A luminescent on-off probe based calix[4]arene linked through triazole with ruthenium(ii) polypyridine complexes to sense copper(ii) and sulfide ions

Article abstract of DOI:10.1039/c9nj01632e

A bis-ruthenium(ii) polypyridyl complex was connected with a p-tert-butyl calix[4]arene platform (Ru₂L) through 1,2,3-triazole and used as a luminescent probe to detect selected ions. Ru₂L acted as a highly sensitive and selective turn off sensor towards Cu²⁺ ions in CH₃CN/10 mM HEPES buffer (pH = 7.4) (8/2, v/v) medium. The detection limit of Cu²⁺ was found to be 4.11 μM. The Cu²⁺ ion binding with Ru₂L showed a paramagnetic signal in EPR analysis. The ensemble (Ru₂L-Cu²⁺) was investigated with a variety of anions, but only sulfide anions enhanced the emission intensity in the presence of other anions. The ensemble works as a turn-on sensor towards S^2-via a metal ion displacement method to form stable CuS. The detection limit of S^2- was estimated from the emission intensity to be 0.35 μM, which is an excellent result with respect to supramolecular platform based sensors in an aqueous medium. Ru₂L also showed low cytotoxicity against human lung cancer cell line A549 and hence can be used in cell imaging.

Full text of DOI:10.1039/c9nj01632e

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Luminescent On-Off Probe based Calix[4]arene Linked through
Triazole with Ruthenium (II) Polypyridine Complexes to Sense
Copper (II) and Sulfide ions
Received 00th January 20xx,
Accepted 00th January 20xx
Mohanraj Ramachandran a, Sambandam Anandan*a and Muthupandian Ashokkumarb
DOI: 10.1039/x0xx00000x
Bis-Ruthenium(II) polypyridyl complex was connected with p-tert-butyl calix[4]arene platform (Ru2L) through 1,2,3-triazole
and used as a luminescent probe to detect selected ions. Ru2L acted as a highly sensitive and selective turn off sensor
towards Cu2+ ion in CH3CN/10mM HEPES buffer (pH = 7.4) (8/2, v/v) medium. The detection limit of Cu2+ was found to be
4.11 M. The Cu2+ ion binding with Ru2L showed paramagnetic signal in EPR analysis. The ensemble (Ru2L-Cu2+) was
investigated with a variety of anions, only sulfide anion enhanced the emission intensity in the presence of other anions.
The ensemble works as a turn-on sensor towards S2- via metal ion displacement method to form stable CuS. The detection
limit of S2- was estimated from the emission intensity is to be 0.35 M, which is an excellent result from supramolecular
platform based sensors in an aqueous medium. The Ru2L also showed low cytotoxicity against Human lung cancer cell lines
A549 and hence can be used in cell imaging.
www.rsc.org/
Introduction
groups, possessing specific conformational properties and
cavities that are capable of encapsulating neutral and ionic
Well designed judicious receptors are necessary for detecting
specific ions and molecules in the area of sensors. Especially,
determining the existence of metal ions and anions is of great
interest in the field of chemical sensors. Copper is one of the
most crucial and third abundant trace elements in the living
biosystem.1,2 Under physiological conditions, the minimum or
maximum level of copper ion variation from normal level can
produce dangerous biological effects.3,4 Sensing of biologically
relevant anions has been one of the most interesting fields,
since monitoring its level would provide resistance to many
diseases. Detection of inorganic sulfide ion (S2-) in living cells is
of great importance. In biological systems, sulfide anions are
generated by sulfur-containing amino acids present in meat
protein as well as microbial reduction of sulfate by bacteria.5,6
The sulfide anions are extensively produced in many industrial
processes and contaminate the enviroment5. The excess level
of sulfide exposure can cause diseases like unconsciousness,
paralysis, and mucosa.7-9 There is a need to develop a sensor
for the selective detection of sulfide ions.
species.10-11 Calix[4]arene based sensor is used to detect the
availability of cation and anion using different methods, for
example, photophysical, electrochemical, nanometal, self-
assembled monolayer.11,12-14
The photophysical method is an emerging technique for
analyzing and detecting specific ions and molecules. The
mechanisms involved in the photophysical method include
Photoinduced Electron Transfer (PET), Photoinduced Charge
Transfer (PCT), Aggregation-Induced Emission (AIE), and
Forster Resonance Energy Transfer (FRET).12,15
Recently many sensors have been developed using
supramolecular
architectures.
Calix[4]arene
is
a
supramolecular platform. For instance, Cyclodextrin,
Calixarene, Rotaxane. Calix[4]arenes are cyclic oligomers
composed of phenol units associated through methylene
a.Department of Chemistry, National Institute of Technology, Tiruchirappalli-620
015, India
b School of Chemistry, University of Melbourne, Vic 3010, Australia.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
Scheme 1. (A) Synthetic route for 5,11,17,23-tetra-t-butyl-25,27-bis(O-
propargyl)calix[4a]rene (1) and (B) Synthetic route for 4-azido-2,2’-bipyridyl
(2d)
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Scheme 2. Synthetic route for L and Ru2L
As per the PET mechanism, the fluorophore provides a signal Cu2+ ensemble. This is a novel observation to recognize sulfide
upon ion binding with the ionophore. anion by employing a metal displacement method, using
Many designed molecules with a variety of fluorogenic copper coordinated triazole moiety.
moieties such as Pyrene, BODIPY, Ru(II) complexes are used for
the PET process.16-18 Ru(II) polypyridyl complexes have been Result and discussion
extensively accepted as a chromosphere due to their immense
photochemical and electrochemical properties. Ru(II) Synthesis and Characterization of 5,11,17,23-tetra-t-butyl-
polypyridyl complexes have been used in many applications 25,27-[bis(O-methyl)-2H-triazole-4-2,2’bipyridyl]calix[4]arene
such as DNA interaction, anticancer agent, photodynamic (L)
and
Bis-ruthenium
polypyridyl-triazole-4-2,2'-
therapy, water oxidation catalysts and as a fluorophore in bipyridylcalix[4]arene (Ru2L)
sensors.19-22 Particularly in the growing field of anticancer
activity, ruthenium-based complexes with wide stable The synthetic procedure of 1, 2d and L, Ru2L is shown in
oxidation states (II, III, and IV) under physiological condition Scheme 1 and Scheme 2. The characterization results are
take part as an alternative for platinum complexes.23-28 Many included in supporting information (S1-S17). 4-azido-2,2'-
toxic heavy metal ions and biologically important anions are bipyridine was synthesized from 4-nitro-2,2-bipyridine. Three
selectively monitored by using diverse luminescent Ru(II) steps were involved in synthesizing of 4-nitro-2,2'-bipyridine.
