Experimental and theoretical studies of imidazole based chemosensor for Palladium and their biological applications

Article abstract of DOI:10.1016/j.jphotochem.2019.112092

An imidazole derivative (probe L1) bearing a thiophene group was developed as an ON-OFF fluorescent chemosensor for palladium ions. The UV–vis absorption and fluorescence spectral behavior of probe L1 towards various cations were investigated in HEPES buffer aqueous solution. The absorbance intensity of probe L1 considerably enhanced whereas the fluorescence emission intensity was quenched in the presence of Pd²⁺ ions, while the presence of other metal ions had no notable interference. The stoichiometry of the complex was determined using Job's plot and a plausible recognition mechanism of probe L1 and Pd²⁺ is proposed. Density Functional Theory (DFT) calculations were used to study the electronic properties and to optimize the structure on the selectivity of Pd²⁺ ions and are correlated with the experimental results. The intracellular Pd²⁺ ion detection in living cells was performed using probe L1 on brine shrimp nauplii (Artemia salina) up to 20 μg/mL.

Full text of DOI:10.1016/j.jphotochem.2019.112092

Journal Pre-proof
Experimental and theoretical studies of imidazole based chemosensor
for Palladium and their biological applications
Shanmugam Suresh, Nanjan Bhuvanesh, Arunachalam Raman,
Paramasivam Sugumar, Dhanasekaran Padmanabhan,
Shanmugam Easwaramoorthi, Mondikalipudur Nanjappagounder
Ponnuswamy, Subbiah Kavitha, Raju Nandhakumar
PII:
S1010-6030(19)30682-3
DOI:
https://doi.org/10.1016/j.jphotochem.2019.112092
JPC 112092
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Accepted Date:
22 April 2019
11 September 2019
12 September 2019
Please cite this article as: Suresh S, Bhuvanesh N, Raman A, Sugumar P, Padmanabhan D,
Easwaramoorthi S, Ponnuswamy MN, Kavitha S, Nandhakumar R, Experimental and
theoretical studies of imidazole based chemosensor for Palladium and their biological
applications, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2019),
doi: https://doi.org/10.1016/j.jphotochem.2019.112092

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Experimental and theoretical studies of imidazole based chemosensor for Palladium and
their biological applications
Shanmugam Suresha, Nanjan Bhuvanesha, Arunachalam Ramanb, Paramasivam Sugumarc,
Dhanasekaran Padmanabhand, Shanmugam Easwaramoorthi٭,b, Mondikalipudur
Nanjappagounder Ponnuswamy c, Subbiah Kavitha*,d and Raju Nandhakumar*,a
aDepartment of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore – 641 114, India. E-mail: nandhakumar@karunya.edu
bInorganic & Physical Chemistry Laboratory, CSIR-Central Leather Research Institute, Adyar,
India. Email: moorthi@clri.res.in
cCentre of Advanced Studies in Crystallography and Biophysics, University of Madras, Guindy
Campus, Chennai 600 025, India.
dDepartment of Biotechnology, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore – 641 114, India. E-mail: kavibiotec@karunya.edu
Graphical Abstract

Highlights
 Imidazole derivative bearing a thiphene group (L1) senses Pd2+ sensor via turn on-off
mechanism without any influence of other metal ions
 The absorbance intensity of probe L1 considerably enhanced whereas the fluorescence
emission intensity was quenched in the presence of Pd2+ ions

Density Functional Theory (DFT) calculations were used to study the electronic
properties

The intracellular Pd2+ ion detection in living cells was performed using probe L1 on brine
shrimp nauplii (Artemia salina)
Abstract
An imidazole derivative (probe L1) bearing a thiophene group was developed as an ON-
OFF fluorescent chemosensor for palladium ions. The UV-Visible absorption and fluorescence
spectral behavior of probe L1 towards various cations were investigated in HEPES buffer
aqueous solution. The absorbance intensity of probe L1 considerably enhanced whereas the
fluorescence emission intensity was quenched in the presence of Pd2+ ions, while the presence of
other metal ions had no notable interference. The stoichiometry of the complex was determined
using Job’s plot and a plausible recognition mechanism of probe L1 and Pd2+ is proposed.
Density Functional Theory (DFT) calculations were used to study the electronic properties and to
optimize the structure on the selectivity of Pd2+ ions and are correlated with the experimental
results. The intracellular Pd2+ ion detection in living cells was performed using probe L1 on
brine shrimp nauplii (Artemia salina) up to 20 µg/mL.
Keywords: Chemosensor, Pd2+ ion, colorimetric, fluorescence, DFT, antimicrobial,
molecular docking, Bio imaging
1. Introduction

