varied accordingly to
result in requisite mole ratios of metal ion to L, and the total volume of
the solution was maintained at 3 mL in each case by addition of
aqueous ethanol. For absorption studies, the final concentration of [L]
was kept constant at 10 μM, and the procedure used for the titrations
was the same as that used for fluorescence titrations. The in situ
prepared zinc complex of L (L:Zn2+ = 1:2) has been used for the
titration of [ZnL] with thiols. The mixture of {[DTT/Cys + Zn2+] +
L} was again subjected to titration with Cd2+, Hg2+, and H2O2 (30%).
The H2O2 (30%) {1 mL in 2 mL buffer} has been diluted by 2-fold
with buffer and used for the oxidation titrations by adding directly.
Experimental for Computational Calculation of L and
[ZnL]. The computational calculations were carried out to find the
mode of complexation of Zn2+ with L. As the formation of 1:1
{L:Zn2+} species has already been established by the ESI-MS and
supported by the Job’s plot, the species was optimized by
computational calculations based on DFT using Gaussian 03 package
(Supporting Information). The initial model for L was prepared from
the published11h crystal data of triazole-linked calix[4]arene conjugate
by removing Zn2+ followed by the protonation of the salicyl OH
moieties and further by introducing methylthiophene group in the
place of n-butyl group in each of the arm. This is then optimized by
going through a cascade process of computational calculations. Thus,
L was optimized by going through PM3 → HF/STO-3G → HF/3-
21G → HF/6-31G → B3LYP/3-21G → B3LYP/6-31G in a cascade
manner, and the complex was optimized using B3LYP/6-31G(d,p).
The output obtained at every stage was given as the input for the next
higher level of calculations. In order to make the neutral complex of L
with Zn2+, the salicyl OH groups were deprotonated and the resulting
L2− was used for further studies. In order to reduce the computational
times, the L2− was converted to L′2− by replacing the tertiary butyl
moieties with hydrogen. The Zn2+ complex of L′2− was made by
placing the Zn2+ far away from this. After the optimization of [ZnL],
NBO charges and single-excitation time-dependent DFT (TDDFT)
calculations were performed.
1
63%). H NMR (CDCl3, δ ppm): 1.25 (s, 9H,), 6.86 (s, 1H, J = 9.1
Hz), 7.44 (d, 1H, J = 2.4 Hz), 7.52 (dd, 1H, J = 2.4 Hz), 9.82 (s, 1H),
10.80 (s, 1H).
Synthesis and Characterization of 2.14 To a mixture of 1 (5.0
g, 28 mmol) and 35% formaldehyde (25 mL) was added 125 mL of
concentrated HCl and stirred the solution for 2 days at room
temperature. Water was added to the mixture, the product was
extracted into CH2Cl2 (3 × 40 mL), and the organic phase was dried
over anhydrous Na2SO4. The CH2Cl2 was then evaporated under
reduced pressure to afford the pure product as yellow liquid. Yield (5.2
g, 82%). 1H NMR (CDCl3): 1.34 (s, 9H), 4.70 (s, 2H), 7.52 (d, 1H, J
= 2.4 Hz), 7.67 (d, 2H, J = 2.5 Hz), 9.90 (s, 1H), 11.28 (s, 1H).
Synthesis and Characterization of L2.11h Compound 2 (2.65 g,
11.89 mmol) was added to a solution of sodium azide (1.519 g, 23.37
mmol) in 30 mL of dimethylformamide under stirring for 12 h. After
completion of reaction mixture was diluted with 100 mL of water and
ethyl acetate. The organic layer was separated and washed with water
and brine. A yellow liquid was obtained upon evaporation of organic
solvent. Yield: 89%. 1H NMR (CDCl3, 400 Hz): 11.2 (br s, 1H), 9.99
(s, 1H), 7.55 (dd, 1H), 7.59 (s, 1H), 4.48 (s, 2H), 1.35 (s, 9 H). IR:
νmax = 3471, 2961, 2686, 2104, 1676.