complexes.29-31 Triazole, amide, and ester are different types Initially, selective oxidation takes place on one of the nitrogens
of linkers used to connect the Ru(II) polypyridyl fluorophore in 2,2'-bipyridine by H2O2/TFA and a quantitative conversion of
with a binding core of the analyte molecule. Triazole unit has 2,2'-bipyridine-N-oxide was observed. When nitration takes
attracted much attention due to its electron rich and electron place on 2,2'-bipyridine-N-oxide using a nitrating mixture (as
deficient nitrogens.30,32,33 The bridging triazole, which acts as a fuming HNO3/H2SO4), 40% yield of 4-nitro-2,2'-bipyridine-N-
linker in between the fluorophore and calix[4]arene, was used oxide was observed. Further 4-nitro-2,2'-bipyridine-N-oxide
in sensors to sense metal ions as well as anions.11,34-37 Various was reduced with phosphorous trichloride to obtain 4-nitro-
signaling units were placed in the sensory probe with triazole 2,2'-bipyridine. Using CuAAC click reaction, the ligand (
L) was
modified calix[4]arene.12,38-41
prepared (Yield 41%) from and 2d with CuSO4.5H2O and
1
Several common types of ON-OFF, OFF-ON, ON-OFF-ON sodium ascorbate in DCM:Water (1:1 v/v) medium. The singlet
fluorescent tactics were used in sensing of ion and peak at 9.04 ppm and a new band at 3156 cm-1 are the
molecules.42-45 Many receptors are reported based on copper- indications for triazole formation from 1H NMR and FT-IR
organic fluorophore ensembles46-49
,
copper-nanoparticles spectra, respectively (See figure S1-S17). The complex Ru2L
ensembles50,51 and a few analytes in Ru-Cu ensembles are also was synthesized by the reaction of cis-Ru(bpy)2Cl2 with a
available.52-54 Herein, we report on the design and application corresponding amount of
in CH3OH. The proton NMR
L
of Ru(II) polypyridyl complex (Ru2L) as a luminescent probe spectrum of Ru2L clearly shows by two peaks that calix[4]arene
conjugated through triazole with the help of the calix[4]arene retained its cone conformation. The peaks appeared at 3.24,
platform. The biological activity of the developed sensor was 4.09 ppm (for bridging methylene of calix[4]arene) and the
evaluated using human lung cancer (A549) cells. The probe peaks at 0.99, 1.34 ppm (for tert-butyl group containing
developed showed selectively towards Cu2+ ion. The emission hydrogen) (See figure S1-S17) also confirmed the cone
was quenched only with that paramagnetic Cu2+ ion, without conformation of calix[4]arene in the solution state.36,55 The
1
interference from other metal ions. Ru2L luminescence could formation of
be reverted by incremental addition of sulfide anion to Ru2L-
L
and Ru2L was also confirmed by H, 13C NMR,
FT-IR, Mass spectra and Elemental analysis (See figure S1-S17).
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However, under UV light illumination, a ViewreAdrt-icoleraOnnlginee
luminescence of Ru2L was turned off upon the addition of
copper ions (Fig. S.19A). Upon excitation at the MLCT band
region, Ru2L can emit intense luminescence at 637 nm in
DOI: 10.1039/C9NJ01632E
CH3CN/10 mM HEPES buffer (pH = 7.4) (8/2, v/v) at room
3
temperature due to MLCT dπ(Ru)
 dπ* transition (Fig. 1).
The luminescence behaviour of Ru2L was examined with a
wide range of metal ions (toxic metals and metals present in
biological organisms) up to 10 equiv. ratio (Fig. 2A).
Interestingly, the emission intensity of Ru2L is significantly
quenched only with 4 equiv. of Cu2+ ions (Fig. 2B) whereas the
emission intensity is only slightly affected upon the addition of
some other metal ions.53,56 The photoelectron transfer (PET)
mechanism (photoelectron transferred from excited state of
Ru moiety to paramagnetic Cu2+ ion) was the reason for
luminescence quenching.57 Nearly 80% of luminescence
intensity is quenched by Cu2+ ions whereas about 22-25% of
the intensity was quenched by Fe2+ and Ni2+. The binding
constant value (2.31 x 104) was calculated from the emission
spectra for Ru2L interaction with copper (II) ion which is good
agreement with already reported values.53 The detection limit
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Fig.1. Combined UV-Vis and Fluorescence Spectra of Ru2L (10 ) in
CH3CN/HEPES Buffer (pH = 7.4) (8/2, v/v).
Luminescence behaviour of Ru2L towards metal ions
UV-Vis absorption spectrum of Ru2L shows three bands (Fig. 1) value is found to be 4.11 x 10-6 M from emission spectra, which
in CH3CN/10 mM HEPES buffer (pH = 7.4) (8/2, v/v). Intense is convincingly higher selectivity compared to the level of
higher energy transitions corresponding to n-

* and

-

*
detection by the U.S. environmental protection agency (20 x
bands appear at 241 and 288 nm, respectively. Another 10-6 M of copper ion in drinking water).53 Other metal ions
intense band appears at 457 nm from low energy singlet state interference of Ru2L-Cu2+ study with copper (II) ion was carried
metal to ligand charge transfer (1MLCT). At room temperature, out and its luminescence intensity can be seen from Fig. 3A
UV-Vis spectrum of Ru2L was screened towards various types that there is insignificant interference by other metal ions. A
of metal ions: Ag+, Al3+, Fe2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ linear dependency of fluorescence intensity on Cu2+ ion
Zn2+, Fe3+, Ca2+ and Co2+ (10 equiv.) and Cu2+ (4 equiv.) in concentration (r = 0.9948) was obtained (Fig. 3B). From these
CH3CN/10 mM HEPES buffer (pH = 7.4) (8/2, v/v) (Fig. S.18A). results, Ru2L can be found to be a highly reliable probe for
There is no significant difference in the UV-Vis spectra among detecting Cu2+ ions in the presence of other cations. No
these ions. However, a hyperchromic effect is observed upon significant change was noticed in the luminescence behaviour
addition of different concentrations of Cu2+ ion (0-4 equiv.) upon moving to acidic to neutral pH in the presence of Cu2+
(Fig. S.18B). No colour change was noticed by naked eye ions. Whereas moving from neutral to basic pH in the presence
detection while adding copper ions.
of Cu2+ ions, the emission intensity was found to decrease (Fig.
S.20B) probably due to the deprotonation of the two hydroxyl
Fig.2. (A) Emission spectra of Ru2L (10 ) with various types of metal
ions (10 equiv.) and Cu2+ ion (4 equiv.) in CH3CN/HEPES buffer (pH =
7.4) (8/2, v/v).
Fig.2. (B) Incremental addition of Cu2+ ion (0-4 equiv.) to Ru2L (10 M)
in CH3CN/HEPES buffer (pH = 7.4) (8/2, v/v).
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Fig.3. (A) Interference study of bar diagram of Ru2L+Cu2+ (10 M) with
various types of metal ions (10 equiv.).
Fig.3. (B) Linear plot of incremental addition of Cu2+ ion to Ru2L in
CH3CN/HEPES buffer (pH = 7.4) (8/2, v/v).
groups of calix[4]arene. Even at higher pH range in the potential was obtained at +0.76 V, due to the tertiary amine
absence of Cu2+ ions, there is no luminescent quenching oxidation process. The Ru2L-Cu2+ exhibits three reversible
because t-butyl substituent of p-tertbutyl calix[4]arne moiety oxidation potentials at +1.17, +0.76 and +0.47 V in the
can effectively perturb the formation of quinone (from phenol) particular window range (-0.5 to 1.6 V). First, two potentials
moiety which is
complexes.