Recently, development of selective and sensitive fluorescent diagnostics for important
transition metal ions such as palladium is of huge curiosity due to the plentiful usage in various
physical and profitable applications [1-3]. For example, it is extensively used as a catalyst in
synthesis because of its brilliant catalytic ability in pharmaceutically valuable products [4,5].
Palladium plays a significant role in the manufacture of medical devices, dental, jewelry,
catalytic convertor for automobiles, connecting plating for electronics and as essential catalysts
in many organic reactions [6,7]. Palladium can be found in biological materials and augmented
by the food chain to cause an imminent fitness problems. After a thorough investigation, World
Health Organization (WHO) has confirmed that the deadly dose of palladium is 5-10 ppm, [8–
10] and uptake may be controlled to ~ 1.5 – 15 µg/day. Besides the need, palladium causes eye,
skin, breathing tract irritation and the complex of Pd (II) are regarded highly toxic and
carcinogenic. For example, it can coordinate with bimolecular DNA, proteins consisting of thiol
or thiol – ether group containing amino acid, vitamin B6 etc. and are active energy driving
centers in living cells [7,11–13]. Analytical instrumentation (plasma emission spectroscopy (e.g.
ICP-MS and ICP-AES), atomic absorption spectrometry (AAS), solid phase microextraction-
high performance liquid chromatography and X-ray fluorescence) for the quantification of Pd
(II) generally costs high price for the instrumentation and tedious complex procedures.
Therefore, the development of palladium detecting probes, especially for imaging in living
systems and to detect the trace level of the metal remaining in air, water, food product, beverages
etc., are in high demand. Nevertheless, there are some reported analytical methods for the
recognition of Pd2+ ion; however, fluorescent based detection has great attention due to its
extreme sensitivity and selectivity and to some extent suitability to be active in biological
samples. As a consequence, development of fluorescent chemosensors for palladium ion is
highly needed. We herein report a heterocycle based small molecule, imidazole thiophene
conjugate L1 coupled by a freely rotatable carbon-carbon single bond for the selective detection
of palladium. To the better of our understanding this is the first report where there is an
absorption enhancement and emission quenching during the sensing of palladium by the
chemosensor L1 as a dual mode detection method in semi aqueous media. In addition, imidazole
based compounds play an important role as anticancer, antifungal, antibacterial and anti-
inflammatory agents [14–16]. Hence, the chemosensor L1 and its palladium complex are further
screened for various biological applications including bio-imaging of Artemia Salina.

2. Experimental
2.1. Materials & Instruments
All the chemicals and reagents such as 1,2-phenylenediamine, thiophene-2-
carboxaldehyde, absolute ethanol, sodium hydroxide, high pure HEPES (99.0 %), Na2EDTA
(98.0%) etc., were purchased from commercially available sources like Himedia, Loba Chem.
Ltd., Sigma Aldrich etc., were used after being purified by standard techniques. TLC plates were
purchased from Merck pre-coated alumina (Aluminium oxide) sheets of 60 F-254. Melting point
was recorded using a Buchi Melting Point B-545, an electrothermal apparatus using capillary
tubes and are uncorrected. The nitrate, acetate and chloride salts of K+, Ca2+, Na+, Mg2+, Cr3+,
Mn2+, Co2+, Ni2+, Cu2+, Hg2+, Cd2+, Fe3+, Zn2+, Al3+, La2+, Bi3+, Sr2+, Ag+, Ba2+, Zr2+, Pb2+, Fe2+,
Ce3+ and Pd2+ were used as metal salts for recognition. EDTA solution was prepared with
ethylenediaminetetraacetic acid disodium salt (Na2EDTA) (100 equiv.) in double distilled water
(25 mL). Double distilled water was prepared in the laboratory using a water treatment device
and used throughout the work. 1H NMR and 13C NMR spectra were verified on a Bruker 400 and
100 MHz high-resolution NMR spectrometer, respectively, utilizing DMSO-d6 solution with
TMS as an internal standard and coupling constants (J) were measured in Hertz. LC-MS and ESI
mass were determined on a Shimadzu Lab Solutions Data Report. UV-Visible absorption
spectrum was taken at ambient temperature using a Shimadzu UV-Vis spectrophotometer using 1
cm path length quartz cuvette. Fluorescence emission spectra were recorded on a Jasco FP-8200
spectrofluorometer with quartz cuvette 4.5 cm height of 1 cm path length. The excitation and
emission slot widths recorded were 5.0 nm. All cation absorption and emission spectra were
recorded at 24±1°C. Scanning Electron Microscopy (SEM) images and EDAX spectrum were
carried out using SEM instrument (Model: JOEL-6390, Japan) whereby the samples are
dispersed on a carbon tape. Docking calculations were done using Docking Server (Bikadi,
Hazai, 2009), needed hydrogen atoms, Kollman charges, and solvation parameters were added
with AutoDock tools.[17] Molecular docking simulations were performed using the Lamarckian
genetic algorithm (LGA) and the Solis & Wets local search technique.[18]
2.2. Synthesis of 2- (thiophen-2-yl)-1H-benzo [d] imidazole (Probe L1)

