A novel biphenolic ligand for selective Mg2+ and Zn2+ ions sensing followed by colorimetric, spectroscopic and cell imaging methods

Article abstract of DOI:10.1016/j.ejps.2018.01.025

The (E)-2-((2-hydrohy-5-methylphenylimino) methyl) phenol ligand was synthesized. The receptor was characterized by IR, ¹H and ¹³C NMR and CHN analysis. The ligand exhibits colorimetric and fluorometric sensing of Zn²⁺ and

Full text of DOI:10.1016/j.ejps.2018.01.025

Accepted Manuscript
A novel biphenolic ligand for selective Mg2+ and Zn2+ ions
sensing followed by colorimetric, spectroscopic and cell imaging
methods
Palanisamy Uma Maheswari, Duraisamy Renuga, H.L. Jeeva
Kumari, Kandasamy Ruckmani
PII:
DOI:
Reference:
S0928-0987(18)30032-0
https://doi.org/10.1016/j.ejps.2018.01.025
PHASCI 4375
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
30 April 2017
20 December 2017
11 January 2018
Please cite this article as: Palanisamy Uma Maheswari, Duraisamy Renuga, H.L. Jeeva
Kumari, Kandasamy Ruckmani , A novel biphenolic ligand for selective Mg2+ and Zn2+
ions sensing followed by colorimetric, spectroscopic and cell imaging methods. The
address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Phasci(2017), https://doi.org/10.1016/j.ejps.2018.01.025
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ACCEPTED MANUSCRIPT
Submitted to: Eur.J. Pharm. Sci. 2017
Article type: Full Paper
A novel biphenolic ligand for selective Mg2+ and Zn2+ ions sensing
followed by colorimetric, spectroscopic and cell imaging methods.
Palanisamy Uma Maheswari,a* Duraisamy Renuga,b H.L.Jeeva Kumaric and
Kandasamy Ruckmani c*
a Department of Chemistry, National Institute of Technology, Tiruchirappalli – 620015, Tamilnadu, India.
b Department of Chemistry, Selvamm College of Technology, Namakkal – 637003, Tamilnadu, India.
c Department of Pharmaceutical Technology, Centre for Excellence in Nanobio Translational Research (CENTRE),
Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620024, Tamil Nadu, India
Key words: MgCl2, ZnCl2, colorimetric, fluorescence microscopy, live cell imaging.

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1. Introduction
The design and synthesis of chemo sensors for biologically important metal ions have emerged
as the most important topic in the field of medicinal chemistry and live cell imaging. (Kyle et al.
2014). The chemo sensors are expected to be selective, easy to prepare, with good ADME
profile, low cytotoxicity during the imaging duration and good association constant on binding to
the metal ions (Lok Nath et al., 2016). The chemo sensors are widely used for calorimetric,
fluorometric detection of metal ions, which can be observed by naked eye, fluorescence
spectrophotometer or by a fluorescence microscopy. The chemo sensors have been extrapolated
with intracellular or extracellular applications for various metal ions, whether they are present in
trace or bulk concentrations (Yifan et al., 2016). The sensor absorption and emission profile
should be appreciable especially after binding to metal ions with good quantum yield. The
intense emissive property of the chemo sensors on binding to metal ions are widely used in live
cell imaging of biologically important metal ions to follow the metabolism often. The chemo
sensors are usually designed by appropriate coordination sites suitable for the detection of the
respective metal ions based on their size and appending a fluorophore to the designed receptor
for fluorometric detection in-vitro and in-vivo (Lourdes et al., 2007). This method is
advantageous over atomic absorption spectroscopy, ion-selective electrodes etc as this can be
applicable to living cell and physiological conditions. The chemo sensors have been used to
detect Hg, Pb, Mg, Ca, Cu, Zn and many other metal ions through live cell imaging (Yong Sung
et al., 2016; Kim et al., 2014; Tayade et al., 2014; Tayade et al., 2014; Liu et al., 2012).
Magnesium ions has major role in the biological systems and considered as an important
factor participating in many biochemical reactions, cell growth, in stabilizing the double stranded
DNA conformation and even in regulating the calcium ion channels (Cowan et al., 2002). Mg2+
is also present in chlorophyll and involved in photosynthesis of carbohydrates. Mg2+ is a bulk
metal in need for muscle coordination and bone health, for functioning of nerves and immune
systems and reason for many congestive cardiac diseases (Wolf et al., 2003). The concentration
of Mg2+ ions in cells vary from 1 mM to 6 mM. Thus, Mg2+ estimation in-vivo can be an
important tool to follow the function or dysfunction of human body, appropriately. The sample
can be as simple as the blood serum for detection. Zinc metal ions also has role in muscle
strengthening, as a co- factor in many hydroxylation enzymes, part of zinc fingers and help
towards many bio-transformations and signaling (Li et al., 2014; Zhipeng et al., 2014).