a
quencher to ruthenium polypyridyl were assigned to RuIII/RuII redox process, a tertiary amine
oxidation process and the third one +0.47 V was assigned to
the copper ion (CuII/CuI vs NHE) redox potential process.60 The
EPR and Electrochemical studies
luminescent lifetime decay profile diagram of Ru2L and the
ensemble (Ru2L-Cu2+) shows a bi-exponential decay and were
To further confirm the copper ion binding, EPR measurements represented in Fig. S.22B. In the absence of copper (II) ion,
were made. The spectra were recorded with a microwave Ru2L shows a bi-exponential decay with lifetime 2 = 38 ns at
frequency of 9.8 GHz in CH3CN at room temperature and liquid room temperature. The lifetime of Ru2L-Cu2+ ensemble was
nitrogen temperature. At room temperature, there is no EPR decreased by twenty nanoseconds (from 2 = 38 to 18 ns)
signal for Ru2L due to the diamagnetic character of Ru2+ ions. when compared to Ru2L. A small lifetime change is attributed
Addition of Cu2+ ions to Ru2L a new signal around 312 mT to the decay of the excited state quenched by photoinduced
which corresponds to the binding of Cu2+ ion on Ru2L Ru2L- electron transfer (PET).60
(
Cu2+) (Fig. S.21A) was observed. The hyperfine splitting pattern
(peak at 2.34 and 2.04 responded to average g∥ and g⊥ values, Luminescence behaviour of Ru2L-Cu2+ towards anions
respectively) was observed clearly at liquid nitrogen
temperature (Fig. S.21B). From the above findings, it is The ensemble (Ru2L-Cu2+) contains supramolecular platform, it
observed that g∥ > g⊥ > 2.003 for Ru2L-Cu2+ which illustrate the could be formed 1:1 ratio from Ru2L and Cu2+ ion in
characteristic of d9 copper (II) and a ground state doublet CH3CN/10mM HEPES buffer (pH = 7.4) (8/2, v/v). Only
2
2
−y
representing the existence of an unpaired electron in the dx
negligible changes occurred in absorption spectra of the
ensemble with sulfide anion (7 equiv.) whereas no changes
orbitals.57,58
The electrochemical properties of Ru2L and Ru2L-Cu2+ were were observed even the addition of 10 equiv. of various kind
studied using three electrode cyclic voltammetric system in of anions like CH3COO-, CN-, CO32-, Cys, F-, HPO4 , HSO4 , NO2 ,
-
-
-
-
-
2-
degassed and dry CH3CN solvent with tetrabutylammonium NO3 , OH, SO3 and GSH in CH3CN/10mM HEPES buffer (pH =
hexafluorophosphate as supporting electrolyte (Fig. S.22A). 7.4) (8/2, v/v). The luminescence behaviour of Ru2L-Cu2+ was
[Ru(bpy)3]2+ was taken as
a reference complex, which checked with 10 equiv. of above mentioned various anions in
generates one positive potential from RuII/RuIII redox process CH3CN/10mM HEPES Buffer (pH = 7.4) (8/2, v/v) (Fig. 4A). From
and three negative potentials from bipyridine moiety.59 Ru2L these scanning results, it can be seen that sulfide anion
exhibits two reversible oxidation potentials and one quasi- behaves different to other anions in enhancing the emission
reversible process occurring at +1.08 V vs Ag/AgCl which is intensity of the ensemble. The emission intensity of the
responsible from RuIII/RuII process of dinuclear ruthenium ensemble was increased gradually at 637 nm upon addition of
complexes. The peak value of dinuclear ruthenium complex 0-7 equiv. of S2- anion (Fig. 4B). This is caused by the formation
was shifted towards negative side compared to mononuclear of stable copper sulfide or the development of CuxSx rings in
ruthenium complex due to two one-electron RuIII/RuII redox solution.61 Sulfur ion has more affinity towards Cu2+ ion
process, but only one oxidation potential is obtained for (compared to nitrogen and oxygen) and for this reason, its
dinuclear ruthenium complex.31 The second reversible
luminescence properties of Ru2L is retained.
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Fig. 4.(B) Incremental addition of S2- anion (0-7 equiv.) to Ru2L-Cu2+ in
CH3CN/HEPES buffer (pH = 7.4) (8/2, v/v).
Fig.4.(A) Emission spectra of Ru2L-Cu2+ (10 M) with different types of
anions (10 equiv.) and S2- anion (7 equiv.)
Fig.5.(A) Interference study from the emission spectra of Ru2L-Cu2++S2-
with different types of anions (10 equiv.).
Fig.5.(B) Linear plot from incremental addition of S2- ion to Ru2L-Cu2+
in CH3CN/HEPES buffer (pH = 7.4) (8/2, v/v).
In naked eye view, luminescence behaviour of the ensemble Proposed Sensing Mechanism
was investigated with all anions under UV-light illumination.
Only sulfide anion responded by showing the emission The target complex (Ru2L) is soluble in aqueous medium due
enhancement (Fig. S.19B). The reason may be that the sulfide to the presence of bis-ruthenium polypyridyl unit. The p-
anion act as a turn-on sensor (as restored the emission tertbutyl calix[4]arene has been found to be a successful
intensity of Ru2L) from turn-off (Ru2L-Cu2+). The maximum supramolecular platform due to the formation of cone
emission intensity from the ensemble was retained (98%) conformation modified with bis-ruthenium polypyridyl
when S2- anion was added. Interference of sulfide anion was complex through triazole linker. Two hydroxyl groups would
tested in the presence of other anions with Ru2L-Cu2+, and in marginally assist triazole moiety for coordination with a metal
this competitive experiment, sulfide anion showed selectivity ion and also render the varaible luminescent behaviour of
towards copper ion in the presence other anions (Fig.5A). A Ru2L. Triazole moiety acts as an excellent linker and bind
good linear relationship (r = 0.9918) was obtained while efficiently with metal ions through due to the presence of
incremental addition of sulfide anion to Ru2L-Cu2+ (Fig.5B). The other atoms capable of coordinating. The cavity of
detection limit of S2- was estimated from the emission calix[4]arene, conformation of the platform, N2O2 (two
intensity of Ru2L-Cu2+ is to be 3.56 x 10-7 M and it is the better nitrogen from triazole moiety and two oxygen from
result from supramolecular platform based-probe in aqueous calix[4]arene) binding core and bis-ruthenium polypyridyl
condition.43 Furthermore, the selectivity of the ensemble was complex take part a major role in sensing Cu2+ ions and anions
examined with sulfide anions in variant pH level. Negligible by fluorescence. Ru2L-Cu2+ ensemble has a very low (5.6 fold
changes were observed (Fig. S.20A). From the above less) luminescence compared to Ru2L, while Cu2+ ion could be
discussions, Ru2L-Cu2+ is found to be an efficient turn on binding with N2O2 core of the probe. The binding ratio (1:1) of
luminescent probe towards sulfide anion in the presence of metal with the probe was confirmed by job plot (Fig. S23A).
other anions.
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Fig.6. Proposed mechanism for Cu2+ ion binding with Ru2L and S2- anion releases Cu2+ ion from Ru2L-Cu2+.