To a stirred of o-phenylenediamine in 10 mL absolute ethanol (0.50 g, 0.005 mol), 2-
thiophene carboxaldehyde (0.57 g, 0.005mol) in absolute ethanol (20 mL) was added slowly.
The reaction mixture was further stirred under room temperature for 8 hrs. After completion of
the reaction, monitored by the TLC, the yellow precipitate formed was filtered and washed
several times with cold ethanol. Finally, recrystallization of the crude product from ethanol gave
the probe L1 in 80% yield. Mp: 152 0C; 1H NMR (400 MHz, DMSO-d6): δ (ppm): 12.96 (s, 1H),
7.84-7.83 (d, 1H), 7.73-7.72 (d, 1H), 7.55 (s, 1H), 7.24-7.18 (m, 3H) (Figure S1). 13C NMR (100
MHz, DMSO-d6): 147.5(1C), 134.18(2C), 129.2(2C), 128.7(2C), 127.1(2C), 122.66(2C) (Figure
+
S2). LC-MS (ESI) m/z calcd. For C11H8N2S (M) 200.26, found 199.726 (M-1) (Figure S3).
(Scheme 1).
Scheme 1: Synthesis of probe L1. Absolute Ethanol, RT, 8 hours
2.3. UV- Visible and fluorescence studies
The UV-Visible and the fluorescence spectra of the chemosensor L1 were recorded at 25 ͦ C in
CH3CN/H2O (1:1 v/v) using 50 mM HEPES buffer at a physiological pH of 7.4. Stock solutions
of the metal ions utilized for the study was prepared (2×10-3 M concentration of probe L1
(CH3CN/H2O (1:1 v/v)), HEPES=50 mM, pH=7.4) prior to the experiment. The solutions of
metal ions were prepared from nitrate salts of Ag+, Al3+, Ba2+, Ca2+, Cd2+, Ce3+, Co2+, Cr3+, Cu2+,
Fe2+, Fe3+, Hg2+, K+, La3+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sr2+, Zn2+, Pd2+ and Zr2+.
2.4. Density Functional Theory (DFT) calculation

The molecular geometry optimization with respect to energy was investigated
computationally by performing Density Functional Theory (DFT) to get way for validation of
experimental results. The ground state geometries of probe L1 and probe L1+Pd2+ were
optimized using Density Functional Theory (DFT) with B3LYP functional and 6-31G (d) basis
set using Gaussian 09 program. To verify the stoichiometric ratio and stability of probe L1 and
its complex with Pd2+, optimized geometry parameters such as ground state energies, bond
length, bond angles, etc., were calculated.
2.5. Anti-bacterial activity
Anti-bacterial activity of the L1, and L1+Pd2+ were tested against the cultures of
Staphylococcus aureus and Escherichia coli by agar well diffusion method [19–21] using
Muller-Hinton Agar (MHA). Sterile cotton swab was dipped in the respective broth culture and
squeezed against the wall of the tube to drain excess liquid. Then it was swabbed on to the
surface-dried MHA plates. Wells (4-6 mm) were made using a sterile cork borer (4 wells/plate;
each for L1, Pd2+, L1+Pd2+ and DMSO a separate plate was used for Ampicillin; 1 plate per
bacteria). 50 µL of each mixture was added to the respective wells, allowed to be absorbed for
20-30 minutes at 4°C and thereafter hatched at 37 °C for 18-24 hours. Diameter of zone of
inhibition (in mm) was measured and tabulated. Briefly, a single colony was used to inoculate 10
mL of Mueller-Hinton broth (Himedia laboratories, Mumbai, India) and incubated overnight at
37 °C. From this overnight culture broth, 0.1% inoculum (10 µL) was used to inoculate a fresh
Muller-Hinton broth (10 mL) and incubated at 37°C until the turbidity of the cultures were
visually equivalent to that of the 0.5 MacFarland Standard tube (To prepare 10 mL of this
standard solution, 0.05 mL of 1% Barium chloride was added to 9.95 mL of 1% Sulphuric acid
and mixed well to uniform turbidity. The absorbance of 0.5 McFarland Standard gives an
absorbance in the range of 0.08 to 0.1 at 625 nm). This gives an approximate bacterial
suspension equal to 1.5 x 108 CFU/ mL. Hence, the probe L1, Pd2+, and L1+Pd2+ were dissolved
in dimethyl sulphoxide (DMSO) at a concentration of 0.1 g/mL (10%), with DMSO and the
antibiotic ampicillin (200 µg/mL) acting as controls.
2.6. Docking Studies