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Detection of both Mg2+ and Zn2+ separately and together by a chemo sensor can be an
important application. The biphenolic receptor prepared from salicylaldehyde and 2-amino-4-
methyl phenol found to detect Mg2+ and Zn2+ selectively and has no specificity for other metal
ions (Scheme 1). The paper describes the calorimetric, absorption and emission spectroscopic
detection of Mg and Zn ions by the receptor and its live cell imaging of the same metal ions, in
vitro.
2. Materials and Methods
2.1. Materials
The chemicals and solvents were all of analytical grade were used without further
purification. Salicylaldehyde, 2-amino 4-methyl phenol and metal salts were purchased from
Sigma-Aldrich, Bangalore and used as such. HeLa cells were obtained from National Centre for
Cell Science (NCCS), Pune, India. Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum (FBS) were procured from Invitrogen, USA. 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was purchased from Hi media, Mumbai, India. Milli Q
water was used to prepare solutions and buffers.
2.2. Methods
1
IR spectra were obtained on a Perkin-Elmer spectrophotometer. H and 13C NMR spectra
were recorded on a Bruker 400 MHz spectrometer with DMSO-d6 as solvent. Varian
spectrophotometer was used to record absorption and fluorescence emission spectra. For
calorimetric, absorption and emission titration, a stock solution of the ZnCl2 or MgCl2 (1.5× 10-
3M) was prepared in pure water. The receptor solution (5×10-5M) was prepared in DMSO. For
each titration, 3 ml of receptor was titrated with 0.1 eq (10 μL) – 1 eq (100 μL) of the respective
Zn2+ or Mg2+ ions in DMSO/water medium. For cyclic voltammetric studies, EG and G
potentiostat was used. The receptor solution was prepared in 1 mM concentration for about 20
mL of volume with CH3CN as solvent. The supporting electrolyte was tetrabutylammonium
hexafluorophosphate. The respective metal ions were added in 2 equivalents to the receptor
concentration to follow the metal ion detection. Platinum plate and platinum wire were used as a
working and counter electrode. Ag+/Ag was used as the reference electrode.