Cytotoxicity, AO/EB staining and Hoechst 33528 staining
The metal ion exchange experiment was carried out using studies towards Ru2L
three different pH, this result reveals that some negligible
changes were obtained (Fig. S25). Using the emission Few ruthenium compounds have worked as a potential
intensities and the molar extinction coefficients at 457 nm, we anticancer activity in clinical trials.25,26 However, there are
have calculated the quantum yields of Ru2L is 0.033 whereas some disadvantages such as uncontrolled reactions with
quantum yields is 0.062 upon using the standard [Ru(bpy)3]2+. proteins, high cytotoxicity, and poor water solubility. MTT
However, upon addition of Cu2+ ion to Ru2L, the quantum yield assay method was used to examine the cytotoxicity of Ru2L
was decreased from 0.033 to 0.010 whereas the quantum yield against human lung cancer (A549) cell lines. Inhibition of
was retained back to 0.031 for the Ru2L+Cu2++S2- (i.e., the cellular viability was tested after 24 h incubation with different
addition of S2-.54
concentration of the complexes (Ru2L). As a result, the
Metal displacement approach is one of the tactics in inhibition level increased gradually from 0 to 88% when
luminescent anion sensors. In this approach, a specific anion increasing the concentration of the complex from 0 to 200
displaces the metal ion selectively at optimum condition. The
sulfide anion reacts with the ensemble to enhance toxicity towards A549 cell lines compared to cis-platin (IC50
luminescence due to metal displacement method (Fig. 6). The 18.5 ± 1.4 M). Apoptosis is the process of linking a series of
g/ml (Fig. S26). Ru2L (IC50 = 106.5 ± 0.5 M) showed low
=

emission intensity of Ru2L was retained at 98% from ensemble biochemical actions important to characteristic cell
by metal displacement method through sulfur anion (Fig. morphology modification and finally controlled cell death. The
S23B). Moreover, the sulfur ion has more coordinating power necrosis is another form of uncontrolled cell death (autolysis)
(compared to nitrogen and oxygen) towards copper (II) ion to divergent from apoptosis where uncontrolled cell death leads
generate copper sulfide and to enhance fluorescence by thus to lysis of cells.62 Initially, A549 cell lines were incubated with
reformed Ru2L molecule.
Ru2L for 24 h. After that, the treated cell lines were stained
Fig.7. AO/EB staining of A549 cells with the control (a), incubated with Ru2L complex (b) showing the variations in nuclear morphology and Hoechst
33258 staining of A549 cells with the control (c), treated with Ru2L complex (d) screening the changes in nuclear morphology.
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luminescent was retained upon addition of S2- (FiVgi.ew8A).rtiFcluerOtnhliener
studies are required to understand the interaction of
DOI: 10.1039/CR9NuJ20L1w63i2thE
cellular organelles.
6
7
Conclusions
8
9
Supramolecular based Ru2L has been synthesized and
converted to an aqueous soluble probe using hydrophobic p-
tertbutyl calix[4]arene and bis-ruthenium polypyridyl
complexes through triazole linker. In the presence of a wide
range of metal ions, the emission intensity of Ru2L was
marginally quenched only with paramagnetic Cu2+ ion, due to
photoinduced electron transfer mechanism. The binding mode
1:1 was further confirmed by the Job’s plot and the binding
constant was estimated to be 2.31 x 104 M-1 from the emission
spectra. Upon the addition of sulfide anions to Ru2L-Cu2+, the
weak luminescence of Ru2L-Cu2+ significantly enhanced
showing the ‘turn-on’ sensitivity. Thus, Ru2L-Cu2+ could be an
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Fig.8. The controlled A549 cells (a and e) treated with Ru2L (10 M) for
1 h (b and f) and the cells were incubated with two different
concentration of Cu2+ ion such as 10 M (c), 20 M (g) for 60 min and
then Ru2L-Cu2+ loaded cells were further treated with two different
concentration of sulfide anion 20 M (d) and 40 M (h).
with acridine orange (AO) and ethidium bromide (EB) solution. excellent turn-on luminescent sensor towards discriminating
Morphological changes at the time of cell death are significant sulfide anions in the presence of other anions. From biological
criteria in apoptosis that can be studied via AO/EB staining. studies, Ru2L complexes showed low toxicity against A549 cell
The control or viable cells became visibly green having uniform lines compared to cis-platin and ruthenium polypyridine based
chromatin with the inviolate cell membrane. Early apoptotic complexes reported in the literature. Furthermore, Ru2L
features such as cell shrinkage, condensation, fragmentation complexes have been used in fluorescence imaging studies
were spotted mostly in Ru2L treated cells. Further, a small where it clearly differentiates the apoptosis and necrosis cells
fraction of cells show the morphology of blebbing, apoptotic from control cells using cellular uptake, AO/EB staining and
bodies and a very few necrosis modes of cell death also Hoechst 33528 staining methods.
appeared in orange-red colour (Fig. 7). Human lung cancer cell
lines were incubated with IC50 dose of the method and
monitoring the changes in cytology of the cells (with special
Experimental Section
reference to the nuclei and cytoplasm). It is observed that the
All synthetic procedures were carried out under an inert
initial apoptotic characteristics such as cell shrinkage,
atmosphere. cis-Ru(bpy)2Cl2 was prepared according to the
chromatin condensation and fragmentation have been mostly
literature.63 All chemicals were, from Alfa Aesar and Sigma
were also noticed. The Hoechst 33528 staining (Fig. S27B)
Aldrich and used without further purification. The solvents
illustrates the different percentage of normal and abnormal
CH3CN and CH3OH were used after distillation. Metal
cells from control and Ru2L treated cells. Hence, the findings
perchlorate salts procured from Merck were used as the
from AO/EB staining agreed with Hoechst staining results,
source for metal ions and all anions used were tertbutyl
which induce apoptosis (cell death in A549 cell lines) by the
ammonium (TBA) salts. UV-Vis and fluorescence spectra were
complex (Ru2L).
recorded on
spectrophotometer
a
Specord
and
S
600 diode-array UV-Vis
Shimadzu RF-5301 PC
Live cell Imaging studies
spectrofluorophotometer, respectively at room temperature.
The FT-IR spectra were recorded on a Thermo Scientific Nicolet
iS5 FT IR spectrometer. The cyclic voltammetric analysis was
performed on a Autolab three electrode system, where
The cellular uptake was measured in terms of intracellular
fluorescence intensity displayed by the treatment of Ru2L with
live cells. The Ru2L (10 M) treated with A549 cell lines for 1h
Ag/AgCl as
a reference electrode, glassy carbon as a
described their localization. It is clear from the images that,
Ru2L is mainly present on the cytoplasm of the cells and some
of them are even accumulated in the nucleus membrane.
Subsequently, the Ru2L-loaded cells were further treated with
working electrode and platinum wire as a counter electrode
in
an
acetonitrile
solvent
containing
0.1
M
-
tetrabutylammonium hexafluorophosphate (tBu4N+PF6 ) as the
supporting electrolyte with 100 mV scan rate and the system
was calibrated using ferrocene/ferrocenium ion (Fc/Fc+)
standard. The matrix-assisted laser desorption/ionization-time
of flight (MALDI-TOF) mass spectrometry was used to
two different (10
M, 20 
M) concentrations of Cu2+ for
another 60 min at room temperature. Ru2L complex loaded
cells contain bright intracellular red luminescent, which clearly
illustrates that while increasing Cu2+ ion concentrations, the
red luminescent gradually weaken. Ru2L-Cu2+ loaded cells were
incubated furthermore for one hour with two different sulfide
determine the mass of the sample. H and 13C NMR spectra
1
were recorded with Bruker Avance 500 and AMX 400
spectrometers using CDCl3 and DMSO-d6 solvent (TMS as
internal standard) in the ppm range. The luminescence lifetime
anion concentrations (20 and 40
M) and recorded the
images. It is seen that the maximum bright intracellular red
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was carried out with the Time-Correlated Single Photon heated at 110°C for 5 hours. Once cooled, the solution was
View Article Online
Counting method (TC-SPC) in
a
Horiba FluoroMax-4 poured into ice (150 g) and adjustedDtOoI: 1p0H.10389/uCs9iNnJg0136382%E
instrument. EPR were recorded using Bruker EMX Plus X-band aqueous NaOH. The light yellow precipitate was filtered and
(9.8 GHz) spectrometer.