Synthesis and Characterization of L1.11h 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 the
above stirred mixture over a period of 30 min. The reaction mixture
was refluxed for 24 h and was then allowed to cool to room
temperature. The reaction mixture was filtered over Celite to remove
insoluble particles, and the filtrate was concentrated by a rotatory
evaporator. 100 mL of 2 M HCl was added to the concentrated
reaction mixture, and the product was extracted with dichloromethane
(3 × 100 mL). The combined organic extract was successively washed
with water and brine (100 mL), dried over anhydrous Na2SO4, filtered,
and evaporated to dryness in vacuo. The crude product was
10047
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
recrystallized from CH2Cl2/CH3OH to afford 2 as a white solid (9.10
g, 82% yield). H NMR (400 MHz, CDCl3) δ (ppm): 7.07 (s, 4H),
6.73 (s, 4H), 6.50 (s, 2H), 4.74 (d, J = 2.4 Hz, 4H), 4.37 (d, J = 13.4
Hz, 4H), 3.33 (d, J = 13.4 Hz, 4H), 2.54 (t, J = 2.4 Hz, 2H), 1.30 (s,
18H), 0.90 (s, 18H). HRMS m/z Calcd for C50H60O4: (M + H)
725.4570. Found 725.4576.
Mol. Life Sci. 2002, 59, 627. (d) Palmiter, R. D. Proc. Natl. Acad. Sci. U.
S. A. 2004, 101, 4918. (e) Ercal, N.; Gurer-Orhan, H.; Aykin-Burns, N.
Curr. Top. Med. Chem. 2001, 1, 529.
(3) (a) Colvin, R. A.; Holmes, W. R.; Fontaine, C. P.; Maret, W.
Metallomics 2010, 2, 306. (b) Aizenman, E.; Stout, A. K.; Hartnett, K.
A.; Dineley, K. E.; McLaughlin, B.; Reynolds, I. J. J. Neurochem. 2000,
75, 1878.
(4) (a) Chang, C. J.; Jaworski, J.; Nolan, E. M.; Sheng, M.; Lippard,
S. J. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 1129. (b) Maret, W.
Biochemistry 2004, 43, 3301.
(5) (a) Flora, S. J. S.; Mittal, M.; Mehta, A. Indian J. Med. Res. 2008,
128, 501. (b) Hall, J. L. J. Exp. Bot. 2002, 53, 1. (c) Maret, W.
Biometals 2010, 24, 411. (d) Ejnik, J.; Shaw, C. F.; Petering, D. H.
Inorg. Chem. 2010, 49, 6525.
1
Synthesis and Characterization of L3.11h Compound L1 (3.0 g,
4.14 mmol) was added to the solution of L2 (2.12 g, 9.53 mmol) in
100 mL of dichloromethane and water (50:50) mixture. To this
solution, CuSO4·5H2O (124.04 g, 0.50 mmol) and sodium ascorbate
(328.0 mg, 1.70 mmol) were added. The resulting solution was stirred
for 12 h at room temperature. Upon completion of the reaction as
checked based on 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
× 100 mL) and dried over anhydrous Na2SO4, and the solvent was
removed under vacuo. The crude product was purified by triturating
(6) (a) Smirnova, I. V.; Bittel, D. C.; Ravindra, R.; Jiang, H.;
Andrews, G. K. J. Biol. Chem. 2000, 275, 9377. (b) Haase, H.; Rink, L.
Annu. Rev. Nutr. 2009, 29, 133.
1
with hexane followed by filtering the precipitate. Yield: 89.91%. H
(7) (a) Li, Y.; Hawkins, B. E.; DeWitt, D. S.; Prough, D. S.; Maret,
W. Brain Res. 2010, 1330, 131. (b) Sensi, S. L.; Paoletti, P.; Bush, A. I.;
Sekler, I. Nat. Rev. Neurosci. 2009, 10, 780. (c) Mei, Y.; Frederickson,
C. J.; Giblin, L. J.; Weiss, J. H.; Medvedeva, Y.; Bentley, P. A. Chem.
Commun. 2011, 47, 7107. (d) Tomat, E.; Lippard, S. J. Curr. Opin.
Chem. Biol. 2010, 14, 225. (e) Domaille, D. W.; Que, E. L.; Chang, C.