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2.3. Synthesis of the receptor
The receptor was prepared by a simple condensation method. Salicyaldehyde (5 mmol,
0.6106 g) was dissolved in 20 mL of warm ethanol and 2-amino-4-methyl phenol (5 mmol,
0.6157g) was added to the ethanol solution dropwise. The mixture was slowly kept under reflux
for 6 h at 75°C. On cooling, a red brown coloured solid separated out, which was filtered and
washed with ethanol and then dried in vacuum. Yield was 0.9787g (84%). The structure of the
1
receptor was confirmed by NMR spectroscopy. H NMR (400MHz, DMSO-d6): δ 3.3 (1H, s), δ
6.8 – δ 7.6 (4H, m), δ 8.9 (1H, s), δ 9.5 (1H, s), δ 13.8 (1H, s).; 13C NMR (100MHz, DMSO-d6):
δ 21.0, δ 117.2, δ 117.5, δ 119.6, δ 120.4, δ 120.7, δ129.2, δ 133.1, δ133.6, δ135.3. CHN
analysis for the formula C14H13N1O2. Found (calculated); C; 73.56 (73.99%); H; 5.40 (5.77%);
N; 6.02 (6.16%).
2.4. Cytotoxicity studies
HeLa cancer cells were maintained at 1 x 106 cells/mL in DMEM, supplemented with 10% heat-
inactivated FBS and incubated at 37 °C in an atmosphere with 5% CO₂. Cells were maintained in
a 96-well plate at a density of 2 x 10⁴ cells/well and kept to attach over the night. The medium
was discarded and the cells were incubated with different concentrations (6.25-200 μM) of the
receptor sample for 24 h. The sample was dissolved in dimethyl sulfoxide (DMSO) at a stock
concentration of 32 mM. The final concentration of DMSO was not exceeded 0.625 % for every
treatment, and it was used as a negative control. The assays were performed in triplicate
(Sudeshna et al., 2008). MTT dye reduction assay was performed to determine the percentage of
cell viability and the anticancer effect of sample at various concentrations. The assay depends on
the reduction of MTT by mitochondrial dehydrogenase, an enzyme present in the mitochondria
of viable cells to a purple coloured formazan product. After 24 h incubation at 37 °C in 5% CO2
atmosphere, the cells were washed with phosphate-buffered saline (PBS). The morphology of the
cells was observed under an inverted microscope and the phase-contrast images were acquired at
20 X magnification. 20 μL of MTT (5 mg /mL) was then added and incubated for 4 h. The
medium was discarded and 100 μL of DMSO was added to dissolve the formazan crystals. The
absorbance was read at 570 nm in a microplate reader. The percentage of cell viability and the
percentage of inhibition at various concentrations were calculated using the following formulae:

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% viability = (At / Ac) x 100 ………………………(1)
% inhibition = (Ac – At)/Ac x 100 …………………(2)
where, At is the absorbance of the test sample and Ac is the absorbance of the control.
A graph was plotted between the concentration of the sample and percentage of inhibition,
thereby determining the IC50 value of sample which is the maximal concentration of the drug to
cause 50 % inhibition of biological activity of cancer cells.
2.5. Live-cell imaging studies
HeLa cells were seeded in a 24-well plate in DMEM at a density of 3 x 105 cells/well
incubated at 37 °C at 5% CO₂. After an overnight incubation, the medium was discarded and the
cells were incubated with different concentrations (6.25 - 200 μM) of receptor for 30 min at
37°C. The cells were washed thrice with PBS and observed under an inverted fluorescence
microscope for a control. The receptor concentration was varied as 6.25, 12.5, 25, 50, 100 and
200 μM concentrations for each set of experiment. After acquiring the images of HeLa cells
incubated with varying concentrations of receptor for 30 min, addition of individual metal ions
(Mg2+, Zn2+, Ca2+) to each set of experiment with mentioned concentrations were performed
simultaneously and also one set was a mixture of Mg2+ and Zn2+ ions. The bright field and
fluorescence images were acquired at 20 X magnification for each set of experiment
(Udhayakumari et al., 2014). The mean fluorescence intensities of treated samples with
individual metal ions (Mg2+, Zn2+, Ca2+) and combination (Mg2+ and Zn2+) were compared using
NIS-Elements D software (version 4.13), Ti-E, Nikon, Japan, and a graph was plotted with
concentration of the sample vs. fluorescence intensity for evaluation.
3. Results and Discussion
3.1. Colorimetric titrations
The sensing property of the receptor towards various cations was studied by calorimetric
method. In the calorimetric experiments, 3 mL of receptor solution in DMSO/H2O (5 x 10-5 M)
was titrated with the respective metal ion solutions in H2O medium. With 1 equivalents of ZnCl2
or MgCl2 (1.5 x 10-3 M) the color change was observed. The color changes are as shown in the
Figs. 1 and 2. The receptor solution changed to bright yellow from colorless solution for Zn2+,
immediately. The Mg2+ ions on interaction with the receptor produce yellow color change, after