washed with water. The solid was dissolved in methylene
chloride, the mixture was extensively extracted with water and
the combined organic layers were dried over sodium sulfate,
filtered and concentrated to yield 1.52 g (7.0 mmol, 40%) of 4'-
5,11,17,23-tetra-t-butyl-25,27-bis(O-propargyl)calix[4]arene
(1)64
p-tert-butylcalix[4]arene (10.0 g, 15.43 mmol, 1 eq.) in dry nitro-2,2'-bipyridine-N-oxide as a beige solid. 1H NMR (CDCl3) δ
acetone (200 mL) was stirred with potassium carbonate (5.10 (ppm): 9.16 (d, J = 3.4 Hz, 1H), 8.89 (dt, J = 8.1 & 0.8 Hz, 1H),
g, 36.72 mmol, 2.37 eq.) at room temperature for 1 h. A 8.79 (ddd, J = 4.8 & 0.9 Hz, 1H), 8.36 (d, J = 7.3 Hz, 1H), 8.06
solution of propargyl bromide (6.49 g, 30.86 mmol, 2 eq.) in (dd, J = 7.3 & 3.3 Hz, 1H), 7.88 (td, J = 7.8 & 1.7 Hz, 1H), 7.43
dry acetone (50 mL) was added dropwise into the above- (ddd, J = 6.2, 4.8 & 1.22 Hz,1H). (13C NMR, 100 MHz) δ (ppm):
stirred mixture over a period of 30 min. The reaction mixture 149.8, 148.2, 147.5, 142.4, 141.9, 136.6, 125.3, 125.0, 122.5,
was refluxed for 24 h and was then allowed to cool to room 118.8.
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temperature. Using a celite pad, the reaction mixture was
filtered to remove insoluble particles, and the filtrate was 4-Nitro-2,2'-bipyridyl (2c)65
concentrated by a rotatory evaporator. 100 mL of 2 M HCl was
added to the concentrated reaction mixture, and the product In an ice cooled condition, phosphorous trichloride (3.6 ml,
was extracted with dichloromethane (3 × 100 mL). The 41.1 mmol, 3 eq.) was added to 4-nitro-2,2'-bipyridyl-N-oxide
combined organic extract was consecutively washed with (2b) (3.00 g, 13.6 mmol, 1 eq.) in dry DCM (30 ml) and the
water and brine (100 mL), dried over anhydrous Na2SO4, mixture was heated at reflux for overnight. After cooling the
filtered and evaporated to dryness in a vacuum. The crude reaction mixture was carefully poured into crushed ice and
product was recrystallized from DCM/Methanol to afford 1 as basified with 38% sodium hydroxide. The aqueous layer was
1
a white solid (9.10 g, 82% yield). H NMR (CDCl3) δ (ppm): 7.07 exhaustively extracted with dichloromethane and the
(s, 4H), 6.72 (s, 4H), 6.47 (s, 2H, OH), 4.75 (d, J = 2.4 Hz, 4H), combined organic solutions were washed with water, then
4.37 (d, J = 13.4 Hz, 4H), 3.33 (d, J = 13.4 Hz, 4H), 2.53 (t, J = 2.4 dried over sodium sulfate and concentrated to yield a pale
Hz, 2H), 1.30(s, 18H), 0.89 (s, 18H). 13C NMR (CDCl3, 100 MHz) yellow solid. (2.21 g, 80 %) 1H NMR (CDCl3) δ (ppm): 9.18 (d, J =
δ (ppm): 150.2, 149.4, 147.4, 141.7, 132.7, 128.0, 125.5, 125.0, 2.2 Hz, 1H), 8.97 (d, J = 5.3 Hz, 1H), 8.77 (d, J = 4.3 Hz, 1H), 8.49
78.7, 63.2, 33.9, 33.8, 32.0, 31.6, 30.9. FT-IR: 2959, 2120 cm-1.
(dt, J = 8.0 & 0.9 Hz, 1H), 8.04 (dd, J = 5.4 & 2.2 Hz, 1H), 7.90
(td, J = 7.6 & 1.8 Hz, 1H), 7.43 (ddd, J = 6.2, 4.9 & 1.2 Hz, 1H).
(13C NMR, 100 MHz) δ (ppm): 159.4, 155.0, 153.9, 151.2, 149.5,
137.8, 124.9, 121.4, 115.7, 113.7.
2,2'-Bipyridine-N-oxide (2a)65
The mixture of 2,2'-bipyridine (7.8 g, 0.05 mol, 1.0 eq.) and
30% hydrogen peroxide (8.5 mL, 2.6 g, 0.075 mol, 1.5 eq.) 4-azido-2,2'-bipyridyl (2d)66
were stirred for 6 hours in trifluoroacetic acid (40 mL) at room
temperature. After that, neutralised using aqueous 6N NaOH 4-nitro-2,2'-bipyridyl (0.57 g, 0.4 mmol, 1 eq.) with NaN3 (1.50
and extracted with chloroform (4 × 50 mL). The combined g, 3.85 mmol, 8.95 eq.) was heated in DMF (10 mL) for 5 hours
organic layers were washed with aqueous saturated sodium at 100 °C. The solvent was removed under vacuum and water
chloride, dried over Na2SO4, filtered and concentrated to (40 mL) was added to the yellow solid, the aqueous phase was
afford colourless oil, which solidified under vacuum into a extracted with dichloromethane. The combined organic phase
white solid. (7.57 g, 0.044 mol, 88 %). 1H NMR (CDCl3) δ (ppm): was dried over anhydrous sodium sulphate. The crude product
8.88 (ddd, J = 8.1,1.0 & 0.7, Hz 1H), 8.70 (ddd, J = 4.8, 1.7 & 0.9 was purified by flash silica column chromatography using
Hz 1H), 8.30 (dd, J = 6.4 & 1.1 Hz, 1H), 8.11 (dd, J = 7.8 & 2.4 dichloromethane/ethyl acetate (1:1) mixture. The yellow
Hz, 1H), 7.80 (ddd, J = 8.0, 7.2 & 1.6 Hz, 1H), 7.34 (ddd, J = 8.0 , colour solid was obtained as 4-azido-2,2'-bipyridyl (yield,
1
7.2 & 1.2 Hz, 1H), 7.32 (ddd, J = 7.5 , 5.0 & 0.8 Hz, 1H), 7.25 0.52g, 93.54 %). H NMR (CDCl3 ) δ (ppm): 8.69 (ddd, J = 4.8,
(ddd, J = 7.5 , 4.0 & 2.2 Hz, 1H). (13C NMR, 100 MHz) δ (ppm): 1.8 & 0.9 Hz, 1H, H-6’), 8.58 (d, J = 5.4 Hz, 1H, H-6), 8.40 (dt, J
149.4, 149.2, 147.0, 140.0, 136.1, 127.6, 125.8, 125.3, 125.2, = 7.8 & 0.9 Hz, 1H, H-4’), 8.15 (d, J = 2.3 Hz, 1H, H-3), 7.82 (td,
124.1.
J = 7.8 & 1.8, 1H, H-5’), 7.34 (ddd, J = 7.5, 4.8 & 1.2, 1H, H-3’),
6.93 (dd, J = 5.4 & 2.3 Hz, 1H, H-5). (13C NMR, 100 MHz) δ
(ppm): 162.5, 149.4, 148.8, 141.8, 138.4, 136.4, 125.6, 124.7,
117.5, 116.1. FT-IR (ATR): 2115 cm-1 (s).