J. Nat. Chem. Biol. 2008, 4, 168. (f) Komatsu, K.; Urano, Y.; Kojima,
H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447.
(8) (a) Steed, J. W. Science 2002, 298, 976. (b) Reinhoudt, D. N.;
Crego-Calama, M. Science 2002, 295, 2403. (c) Izzet, G.; Zeitouny, J.;
Akdas-Killig, H.; Frapart, Y.; Menage, S.; Douziech, B.; Jabin, I.; Le
Mest, Y.; Renaud, O. J. Am. Chem. Soc. 2008, 130, 9514. (d) Le Poul,
N.; Douziech, B.; Zeitouny, J.; Thiabaud, G.; Colas, H.; Conan, F.;
Cosquer, N.; Jabin, I.; Lagrost, C.; Hapiot, P.; Reinaud, O.; Le Mest, Y.
J. Am. Chem. Soc. 2009, 131, 17800. (e) Seneque, O.; Rager, M.-N.;
Giorgi, M.; Prange, T.; Tomas, A.; Reinaud, O. J. Am. Chem. Soc. 2005,
127, 14833. (f) Perret, F.; Lazar, A. N.; Coleman, A. W. Chem.
Commun. 2006, 2425.
(9) (a) Cacciapaglia, R.; Casnati, A.; Mandolini, L.; Reinhoudt, D.
N.; Salvio, R.; Sartori, A.; Ungaro, R. J. Am. Chem. Soc. 2006, 128,
12322. (b) Cacciapaglia, R.; Casnati, A.; Mandolini, L.; Reinhoudt, D.
N.; Salvio, R.; Sartori, A.; Ungaro, R. J. Org. Chem. 2005, 70, 624.
(c) Cacciapaglia, R.; Casnati, A.; Mandolini, L.; Reinhoudt, D. N.;
Salvio, R.; Sartori, A.; Ungaro, R. J. Org. Chem. 2005, 70, 5398.
(d) Bakirci, H.; Koner, A. L.; Dickman, M. H.; Kortz, U.; Nau, W. M.
Angew. Chem., Int. Ed. 2006, 45, 7400. (e) Ozturk, G.; Akkaya, E. U.
Org. Lett. 2004, 6, 241. (f) Park, S. Y.; Yoon, J. H.; Hong, C. S.;
Souane, R.; Kim, J. S.; Matthews, S. E.; Vicens, J. J. Org. Chem. 2008,
73, 8212. (g) Senthilvelan, A.; Ho, I.-T.; Chang, K.-C.; Lee, G.-H.; Liu,
Y.-H.; Chung, W.-S. Chem.Eur. J. 2009, 15, 6152. (h) Bagatin, I. A.;
de Souza, E. S.; Ito, A. S.; Toma, H. E. Inorg. Chem. Commun. 2003, 6,
288. (i) Jiang, P.; Guo, Z. Coord. Chem. Rev. 2004, 248, 205.
(j) Kumar, M.; Kumar, R.; Bhalla, V. Org. Lett. 2011, 13, 366.
(10) Valeur, B.; Leray, I. Coord. Chem. Rev. 2000, 205, 3. (b) Leray,
I.; Valeur, B. Eur. J. Inorg. Chem. 2009, 3525. (c) Kim, J. S.; Lee, S. Y.;
Yoon, J.; Vicens, J. Chem. Commun. 2009, 4791. (d) de Silva, A. P.;
Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C.
P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515.
(e) Chang, K.-C.; Su, I.-H.; Lee, G.-H.; Chung, W.-S. Tetrahedron Lett.
2007, 48, 7274. (f) Chang, K.-C.; Su, I.-H.; Senthilvelan, A.; Chung,
W.-S. Org. Lett. 2007, 9, 3363. (g) Chang, K.-C.; Su, I.-H.; Wang, Y.-
Y.; Chung, W.-S. Eur. J. Org. Chem. 2010, 4700.