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30 minutes. Other cations like K+, Na2+ and Ca2+ (up to 10 equivalents), transition metal ions like
Cr2+, Fe2+, Ni2+, Co2+, Cu2+ and heavy metal ions like Sn2+, Pd2+, Ce2+, Hg2+, Cd2+ do not show
any color changes on interaction with the recptor.
3.2. UV-Visible spectroscopic studies
The sensing behavior of the receptor towards Zn2+ and Mg2+ ions were also followed by UV-
Visible spectroscopy. The titration involves 1 mL of the receptor solution (5 x 10-5 M) and
incremental addition of respective metal ions upto 2 eq (1 x 10-3 M) in DMSO/H2O medium. The
pure receptor in DMSO/H2O medium showed a prominent absorption band at 360 nm. This band
on Zn2+ coordination decreases in intensity and a new band at 450 nm found to be increasing in
intensity. The 360 nm band was assigned to π to π* transition and the new band at 450 nm was
assigned to metal to ligand charge transfer band. The observation of two isosbestic points clearly
depicted a single binding event (Fig. 3). A similar trend was also observed for Mg2+ ions binding
to the receptor (Fig. 4). Both Zn2+ and Mg2+ ions showed 8 to 10 fold increase in the new band
intensity. This ensures the same binding mode for both metal ions to the receptor. The
investigation of absorption spectral changes of the receptor upon addition of various other metal
ions with the same concentrations or even at higher concentrations showed no prominent spectral
changes (Fig. 5). It reveals the selective binding of the receptor to Zn2+ and Mg2+ ions only and
not to any other metal ions. Therefore, it was clear that the receptor can be used for selective
sensing of Zn2+ and Mg2+ over a wide range of cations.
3.3. Fluorescence spectroscopic studies
The receptor binding to Zn2+ and Mg2+ ions binding was also followed by emission spectra.
Titration experiments were carried out similar to absorption spectroscopy with the same
concentrations and volume. When the pure receptor is excited at 450 nm, it gave a very weak
emission band around 525 nm but after the addition to Zn2+ ions, it gave a strong emission band
at 560 nm with a red shift (Fig 6). The same titration of the receptor with Mg2+ ions, showed
similar emission trend but with 100 and above fold of increase in emission intensity at the band
around 560 nm (Fig. 7). The K (binding constant) values are determined using scatchard plots
and fluorescence data and were found to be 4.28 ± 0.62 for Zn2+; and 15.14 ± 0.24 for Mg2+. The
many fold increase in emission intensity on binding of Mg2+ ions compared to Zn2+ ions shows

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the facile binding event and the much increase in fluorescence emission. As observed in the
absorption spectroscopy, the emission increase was selectively observed only in the case of Zn2+
and Mg2+ ions and other metal ions did not show any prominent increase in the emission
intensity (Fig. 8).
3.4. Job’s plot studies
The Job’s plot study was done as described elsewhere (Renuga et al., 2012), with the receptor
and the Zn2+ or Mg2+ ions, with the concentration fixed at 5×10-3M in DMSO/H2O medium (Fig
9). The volume of the solution was 3 mL and kept constant through out the study. Absorption at
450 nm was chosen for the observation. The studies revealed the binding stoichiometry as 2:1 for
receptor: Zn2+ or Mg2+ ions as shown in the Scheme 2.
3.5. Binding sites of the receptor to Zn2+ or Mg2+ ions probed by 1H NMR
The pure receptor, in DMSO-d6 showed the two phenolic protons at δ8.9 (Fig. 10, 1) and 9.5
(Fig. 10, 2). On binding to Zn2+ or Mg2+ ions these two protons are highly perturbed than the
imine proton at δ13.7 (Fig. 10, 3). After addition of 1 equivalent of Zn2+ or Mg2+ ions, the
phenolic protons are completely deprotonated and the binding occurs through the two ‘O’ donors
as revealed by the NMR spectroscopy. But the imine proton exchange due to the coordination of
the receptor became medium rather than fast or slow for intermolecular interactions, so the
respective NMR peak also became less intense.
3.6. Cyclic voltammetry studies
The cyclic voltammogram of the pure receptor showed an oxidation peak at around 0.7 V
(Fig 11). The scan speed was 100 mV/s and the voltammogram was an average presentation of
five scans. The potential window was kept from -1 to +1 V. The oxidation peak was assigned to
be phenol to phenoxyl radical formation under applied potential. This oxidation peak which
corresponds to phenoxyl radical formation generally on coordination occurs at less potential than
the pure receptor. Thus on addition of 1 equivalent of ZnCl2 the oxidation occurred at 0.68 V and
the same oxidation occurs at 0.5 V on addition of 1 equivalent of MgCl2 (Fig. 12). The oxidation