4'-Nitro-2,2'-bipyridine-N-oxide (2b)65
In an ice bath, 2,2'-Bipyridine-N-oxide (3.0 g, 17.0 mmol, 1 eq.)
was dissolved in concentrated sulphuric acid (19 mL, 34.2 g, 5,11,17,23-tetra-t-butyl-25,27-[bis(O-methyl)-2H-triazole-4-
0.349 mol, 20 eq.) under vigorous stirring. A mixture of fuming 2,2’-bipyridyl]calix[4]arene (L)
nitric acid (30 mL, 43.9 g, 0.697 mol, 40 eq.) in concentrated
sulphuric acid (14 mL, 25.7 g, 0.262 mol, 15 eq.) was added 5,11,17,23-tetra-t-butyl-25,27-bis(O-propargyl)calix[4]arene
1
dropwise over 15 min and then the reaction mixture was (0.435 g, 0.6 mmol, 1 eq.) was added to the solution of 4-
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azido-2,2'-bipyridyl 2d (0.236 g, 1.2 mmol, 2 eq.) in 30 mL of Sample preparation for Absorption and EmissionVTieiwtrAarttiicolenOsnline
dichloromethane and water (50:50) mixture. To this solution,
CuSO4·5H2O (15 mg, 0.06 mmol, 0.1 eq.) and sodium ascorbate 1 mM of the Ru2L was prepared as a stock solution in CH3CN
(23 mg, 0.12 mmol, 0.2 eq.) were added. The resulting solution solvent. The concentration of the solution was fixed at 10 M
DOI: 10.1039/C9NJ01632E
6
was stirred for 12 h at room temperature. Upon completion of [CH3CN/10mM HEPES Buffer (pH = 7.4) (8/2, v/v)] for using
the reaction as confirmed by TLC, the organic layer was absorption and emission measurements. 1mM stock solution
separated and the aqueous layer was extracted with of metal salts and anions were prepared, further, it is diluted
dichloromethane (2 × 25 mL). The combined organic layer was to optimum concentration if lower molar concentration arises.
washed with water and then with brine (2 × 50 mL). The Ten minutes stirring was required for Ru2L with a metal ion,
organic layer was stirred for two hours with aqueous EDTA before monitoring the absorbance and emission titration
solution and then organic layer was separated dried over experiment. In anion sensing, Ru2L+Cu2+ with anions were
anhydrous sodium sulphate, and the solvent was removed stirred for two minutes prior to recording absorbance and
under vacuum. The crude product was purified by column emission titration experiment. For the emission spectrum, the
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chromatography using hexane: ethyl acetate (7:3) to afford excitation wavelength was fixed at 457 nm (such as Cu2+ ion
1
white precipitate (L). Yield: 0.281 g (41%). H NMR (CDCl3):

=
added to Ru2L and S2- anion added to Ru2L-Cu2+).
9.14 (d, 2H), 9.04 (s, 2H), 8.76 (d, 2H), 8.37 (dd, 2H), 8.32 (d,
2H), 7.89 (ddd, 2H), 7.82 (s, 2H), 7.64 (dd, 2H), 7.41 (ddd, 2H), Cell Culture
7.11 (s, 4H), 6.9 (s, 4H), 5.19 (s, 4H), 4.33 (d, 4H), 3.43 (d, 4H),
1.20 (s, 18H), 1.04 (s, 18H). 13C NMR (CDCl3);
 = 158.2, 154.8, The human lung cancer cell line A549 was acquired from
150.8, 150.3, 149.2, 148.0, 146.5, 143.0, 142.5, 137.0, 132.7, National Center for Cell Science (NCCS), Pune, India. The cells
127.8, 125.9, 125.4, 124.4, 121.5, 120.2, 112.4, 108.8, 70.7, were cultured in F-12K medium (Sigma-Aldrich, USA),
34.1, 33.9, 31.6, and 31.0. MALDI-TOF-Ms Calculated for supplemented with 10% fetal bovine serum (Himedia, India)
C70H74N10O4 ([M+Na]+) 1142.4; found, 1142.50. Anal. calcd for and 20 mL of penicillin/streptomycin as antibiotics (Himedia,
C70H74N10O4: C, 75.11; H, 6.66; N, 12.51%. Found: 75.32; H, India), in 96 well culture plates, at 37°C in a humidified
6.81; N, 13.06%.
atmosphere of 5% CO2 in a CO2 incubator (Thermo Scientific,
USA) for 2-3 days earlier to monitoring experiment. All
experiments were carried out using cells from passage 15 or
less.
Bis-rutheniumpolypyridyl-triazole-4-2,2'-
bipyridylcalix[4]arene (Ru2L)
In argon atmosphere, cis-Ru(bpy)2Cl2 (135 mg, 0.28 mmol, 2 MTT assay
eq.) and silver nitrate (95 mg, 0.56 mmol, 4 eq.) were stirred
for 3 hours in methanol (25 mL) under room temperature. The The cytotoxic activities of Ru2L have been screened against the
suspension was filtered in order to remove the silver salt, and A549 human lung cancer cell lines by using MTT assay.67 The
the filtrate was added to 5,11,17,23-tetra-t-butyl-25,27-[bis(O- stock solution of the Ru2L was prepared by dissolving in
methyl)-2H-triazole-4-2,2’ bipyridyl+calix*4+arene
L (159 mg, dimethyl sulfoxide (DMSO) and then diluted separately with
0.14 mmol, 1 eq.) in methanol. The mixture was heated at media to get different concentrations of working solution. Five
reflux condition in an argon atmosphere under dark for thousand A549 cells per well were seeded which is treated
overnight. The reaction mixture was allowed to reach room with 200 l of the Ru2L and incubated for 24 hours. Washed
temperature and the solvent was evaporated. The remaining three times with PBS buffer, 20 µl of MTT solution (5mg/mL in
solid was re-dissolved in a minimum amount of methanol, and PBS) was added to each well and the plate was enfolded with
the desired compound was precipitated by the dropwise aluminum foil and incubated for 4 h at 310 K. The purple
addition of a saturated aqueous solution of ammonium colour formazan dye product was dissolved by addition of 100
hexafluorophosphate. The precipitate was filtered and dried μL of DMSO to each well. The optical density of absorbance
under vacuum to yield 224 mg (82 %) of the desired ruthenium was monitored at 570 nm using a 96-well microplate reader
1
complex Ru2L as a red solid. H NMR (DMSO-d6):
 = 9.30 (d, (Bio-Rad, Hercules, USA). Data were collected for three
2H), 9.27 (s, 2H), 9.00 (d, 2H), 8.83 (m, 8H), 8.23-8.16 (m, 14H), replicates each and used to calculate the respective mean. The
7.89 (ddd, 2H), 7.82 (s, 2H), 7.64 (m, 8H) , 7.59-7.51 (m, 12H), percentage inhibition (PI) was calculated, from this data, using
7.41 (ddd, 2H), 7.11 (s, 4H), 6.9 (s, 4H), 5.19 (s, 4H), 4.08 (d, the formula
4H), 3.28 (d, 4H), 1.19 (s, 18H), 0.99 (s, 18H). 13C NMR (DMSO-
d6);

= 158.0, 156.2, 155.4, 153.4, 152.8, 148.9, 149.0, 148.8, PI (%) = [OD
- OD
/ OD
treated cells untreated cells
untreated cells (control)
145.8, 142.1, 139.2, 138.6, 137.9, 136.5, 132.6, 127.8, 127.6, (control)] × 100%
127.4, 127.0, 126.6, 125.9, 125.7, 125.4, 124.9, 124.7, 123.8,
123.5, 121.1, 116.5, 71.7, 34.1, 33.9, 31.6, and 31.0. MALDI- From the values thus obtained, the IC50 values were monitored
TOF-Ms Calculated for C33H27N9Ru ([M]+) 649.14; found, after 24 h treatment for A549 cells and were deduced from the
649.46. Anal. calcd for C110H106N18O4Ru2: C, 67.88; H, 5.49; N, curves obtained by plotting percentage inhibition against
12.95%. Found: 67.93; H, 5.99; N, 13.19%.
concentration.