Docking calculations were carried out using Docking Server[22]. Gasteiger partial
charges were added to the ligand atoms. Non-polar hydrogen atoms were merged and rotatable
bonds were defined. Docking calculations were carried out on Untitled-1 protein model.
Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were
added with the aid of AutoDock tools. Affinity (grid) maps of 60×60×60 Å grid points and 0.375
Å spacing were generated using the Autogrid program. AutoDock parameter set- and distance-
dependent dielectric functions were used in the calculation of the van der Waals and the
electrostatic terms, respectively. Docking simulations were performed using the Lamarckian
genetic algorithm (LGA) and the Solis & Wets local search method. Initial position, orientation,
and torsions of the ligand molecules were set randomly. All rotatable torsions were released
during docking. Each docking experiment was derived from 10 different runs that were set to
terminate after a maximum of 250000 energy evaluations. The population size was set to 150.
During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were
applied.
2.7. Epifluorescence chemosensor analysis of Artemia Salina
Bio-probing potential of probe L1 was performed on Artemia Salina as described earlier
[23] with little modification. In brief 10-12 hrs post-hatched active nauplii were selected based
on active movement under illumination and transferred to polypropylene PCR tube contain 50
uL of brine solution (3.5 % NaCl w/v), 20 uL of 4 ×10-5 of probe L1 in CH3CN/H2O (1:1 v/v )
pH = 7.4 used as ligand control, 20 µL of Pd2+ ions in100 equiv.CH3CN/H2O (1:1 v/v ) served
as metal control, Probe L1+Pd2+ (1:1) 20 µL was added to study the metal ligand interaction.
50 mM HEPES buffer at 25 ͦ C were used to make up the final volume of 50 uL in all the
experiment tubes, 50 uL of PBS buffer served as experimental control, CH3CN as solvent
control. All the tubes incubated for 60 minutes.at room temperature and washed with 1× PBS
post incubation to remove the unbound L1 and Pd2+ and emission were recorded using Nikon
Eclipse TS100 Epifluorescence microscopy attached with Red and green filter.
3. Results and Discussion

The single step condensation product of o-phenylenediamine and 2-thiophene
carboxyaldehyde in absolute ethanol yielded the desired chemosensor L1. The synthesized
1
13
chemosensor L1 was validated with the help of H-NMR, C–NMR, and LC-MS spectroscopic
techniques. The probe L1 was dissolved in acetonitrile: water (1:1) and scanned in wavelength
range 200-800 nm for UV-Visible absorption spectra plot. In order to consider the Pd2+
recognition abilities of the sensor, we carried out a serious of Host-Guest recognition
experiments in CH3CN/H2O for UV-Visible absorption and fluorescence emission studies. The
probe L1 showed a rapid enhancement in absorbance in the presence of Pd2+ ion. At the same
time, the fluorescence emission of probe L1 was quenched [24]. The absorbance and
fluorescence emission of probe L1 in acetonitrile/water solution are illustrated in Figure 1.
Figure 1. UV-Visible absorption (2×10-5 M) and fluorescence spectra (10 µM) of probe L1 in
CH3CN/H2O (1:1 v/v) pH = 7.4 using 50 mM HEPES buffer solution at 25 ͦ C.
3.1 Selectivity of metal ions by UV-Visible Absorption studies
The UV-Visible absorption spectra of probe L1 was investigated in acetonitrile/water at
an ambient temperature. The solution of probe L1 (2×10-5 M) in CH3CN/H2O [(1:1 v/v) pH = 7.4
using 50 mM HEPES buffer solution at 25
ͦ C] was colorless and exhibited an absorption peak at
320 nm due to the π-π transition. The sensing ability of probe L1 was investigated by adding
̽
various cations, such as Zr2+, Pb2+, Zn2+, Co2+, Ni2+, Ca2+, Mn2+, Cr3+, Ba2+, Ce3+, Mg2+, Fe2+,
Fe3+, Bi3+, Sr2+, Ag+, La2+, K+, Al3+, Cu2+, Cd2+, Hg2+, Na+ and Pd2+ to sensor L1 by UV