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peak thus reveals the facile coordination of the two phenolates of the receptor to Mg2+ rather than
Zn2+ ions.
3.7. Cytotoxicity assay
The morphological assessment of the cells (Fig. 13) reveals that the treatment of receptor at
dose dependent concentration on HeLa cells reduced the cell viability by altering cellular
morphology. The morphological characteristics include shrinkage of cells, reduced cell density,
reduction in cell to cell contact when compared to that of untreated cells (control). Increase in the
concentration of receptor indicates the cytotoxic effect of receptor on HeLa cancer cells.
Negative control did not show any cytotoxicity similar to untreated (control) cells, hence it was
considered negligible. The cytotoxic effect of the receptor is found to increase with increase in
concentration, resulting in a direct dose-response relationship. A maximum of 93.28% inhibition
is observed at 200 μM at 24 hours of incubation time and the IC50 value is found to be 80.74 μM
(equivalent to 18.16 μg/mL) (Fig. 14).
3.8. Live cell imaging studies
The phase contrast and fluorescence images of HeLa cells were captured after being
incubated with receptor, receptor + Mg2+ (Fig. 15), receptor + Zn2+ (Fig. 16) and receptor + Mg2+
+ Zn2+ Fig. 17) for 30 min at 37°C. The receptor alone, at the highest concentration of 200 µM,
on HeLa cells displayed least or no intracellular fluorescence. However, cells exhibited intense
fluorescence on additional treatment with added Mg2+ and Zn2+ separately. Also the cells
exhibited intense fluorescence with the mixture of Mg2+ and Zn2+. These results clearly explain
the intracellular detection of Zn2+ and Mg2+ ions and together by the biphenolic receptor. The
minimum detection level was 12.5 μM for Mg2+ and 25 μM for Zn2+ ions. Like any other Mg
sensors, which competitively detect Ca2+ ions also, the receptor is checked for the competitive
sensing of Ca2+. But as reflected in the absorption and emission studies, Ca2+ was not detected
by the receptor, which makes this receptor to be very unique for Mg2+ sensing, even in the
presence of bulk metal ions like Na+, K+, Ca2+ etc (Fig.18). The Zn2+ concentration inside the
cells will be in nanomolar range which will not be the bulk species available for detection in
comparison to Mg2+. Thus this receptor have the ability to perform dual roles, as a sensor of both
Mg2+ and Zn2+ ions, either singly or simultaneously; and may found application in biological

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monitoring of these metal ions, selectively, because of its relatively low cytotoxicity up to 12 h,
at the indicated dose and time of incubation.
3.9. Discussion
Generally the alkali and alkaline earth metals bind to soft donors like ‘O’ based ligands like
crown ethers rather than hard, ‘N’ based ligands like histidine etc. In transition metal ions also
Zn binds to ‘O’ based ligands like carboxylic acids etc. The biphenolic receptor used for the
current study is thus a simple ligand with phenolic ‘O’ donors, binding to both Mg2+ and Zn2+ in
2:1 ratio, as compatible to the size of metal ions, namely Mg2+ and Zn2+. The colorimetric change
gives yellow color on metal ions binding to the receptor, a complimentary to the absorbed violet
color. The yellow color of observance on the metal ions binding clearly indicates the facile n to
π* electronic transition stimulated in the receptor. The increase in the absorption band between
350 to 450 nm on the metal ions binding event clearly depicts the respective electronic transition.
The unfavorable detection for other metal ions may be due to intermediate binding stoichiometry
or the less symmetric coordination geometry or the less stability constants for the complex
formed. The phenolic ‘O’ donor participation towards coordination of Mg2+ and Zn2+ comes
from the cyclic voltammetry studies which shows the facile nature of the phenolate ligand on
oxidation. The binding constants reveal the high binding affinity of the receptor for Mg2+ rather
than Zn2+ metal ions. The cytotoxicity studies with HeLa cells reveal the dose dependent
anticancer effect of the receptor at 24 hours of incubation. HeLa cells were chosen for
cytotoxicity and live cell imaging studies as it is widely appreciated for fluorescence microscopy
studies. The live cell imaging studies at half an hour incubation clearly depicts the sensing of the
metal ions from the low concentrations of 12.5 μM and 25 μM for Mg2+ and Zn2+ ions,
respectively. This concentration can be extended to the biological macro or micro nutrients
concentration with a high definition microscopy which can observe the metal ions in the sub
cellular organelles also. Thus the biphenolic receptor which gets stimulated with a high intensity
n to π* electronic transition responsible for the enhanced fluorescence with the microscopy
observation can be good choice for the ultimate detection of Mg2+ or Zn2+ ions.