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Acridine orange (AO) and ethidium bromide (EB) staining
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The research was financially supported by the Department of Science
View Article Online
DOI: 10.1039/C9NJ01632E
and Technology, India under Water Technology Initiative scheme
Apoptotic morphology was investigated by AO/EB double (DST/TM/WTI/2K16/258). MR thanks CSIR, New Delhi for a junior
staining method as described by Spector et al. with some research fellowship.
modifications.68 Briefly, the cells were treated with IC50
concentration of the Ru2L for 24 h. After incubation, the cells
References
were harvested and washed with cold PBS. Cell pellets were
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re-suspended and diluted with PBS to a density of 5×105
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1
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S. J. Lippard, J. Berg, Principles of Bioinorganic Chemistry,
University Science Books, Mill Valley, CA, 1994.
cells/mL and mixed with 25 µL of AO/EB solution (3.8 µM of
AO and 2.5 µM of EB in PBS) on clean microscope slide and
immediately examined under fluorescent microscope (Carl
Zeiss, Jena, Germany) with UV filter (450-490 nm). Three
hundred cells for each sample were scored for viable,
apoptotic or necrotic by staining the nucleus structure and
membrane integrity and the percentage of apoptotic and
necrotic cells were calculated accordingly.
E. L. Que, D. W. Domaille, C. J. Chang, Chem. Rev., 2008, 108
,
1517.
D. G. J. Barceloux, D. Barceloux, J. Clin. Toxicol., 1999, 37
,
,
217.
P. Verwilst, K. Sunwoo, J. S. Kim, Chem. Commun., 2015, 51
5556.
World Health Organization, Environmental Health Criteria,
World Health Organization, Geneva, 1981, No. 19.
L. R. Goodwin, D. Francom, F. P. Dieken, J. D. Taylor, M. W.
Warenycia, R. J. Reiffenstein, G. Dowling, J. Anal. Toxicol.,
1989, 13, 105.
L. N. Osbern, R. O. Crapo, Ann. Intern. Med., 1981, 95, 312.
R. J. Reiffenstein, W. C. Hulbert, S. H. Roth, Annu. Rev.
Pharmacol. Toxicol., 1992, 32, 109.
Hoechst 33528 staining
7
8
A549 cells were cultured in separate six-well plates and
incubated with 24 h at IC50 concentrations of Ru2L. After
incubation, the treated and control cells were gathered and
stained with Hoechst 33258 stain (1 mg/mL, aqueous) for 5
min at room temperature.69 A drop of cell suspension was
placed on a glass slide, and a coverslip was laid over to reduce
light diffraction. At random 300 cells (in triplicate) were
observed in the fluorescent microscope fitted with a 355-377
nm filter, and the percentage of cells that reflected
pathological changes was calculated. Data were collected for
three replicates each and used to calculate the means and the
standard deviations.
9
K. A. Brenneman, D. F. Meleason, M. Sar, M.W. Marshall, R.
A. James, E. A. Gross, J. T. Martin, D. C. Dorman, Toxicol.
Pathol., 2002, 30, 200.
10 C. D. Gutsche, Calixarenes: An Introduction, Edition 2, RSC
Publishing, 2008.
11 R. Joseph, C. P. Rao, Chem. Rev., 2011, 111, 4658.
12 J. S. Kim, D. T. Quang, Chem. Rev., 2007, 107, 3780.
13 H. J. Kim, M. H. Lee, L. Mutihac, J. Vicens, J. S. Kim, Chem.
Soc. Rev., 2012, 41, 1173.
14 Y. R. Kim, H. J. Seo, J. W. Oh, H. Lim, T. H. Kim, H. Kim,
Electroanalysis, 2013, 25, 1.
15 Z. Liu, W. He, Z. Guo, Chem. Soc. Rev., 2013, 42, 1568.
16 A. Ghorai, J. Mondal, A. K. Manna, S. Chowdhury, G. K.
Patra, Anal. Methods, 2018, 10, 1063.
17 S. Radunz, H. R. Tschiche, D. Moldenhauer, U. R. Genger,
Sens. and Actuators B, 2017, 251, 490.
18 Q. Gao, W. Zhang, B. Song, R. Zhang, W. Guo, J. Yuan, Anal.
Chem., 2017, 89, 4517.
19 D. R. Boer, L. Wu, P. Lincoln, M. Coll, Angew. Chem. Int. Ed.,
2014, 53, 1949.
Cellular uptake study
Inherent fluorescence characteristics of Ru2L were very much
helpful to learn their internalization into the cells. A549 cells
were seeded on a cover glass and allowed culture to adhere.
Then, cells were treated with Ru2L (10
M) for 1 h. After
20 A. Notaro, G. Gasse, Chem. Soc. Rev., 2017, 46, 7317.
21 F. Heinemann, J. Karges, G. Gasser, Acc. Chem. Res., 2017,
50, 2727.
incubation, the cells were washed with cold PBS immediately
examined under a fluorescent microscope (Carl Zeiss, Jena,
Germany) with UV filters (543-570 nm). Ru2L was then
incubated with two different concentration of Cu2+ (10 and 20
22 L. Tong, R. P. Thummel, Chem. Sci., 2016, 7, 6591.
23 I. Kostova, Curr. Med. Chem., 2006, 13, 1085.

M) for another 60 min. After two times washing with PBS, the
24 T. Ezaki, S. Nishiumi, T. Azuma, M. Yoshida, Toxicol. Res.,
Cu2+ loaded cells (Ru2L-Cu2+) were analysed by fluorescence
imaging measurements. Further, Ru2L-Cu2+ loaded cells were
incubated for one hour with two different concentrations of
2017, 6, 843.
25 M. Groessl, E. Reisner, C. G Hartinger, R. Eichinger, O.
Semenova, A. R. Timerbaev, M. A. Jakupec, V. B.Arion, B. K.
Keppler, J. Med. Chem., 2007, 50, 2185.
sulfide anions (25 and 50
M), then washed using PBS and
26 W. H. Ang, P. J. Dyson, Eur. J. Inorg. Chem., 2006, 2006 (20)
,
captured the fluorescence images.
4003.
27 Du, Y. Kang, Y. Zhao, W. Zheng, Y. Zhang, Y. Lin, Z. Wang, Y.
Wang, Q. Luo, K. Wu, F. Wang, Inorg. Chem., 2016, 55, 4595.
28 B. J. Han, G. B. Jiang, J. Wang, W. Li, H. L. Huang, Y. J. Liu,
Conflicts of interest
RSC Adv., 2014, 4, 40899.