NMR (CDCl3, 400 MHz) δ (ppm): 11.30 (s, 2H), 9.83 (s, 2H), 8.08
(s, 2H), 7.62 (s, 2H) 7.49 (d, 2H), 7.15 (s, 2H), 6.98 (s, 4H), 6.77 (s,
4H), 5.56 (s, 2H), 5.18 (s, 2H), 4.14 (d, J = 13.0 Hz, 4H), 3.17 (d, J =
13.0 Hz, 4H), 1.27 (s, 18H), 1.26 (s, 18H), 0.96). 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) calcd for C74H90N6O8 [M + H]+:
1191.6898; found 1191.6898.
Synthesis and Characterization of L. The mixture of L3 (0.25 g,
0.209 mmol) and the thiophen-2-ylmethanamine (0.047 g, 0.419
mmol) in 5 mL of methanol was stirred for 5 h. A faint yellow
precipitate was observed upon completion of reaction. The precipitate
was filtered to get yellow solid product. Yield: 93%. 1H NMR (CDCl3,
400 MHz): 13.56 (s, 2H), 8.34 (s, 2H), 8.10 (s, 2H), 7.42 (d, 4H),
7.22−7.23 (m, 4H, Sal-H),7.12 (s, 2H), 6.97 (s, 4H), 6.96 (s, 4H),
6.74 (s, 4H), 5.58 (S, 2H), 5.16 (S, 2H), 4.87 (s, 4H), 4.15 (d, 4H),
3.14 (d, 4H), 1.27 (s, 18H), 1.25 (s, 18H), 0.944 (s, 18H). 13C NMR
(CDCl3, 100 MHz) δ (ppm): 165.78, 157.01, 150.58, 149.69, 147.04,
144.01, 141.63, 141.40, 144.30, 132.66, 130.85, 128.93, 127.88, 127.13,
125.75, 125.62, 125.26, 125.04, 124.29, 122.35, 118.18, 69.79, 56.97,
48.91, 34.09, 33.98, 33.88, 31.81, 31.44, 31.08, 30.51. HRMS (ESI)
calcd for C84H100N8O6S2 [M + H]+: 1381.7302; found 1381.7286.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR mass spectral data, absorption data, and
fluorescence data. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 91 22 2576 7162. Fax: 91 22 2572 3480. E-mail:
cprao@iitb.ac.in.
■
ACKNOWLEDGMENTS
■
C.P.R. acknowledges the financial support from DST, CSIR,
and DAE-BRNS. R.K.P. acknowledges CSIR and M.M.
acknowledges UGC for their fellowships.
(11) (a) Kim, S. K.; Lee, S. H.; Lee, J. Y.; Bartsch, R. A.; Kim, J. S. J.
Am. Chem. Soc. 2004, 126, 16499. (b) Schazmann, B.; Alhashimy, N.;
Diamond, D. J. Am. Chem. Soc. 2006, 128, 8607. (c) Kolusheva, S.;
Zadmard, R.; Schrader, T.; Jelinek, R. J. Am. Chem. Soc. 2006, 128,
13592. (d) Filby, M. H.; Dickson, S. J.; Zaccheroni, N.; Prodi, L.;
Bonacchi, S.; Montalti, M.; Paterson, M. J.; Humphries, T. D.;
Chiorboli, C.; Steed, J. W. J. Am. Chem. Soc. 2008, 130, 4105. (e) He,
X.; Yam, W. W. Org. Lett. 2011, 13, 2172. (f) Ni, X.-L.; Wang, S.;
Zeng, X.; Tao, Z.; Yamato, T. Org. Lett. 2010, 13, 552. (g) Diamond,
D. Chem. Soc. Rev. 1996, 15. (h) Pathak, R. K.; Dikundwar, A. G.;
REFERENCES
■
(1) (a) Palmiter, R. D. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 8428.
(b) Blindauer, C. A.; Leszczyszyn, O. I. Nat. Prod. Rep. 2010, 27, 720.
(c) Krezel, A.; Maret, W. Biochem. J. 2007, 402, 551. (d) Nordberg,
M.; Nordberg, G. F. Met. Ions Life Sci. 2009, 5, 1. (e) Krezel, A.; Maret,
W. J. Am. Chem. Soc. 2007, 129, 10911.
(2) (a) Maret, W. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 237.