spectroscopic method. While adding Pd2+ ion to probe L1, there was an enhancement in the
absorption peak with a notable blue shift from 320 nm to 310 nm in the UV-Visible absorption
spectrum and the colorless solution turned to pale yellow (Figure 2). At the same time there were
no significant changes in the absorption spectra during the addition of other cations such as Zr2+,
Pb2+, Zn2+, Co2+, Ni2+, Ca2+, Mn2+, Cr3+, Ba2+, Ce3+, Mg2+, Fe2+, Fe3+, Bi3+, Sr2+, Ag+, La2+, Al3+,
Cu2+, Cd2+, Hg2+, Na+ and K+ to solution of sensor L1. Hence the chemosensor L1 selectively
detects Pd2+ ion compared to other cations.
Figure 2. Absorbance spectra probe L1 (2×10-5 M) in the presence of various metal ions in
CH3CN/H2O solution (1/1 v/v, HEPES 50 mM, pH=7.4). (Inset: naked eye colour change and
under UV illumination of probe L1 and L1+Pd2+)
3.2 Selectivity of metal ions by emission studies
The emission spectra of probe L1 towards the selectivity of metal ions such as the nitrate
and chloride salts of Na+, K+, Al3+, Cu2+, Cd2+, Hg2+, Zr2+, Pb2+, Zn2+, Co2+, Ni2+, Ca2+, Mn2+,
Cr3+, Ba2+, Ce3+, Mg2+, Fe2+, Fe3+, Bi3+, Sr2+, Ag+, La2+ and Pd2+ in CH3CN/H2O (1/1 (v/v)
HEPES = 50 mM, pH=7.4 solution excited at 320 nm were recorded and analyzed. On the
addition of various metal ions (100 equiv.) to chemosensor L1, there was a decrease in
fluorescence intensity at 370 nm for all the metal ions except for Pd2+ ions. Nevertheless, during
the addition of Pd2+ ions to the chemosensor L1, the fluorescence intensity was drastically
quenched,[25],[26] suggesting that the probe explicitly recognize Pd2+ ions over other cations.

This observation clearly indicates that the probe L1 which showed an intense fluorescence
before the addition of Pd2+ ion, was quenched[27],[28] after the addition of Pd2+ ion (Figure 3a).
[29]
3.3 Effect of Counter anions
The effect of the counter anions on the selectivity of Pd2+ ions was then studied which is
shown in Figure 3b. Accordingly, three types of palladium salts were taken i.e. Pd(NO3)2, PdCl2
and Pd(OAc)2 and added with the probe L1 in CH3CN/H2O solution (1/1 v/v, HEPES 50 mM,
pH=7.4), excited at 320 nm. For all the three salts, the fluorescence intensity was quenched
during the addition of the probe L1. These results indicate that the selectivity depends on the
palladium ion ruling out the counter anion effect.
Figure 3. (a) Fluorescence emission spectral changes of probe L1 (10 µM) in CH3CN/ H2O
solution (1/1 v/v, HEPES 50 mM, pH=7.4) (excitation at 320 nm) a) in the presence of various
metal ions. b) Different counter ions 1. Probe L1, 2. Probe L1+ Pd [(NO)3]2 , 3. Probe L1 +
PdCl2, 4. Probe L1 + Pd (OAc)2
3.4 Anti-jamming experiments
To further check the possibility of interference of other metal ions during the Pd2+ ion
detection by the probe L1, anti-jamming experiments were performed by utilizing the absorption
spectroscopic technique. Accordingly, the ability of receptor L1 to detect Pd2+ ion was measured

utilizing 100 equiv. Pd2+ ion with equivalent proportion including Na+, K+, Al3+, Cu2+, Cd2+,
Hg2+, Zr2+, Pb2+, Zn2+, Co2+, Ni2+, Ca2+, Mn2+, Cr3+, Ba2+, Ce3+, Mg2+, Fe2+, Fe3+, Bi3+, Sr2+, Ag+,
and La2+. There were no obvious changes in the fluorescence intensity with all the metal ions,
which suggest that the probe L1 can be utilized for detection of Pd2+ ion in biological and
environmental samples. (Figure 4).[30,31]
Figure 4. Relative absorbance analysis of probe L1 (2×10-5 M) in CH3CN/H2O solution (1/1 v/v,
HEPES 50 mM, pH=7.4) with different competing metal ions (100 equiv.) in the absence and
presence of Pd2+. Black bar represents the absorbance of probe L1 with 100 equiv. of other metal
ions. Red bar represents the absorbance changes during the addition of 100 equiv. of different
relevant competing metal ions to the solution containing the probe L1 and Pd2+
Similarly, another experiment was conducted utilizing the fluorescence technique as
shown in Figure 5. The fluorescence spectral changes of sensor L1 was measured by the
treatment of 100 equiv. Pd2+ ions in the presence of same equiv. of other interfering metal ions
including Na+, K+, Al3+, Cu2+, Cd2+, Hg2+, Zr2+, Pb2+, Zn2+, Co2+, Ni2+, Ca2+, Mn2+, Cr3+, Ba2+,
Ce3+, Mg2+, Fe2+, Fe3+, Bi3+, Sr2+, Ag+, and La2+. These tested interfering metal ions showed no
significant interference with the detection of Pd2+ ions. Therefore, sensor L1 could be used for
the detection of Pd2+ ions as a dual mode detection which includes both the absorption as well as
emission techniques with enhancement and quenching respectively.[32],[33]