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4. Conclusion
The receptor was easily synthesized by simple condensation method of salicylaldehyde and 2-
1
amino-4-methyl phenol. The structure of the receptor was confirmed by CHN, FT-IR, H NMR
13
and C NMR spectroscopic techniques. This receptor is highly selective and sensitive towards
Zn and Mg ions detection through calorimetric response. The binding of the metal ions changes
the colorless solution of the receptor to bright turmeric yellow. Jobs plot studies reveal the
stoichiometry of the complex formed between the receptor and the Zn2+ or Mg2+ ions as 2:1. The
sensing property of the receptor was also probed by absorption and emission spectroscopy. In
absorption spectroscopy, the pure receptor band at 360 nm decreases in intensity and a new band
at 450 nm was observed with increase in intensity for both Zn2+ and Mg2+ ions binding. In
emission spectroscopy, on excitation at 450 nm, both metal ions on binding showed
enhancement in emission intensity with a band around 560 nm. Mg2+ ions showed 100 fold
increase in emission intensity than Zn2+ ions which depicts the facile sensing and coordination of
the Mg2+ ions in comparison with Zn2+ ions. This property is applied for live cell imaging of the
Mg or Zn ions, using HeLa cells, in-vitro. The receptor showed cytotoxicity over 24 hours and
less cytotoxic with in 12 hours. The live cell imaging was done by incubating the receptor with
or without Mg or Zn ions for 30 minutes and were detected through fluorescence microscopy.
The minimum detection level was 12.5 μM for Mg2+ and 25 μM for Zn2+ ions. Thus the receptor
is highly useful for detecting the Mg and Zn ions, separately or together, without any other metal
ions interference, through fluorescence microscopic techniques.
Acknowledgement
Dr. P. U. Maheswari acknowledges DST-SERB for financial support (project: SR/FT/CS-
01/2011(G))
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Abstract
The (E)-2-((2-hydrohy-5-methylphenylimino) methyl) phenol ligand was synthesized. The
1
receptor was characterised by IR, H and 13C NMR and CHN analysis. The ligand exhibits
colorimetric and fluorometric sensing of Zn2+ and Mg2+ ions in semi-aqueous medium (DMSO-
H2O). The receptor was tested with series of transition metal ions (Cr2+, Fe2+, Ni2+, Co2+, Cu2+,
Zn2+) and heavy metal ions (Sn2+, Pd2+, Ce2+, Hg2+, Cd2+) and the essential human body elements
like Ca2+, Mg2+, Na+ and K+ ions. The naked eye colorimetric sensing was absorbed only for Zn2+
and Mg2+. Both ions (ZnCl2 and MgCl2 in H2O), when added to the colorless solutions of the
receptor of about 1 equivalence in incremental additions turn the solution into bright turmeric
yellow. All other ions remain inactive, in colorimetric sensing. Further the Zn2+ and Mg2+ ions
were probed by absorption and emission spectroscopy through incremental addition of respective
metal ions. The in-situ deprotonation of the ligand on both Mg2+ and Zn2+ ions binding was
confirmed by 1H NMR titration studies. The imino nitrogen of the receptor is not coordinated to
the metal ions. The Job’s plot studies reveal the 1: 2 binding ratio of metal ions to the receptor.
The high fold fluorescence output on metal ions binding was positively used to sense the Zn2+
and Mg2+ ions, separately and together in HeLa cancer cells through cell imaging.

Graphics Abstract

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