(b) Carpene, E.; Andreani, G.; Isani, G. J. Trace Elem. Med. Biol. 2007,
21, 35. (c) Coyle, P.; Philcoxa, J. C.; Careya, L. C.; Rofea, A. M. Cell.
10048
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049

The Journal of Organic Chemistry
Article
Guru Row, T. N.; Rao, C. P. Chem. Commun. 2010, 46, 4345.
(i) Pathak, R. K.; Ibrahim, S. M.; Rao, C. P. Tetrahedron Lett. 2009, 50,
2730. (j) Joseph, R.; Rao, C. P. Chem. Rev. 2011, 111, 4658. (k) Kim, J.
S.; Quang, D. T. Chem. Rev. 2007, 107, 3780.
(12) (a) Xu, Z.; Yoon, J.; Spring, D. R. Chem. Soc. Rev. 2010, 39,
1996. (b) Creaven, B. S.; Donlon, D. S.; McGinley, J. Coord. Chem.
Rev. 2009, 253, 893. (c) McGinley, J.; Walsh, J. M. D. Inorg. Chem.
Commun. 2011, 14, 1018. (d) Ni, X.-L.; Zeng, X.; Redshaw, C.;
Yamato, T. J. Org. Chem. 2011, 76, 5696. (e) Zhang, J. F.; Bhuniya, S.;
Lee, Y. H.; Bae, C.; Lee, J. H.; Kim, J. S. Tetrahedron Lett. 2010, 51,
3719. (f) Sahin, O.; Yilmaz, M. Tetrahedron 2011, 67, 3501.
(13) (a) Rivero, I. A.; Gonzalez, T.; Diaz-Garcia, M. E. Comb. Chem.
High. Throughput. Screen. 2006, 9, 535. (b) Sonn, K.; Pankratova, S.;
Korshunova, I.; Zharkovsky, A.; Bock, E.; Berezin, V.; Kiryushko, D. J.
Neurosci. Res. 2010, 88, 1074.
(14) (a) Wang, Q.; Wilson, C.; Blake, A. J.; Collinson, S. R.; Tasker,
P. A.; Schroder, M. Tetrahedron Lett. 2006, 47, 8983. (b) Lambert, E.;
Chabut, B.; Chardon-Noblat, S.; Deronzier, A.; Chottard, G.;
Bousseksou, A.; Tuchagues, J.-P.; Laugier, J.; Bardet, M.; Latour, J.-
M. J. Am. Chem. Soc. 1997, 119, 9424. (c) Shao, N.; Zhang, Y.;
Cheung, S.; Yang, R.; Chan, W.; Mo, T.; Li, K.; Liu, F. Anal. Chem.
2005, 77, 7294. (d) Joseph, R.; Chinta, J. P.; Rao, C. P. Inorg. Chem.
2011, 50, 7050−7058.
(15) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.;
Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.;
Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J.
B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev,
O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.;
Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;
Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.;
Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman,
J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen,
W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02;
Gaussian, Inc.: Wallingford, CT, 2004.
(16) Mameli, M.; Aragoni, M.; Arca, M.; Caltagirone, C.; Demartin,
F.; Farruggia, G.; De Filippo, G.; Devillanova, F.; Garau, A.; Isaia, F.;
Lippolis, V.; Murgia, S.; Prodi, L.; Pintus, A.; Zaccheroni, N. Chem.
Eur. J. 2010, 16, 919.
(17) (a) Ko, C. K.; Wu, J. -S.; Kim, H. J.; Kwon., P. S.; Kim, J. W.;
Bartsch., R. A.; Lee, J. Y.; Kim, J. S. Chem. Commun. 2011, 47, 3165.
(b) Jung, H. S.; Ko, K. C.; Lee, J. H.; Kim, S. H.; Bhuniya, S.; Lee, J. Y.;
Kim, Y.; Kim, S. J.; Kim, J. S. Inorg. Chem. 2010, 49, 8552. (c) Maity,
D.; Manna, A. K.; Karthigeyan, D.; Kundu, T. K.; Pati, S. K.;
Govindaraju, T. Chem.Eur. J. 2011, 17, 11152.
10049
dx.doi.org/10.1021/jo201865x|J. Org. Chem. 2011, 76, 10039−10049