Figure 5. Relative fluorescence intensities of probe L1 (10µM) in CH3CN/H2O solution (1/1 v/v,
HEPES 50 mM, pH=7.4) and then with addition of different metal ions to 100 equiv. Pd2+. Red
bar represents the emission intensity of probe L1 with 100 equiv. other metal ions. Black bar
represents the florescence changes during the addition of 100 equiv. of different relevant
competing metal ions to the solution containing the probe L1 and Pd2+ (100 equiv.).
3.5 Binding analysis of probe L1 with Pd2+
A further understanding of the sensing behaviour of probe L1 to Pd2+, absorption titration
experiment was done by the incremental addition of various amounts of Pd2+ ions to a solution of
L1. As shown in Figure 6, an increase in the absorbance intensity was observed with increasing
Pd2+ concentration (0-50 equiv.). However, the absorbance spectral changes were almost
saturated upon addition of 50 equiv. of Pd2+, revealing that L1 interacts with Pd2+ in 2:1
stoichiometry.[34] During the titration procedure, there was a notable shift of the absorbance
peak intensity from 320 nm to 310 nm (blue shift), indicating the possibility of an intramolecular
charge transfer (ICT) between the imidazole scaffold and thiophene moiety.

Figure 6. UV- visible absorption spectra of probe L1 (2×10-5 mM) (CH3CN/H2O, v/v, 1/1,
HEPES, pH- 7.4) upon addition of different amount of Pd2+ (0-50 equiv.)
Next, a similar titration profile was carried out between the probe L1 and Pd2+ ion using
the fluorescence technique as shown in Figure 7. Accordingly, the fluorescence titration of
sensor L1 with Pd2+ ion was investigated in CH3CN: H2O solution (1:1 v/v, HEPES 50 mM,
pH=7.4). The titration clearly indicates that the fluorescence emission intensity at 370 nm
gradually decreases as the concentration of Pd2+ ion increases. The titration gets saturated during
the addition of 28 equivalents of Pd2+ ions to probe L1. [35,36]

Figure 7. Fluorescence titration of L1 (2×10-5 M) with Pd2+ ion (0-28 equiv.) in CH3CN/ H2O
solution (1:1 v/v, HEPES 50 mM, pH=7.4).
Furthermore, the solubility of the probe L1 and the impact on the absorption and
emission spectra were measured by using different proportions of water and acetonitrile. There
was a gradual increase of intensity at 320 nm and 370 nm in both absorbance and emission
spectra respectively, as the portion of water rises (fw) from 0 to 100%. [37–39] However, there
was precipitate formation occurred after the (1:1) mixture of acetonitrile/water. Therefore, the
(1:1) mixture of acetonitrile/water mixture was selected for all the experiments. In addition,
absence of any spectral shift indicates that there was no aggregation induced emission occurring
in the probe L1. (Figure 8(a) (b))

Figure 8. (a) UV-Visible absorption and (b) Fluorescence emission spectra (λem–370 nm) of
probe L1 (CH3CN: H2O system with different water fraction solution).
3.6 Job’s plot and association constant analysis
To explore the binding mode between L1 and Pd2+ (Host-Guest) complex through
absorption studies, binding analysis were carried out by the Job’s plot method (Figure 9(a)). The
complex showed a maximum mole fraction of L1 is 0.3 at 310 nm, which established a 2:1 (L1:
Pd2+) binding stoichiometry. The binding stoichiometry between L1: Pd2+ was further confirmed
by the Benesi–Hildebrand nonlinear curve fitting method as shown in (Figure 9(b)). The
association constant was determined to be Ka = 1.27 x 104 M-1 for the L1: Pd2+ complex. The
2:1 stoichiometry was further confirmed by LC-MS as shown in the Supporting Information
(Fig. S4). A peak at m/z 542, which corresponds to [2 L1+ Pd + Cl]+ was clearly observed in the
LC-MS. The limit of detection of L1 is found to be 13.3 ×10-6 mol L-1, which is calculated using
3δ/S, where δ was the standard deviation of the blank signal, and S was the slope of the linear
calibration plot.