29 D. L. Ma, V. P. Y. Ma, D. S. H. Chan, K. H. Leung, H. Z. He, C. H.
Leung, Coord. Chem. Rev., 2012, 256, 3087.
30 S. Khatua, M. Schmittel, Org. Lett., 2013, 15, 4422.
31 D. Saha, S. Das, S. Mardanya, S. Baitalik, Dalton Trans., 2012,
41, 8886.
There are no conflicts to declare.
Acknowledgements
32 P. A. Scattergood, P. I. P. Elliott, Dalton Trans., 2017, 46
,
16343.
10 | J. Name., 2012, 00, 1-3
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Page 11 of 12
New Journal of Chemistry
Please do not adjust margins
Journal Name
ARTICLE
1
2
3
4
5
6
7
8
9
33 Z. Du, B. Song, W. Zhang, C. Duan, Y.L. Wang, C. Liu, R. 68 D. L. Spector, R. D. Goldman, L. A. Leinwand, Cell: A
Zhang, J. Yuan, Angew. Chem. Int. Ed., 2018, 57, 3999.
Laboratory Manual, Culture and BiochemicaVl ieAwnAartilcylesiOsnlionef
DOI: 10.1039/C9NJ01632E
34 K. Samanta, D. S. Ranade, A. Upadhyay, P. P. Kulkarni, C. P.
Cells, 1, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York, 1998, p. 34.1.
Rao, ACS Appl. Mater. Interfaces, 2017, 9, 5109.
35 Y. Sun, J. Ma, D. Tian, H. Li, Chem. Commun., 2016, 52, 4602.
36 A. Nehra, D. S. Yarramala, C. P. Rao, Chem. Eur. J., 2016, 22
8980.
37 J. Cho, T. Pradhan, Y. M. Lee, J. S. Kim, S. Kim, Dalton Trans.,
2014, 43, 16178.
69 G. P. A. H. Kasibhatla, D. Finucane, T. Brunner, E. B. Wetzel,
D. R. Green, Protocol: Staining of suspension cells with
Hoechst 33258 to detect apoptosis. Cell: A Laboratory
Manual Culture and Biochemical Analysis of Cells; Vol. 1,
CSHL press, USA, 1998, p. 15.5.
,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
38 P. D. Beer, F. Szemes, P. Passaniti, M. Maestri, Inorg. Chem.,
2004, 43, 3965.
39 S. Y. Park, J. H. Yoon, C. S. Hong, R. Souane, J. S. Kim, S. E.
Matthews, J. Vicens, J. Org. Chem., 2008, 73, 8212.
40 V. Saravanan, A. Kannan, P. Rajakumar, Sens. and Actuators
B, 2017, 242, 904.
41 S. Rahman, Y. Assiri, A. N. Alodhayb, L. Y. Beaulieu, A. K.
Oraby, P. E. Georghiou, New J. Chem., 2016, 40, 434.
42 X. Lou, D. Ou, Q. Li, Z. Li, Chem. Commun., 2012, 48, 8462.
43 R. Kaushik, A. Ghosh, D. A. Jose, Coord. Chem. Rev., 2017,
347, 141.
44 M. Z. Zhang, H. H. Han, S. Z. Zhang, C. Y. Wang, Y. X. Lu, W. H.
Zhu, J. Mater. Chem. B, 2017, 5, 8780.
45 Shaily, A. Kumar, N. Ahmed, Ind. Eng. Chem. Res., 2017, 56,
6358.
46 M. Sun, H. Yu, H. Li, H. Xu, D. Huang, S. Wang, Inorg. Chem.,
2015, 54, 3766.
47 F. Hou, L. Huang, P. Xi, J. Cheng, X. Zhao, G. Xie, Y. Shi, F.
Cheng, X. Yao, D. Bai, Z. Zeng, Inorg. Chem., 2012, 51, 2454.
48 A. K. Mahapatra, S. Mondal, S. K. Manna, K. Maiti, R. Maji, M.
R. Uddin, S. Mandal, D. Sarkar, T. K. Mondal, D. K. Mait,
Dalton Trans., 2015, 44, 6490.
49 H. Li, Anal. Chim. Acta., 2018, 1010, 69.
50 J. Chen, Y. Li, W. Zhong, Q. Hou , H. Wang, X. Sun, P. Yi, Sens.
and Actuators B, 2015, 206, 230.
51 J. Chen, Y. Li , K. Lv , W. Zhong, H. Wang, Z. Wu, P. Yi, J. Jiang,
Sens. and Actuators B, 2016, 224, 298.
52 X. Yue, Z. Zhu, M. Zhang, Z. Ye, Anal. Chem., 2015, 87, 1839.
53 M. Li, Q. Liang, M. Zheng, C. Fang, S. Peng, M. Zhao, Dalton
Trans., 2013, 42, 13509.
54 P. Zhang,Y. Wang,W. Huang, Z. Zhao, H. Li, H. Wang, C. He, J.
Liu, Q. Zhang, Sens. and Actuators B, 2018, 255, 283.
55 M. Bhatt, D. Maity, V. Hingu, E. Suresh, B. Ganguly, P. Paul,
New J. Chem., 2017, 41, 12541.
56 R. Zhang, X. Yu, Y. Yin, Z. Ye, G. Wang, J. Yuan, Anal. Chim.
Acta., 2011, 691, 83.
57 P. Zhang, L. Pei, Yu Chen, W. Xu, Q. Lin, J. Wang, J. Wu, Y.
Shen, L. Ji, H. Chao, Chem. Eur. J., 2013, 19, 15494.
58 N. Queyriaux, E. S. Andreiadis, S. Torelli, J. Pecaut, B. S.
Veldkamp, E. A. Margulies, M. R. Wasielewski, M. C.
Kerlidou, V. Artero, Faraday Discuss., 2017, 198, 251.
59 J. C. H. Acosta, M. A. Méndez, M. D. Scanlon, H. Vrubel, V.
Amstutz, W. Adamiak, M. Opallo, H. H. Girault, Chem. Sci.,
2015, 6, 1761.
60 W. Iali, P. H. Lanoe, S. Torelli, D. Jouvenot, F. Loiseau, C.
Lebrun, O. Hamelin, S. Menage, Angew. Chem. Int. Ed., 2015,
54, 8415.
61 M. Q. Wang, K. Li, J. T. Hou, M. Y. Wu, Z. Huang, X. Q. Yu, J.
Org. Chem., 2012, 77, 8350.
62 F. T. Andón, B. Fadeel, Acc. Chem. Res., 2013, 46, 733.
63 B. P. Sullivian, D. J. Salmon, T. J. Meyer, Inorg. Chem., 1978,
17, 3334.
64 V. V. S. Mummidivarapu, V. K. Hinge, K. Tabbasum, R. G.
Gonnade, C. P. Rao, J. Org. Chem., 2013, 78, 3570.
65 A. Baron, C. Herrero, A. Quaranta, M. F. Charlot, W. Leibl, B.
Vauzeilles, A. Aukauloo, Chem. Commun., 2011, 47, 11011.
66 B. S. Uppal, A. Zahid, P. I. P. Elliott, Eur. J. Inorg. Chem., 2013,
2571.
67 T. Mosmann, J Immunol Methods., 1983, 65, 55.
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The
supramolecular
sensor Ru2L was designed by joining
bis-ruthenium
(II) polypyridyl complex
View Article Online
DOI: 10.1039/C9NJ01632E
butyl calix[4]arene platform through a 1,2,3-triazole linker and used for sensing of copper (II) and sulphide ions by fluorescence.
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