Figure 9. (a) Job’s plot of a 2: 1 complex of Pd2+, where the absorbance at 310 nm was plotted
probe L1 (2×10-5 M) against the mole fraction of Pd2+ (100 equiv.) acetonitrile: water (1:1 v/v,
50mM HEPES buffer) solution. (b) Benesi-Hildebrand plot of probe L1 with Pd2+ ions was in
acetonitrile: water (1:1 v/v, 50 mM HEPES buffer) solution.
Similarly the quenching effect of probe L1 during the addition of Pd2+ ions and their binding
studies using the fluorescence technique were calculated using the Job’s plot method [40]. The
complex showed a maximum mole fraction of L1 is 0.3 which established a 2:1 (L1:Pd2+)
binding stoichiometry (Figure 10(a)). The binding stoichiometry between L1:Pd2+ was further
confirmed by the Benesi–Hildebrand nonlinear curve fitting method (Figure 10(b)). The
association constant was determined to be 1.84×104 M-1 for the L1:Pd2+ complex. The detection
limit was found to be 1.53 ×105 M-1 .[41,42]

Figure 10. (a) Job’s plot of a 2: 1 complex of Pd2+, where the emission at 370 nm was plotted
against the mole fraction of Pd2+. (b) Benesi- Hildebrand plot of probe L1 with Pd2+ ions in an
acetonitrile: water (1:1 v/v, 50mM HEPES buffer) solution.
Furthermore, 1H NMR analysis were performed to corroborate the coordination structures
1
for Pd2+ complex with L1 in a DMSO-d6. (Figure 11) shows the partial H NMR spectra of L1
measured in a DMSO-d6 with and without Pd2+ (0-1 equiv). All chemical shifts were identified
1
by H−1H COSY analysis (Figure 12& 13). Addition of Pd2+ leads to a large downfield shift of
the thiophene-benzimidazole protons (Ha, Hb, Hc, Hd, He, Hf, Hg,). These changes in the chemical
shift value of aromatic protons are due to the decrease in electron density of the thiophene-
benzimidazole moieties by the benzimidazole N coordination. The disappearance of the NH peak
after the addition of palladium may be due to the replacement of exchangeable proton with
deuterium.This indicates that there is a possible Pd2+ coordination with the benzimidazole ‘N’ as
shown in scheme 2.
1
Figure 11. H- NMR (400 MHz) titration of probe L1 (blue line spectra), probe L1+ 0.5 equiv.
Pd2+ (red line spectra), probe L1+ 1.0 equiv. Pd2+ (green line spectra) in DMSO-d6.
3.7 Effect of pH, time and reversibility
For practical applicability of fluorescent probes, the effect of pH and time plays a vital
role in the selectivity of metal ions. Hence, the same was investigated with probe L1 detecting
Pd2+ ions by fluorescence technique, at different pH levels in CH3CN/H2O (1/1 v/v) solution.

The pH was adjusted using 10 mM of NaOH and HCl solutions in double distillated water. The
result showed that the probe L1 displays a strong fluorescence a variety of pH range except a
strong enhancement between 3 and 5 pH ranges under acidic conditions. Similarly, L1-Pd2+
complex also gave the same fluorescence intensity except quenching in the detection process.
(Figure S8 (top panel)). Therefore, the physiological pH of 7.4 was selected as the working
condition through the spectroscopic experiments. The time taken for complete quenching of Pd2+
ion by probe L1 in CH3CN/H2O (1/1, v/v) buffered at pH = 7.4 (50mM) is shown in Figure S8
(b). The result clearly indicates that the detection of Pd2+ ions by the probe L1 is completed
within two minutes and remained steady for further on hour. Hence, probe L1 can be utilized to
detect Pd2+ ions in a short span of time.
The reversibility of L1 to Pd2+ was demonstrated by adding EDTA into a mixture of L1
and Pd2+ (Figure S9 (top panel)). As previously discussed, L1 alone shows a strong fluorescence
and on addition of Pd2+ ion there is a considerable quenching in the fluorescence intensity.
However, upon addition of EDTA to the same solution the emission intensity got enhanced and
peak was identical to that of the probe L1. It was noteworthy that EDTA has a better chelating
ability with Pd2+, and L1+ Pd2+ complex could be easily detached upon complexation of EDTA
with Pd2+ i.e., probe L1 was free from the complex solution [43]. These results indicate that the
Pd2+ recognition process is completely a reversible one, which can be applied for realistic
application. The emission changes were almost reversible even after several cycles with the
sequentially addition of Pd2+ and EDTA as shown in Figure S9 [44]. Thus, these result indicated
that sensor L1 towards Pd2+ could be recyclable simply through using proper reagent such as
EDTA. The SEM images of L1 and L1-Pd2+ complex are displayed in Figure S10 (a), (b). The
morphological changes of L1 with insertion of Pd2+ were analyzed by using SEM analysis.
Receptor L1 shows glassy sheet like structure in the free state. However, on the addition of Pd2+
the glassy structure was changed and the complex molecules were agglomerated because of the
formation of the L1-Pd2+ coordination complex, which further confirms the probe L1 selectivity
towards Pd2+ ions[45]. The chemical composition of the receptor L1-Pd2+ ion complex is
measured utilizing EDAX analysis (Figure S10 (c),(d)) indicates the presence of carbon (C),
nitrogen (N), Sulfur (S) and Palladium (Pd) elements in the receptor L1-Pd2+ ion complex.
3.8. Density Functional Theory calculations

To further understand the absorption and fluorescence behavior of the probe L1 and L1-
Pd2+ complex, we carried out DFT calculations with the 6-31G* basis set using the Gaussian 09
program. The optimized structure of probe L1 shows a planar structure. The bond angle -112.44
of N-C-N in probe L1 slightly becomes increased to 114.5 after coordination with Pd2+ ion and
the charges on the N atom of L1 and L1-Pd2+ is found to be -0.572 and -0.464 reveals that the
electron density on the N atom is decreased after coordination with Pd2+ ion which results in the
increased bond angle. The SCF calculations for NMR studies also carried out for L1 and L1-Pd2+
1
ion in TMS HF/6-31G (d) GIAO as a reference. The H NMR analysis shows that the aromatic
proton (14) shifts to down shield region after coordination with Pd2+ ion which further confirms
the experimental evidences (Figure S13).
Finally, the above results clearly states that the fluorescence quenching spectral responses
of L1-Pd2+ ion arises primarily due to the paramagnetic nature of the Pd2+ ion and the reverse
photoinduced electron transfer also plays a role in the quenching effect. The 2:1 binding
1
stoichiometry was confirmed by H-NMR titration studies, mass spectra, Job’s plot and Benesi-
Hildebrand equations. Therefore a plausible binding mechanism is proposed as shown in scheme
2.
Scheme 2. Proposed binding mechanism of probe L1 with Pd2+ ion
3.9 Biological studies
The results of the antibacterial activity of L1, Pd2+ ion (palladium), L1-Pd2+ ion were
given in Table 1. DMSO, in which all the compounds were dissolved, did not show any
antibacterial activity against S.aureus, but it was inhibited by Pd2+, L1 and L1-Pd2+ ion complex.
Ampicillin also exhibited inhibitory effect on S.aureus. Interestingly, against E.coli, L1 showed

no zone of inhibition whereas the L1-Pd2+ ion complex showed moderate antibacterial activity
(Figure S14). It was also observed that the zone of inhibition for Pd2+ and L1-Pd2+ ion towards
E.coli was lesser that of S.aureus. Overall, the ligand showed better inhibiting activity towards
Gram positive bacteria than Gram negative bacteria. Testing against both bacteria, the
antibacterial activity of the ligand was increased when complexed with palladium but slightly
lower than palladium alone. Our results display the same pattern as showed by Karunaker [46]
who showed that there is an increase in antibacterial and antifungal activities of the ligand-
palladium complex compared to that of free ligand. Joseyphus and Nair [47] also found that the
ligand-palladium complex exert more antibacterial activity than the ligand. They also attributed
this phenomenon of toxicity of the complexes to the increased lipophilic nature of the complexes
due to chelation. Chelation enhances bactericidal activity of the ligand which may be due to the
fact that, the positive charge of the chelated probe L1-Pd2+ ion is partly shared with donor atoms
present in L1 [48]. There is also a possible explanation for the increased inhibiting activity of the
complex as both ligand and the metal ion may interfere in diverse manner during the growth of
the microbe [49]. Thus it may possible that the sensor L1 and their metal probe L1-Pd2+ ion have
the potential to be employed as antibacterial agents in human [50]. The zone of inhibition formed
by the ligand and the complex were comparable to that of the standard drug Ampicillin.
Table: 1. In vitro anti-bacterial activity of L1 and L1-Pd2+ ion
Zone of Inhibition (mm in diameter)
Compound
Staphylococcus aureus
Escherichia coli
Pd(NO3) ion
(Palladium)
20
23
L1
16
18
No zone
13
L1-Pd2+ ion
DMSO
No zone
22
05
Ampicillin
24
3.10 Molecular docking studies
High-resolution crystal structures of Staphylococcus aureus methionine aminopeptidase
L1 in L1-Pd2+ ion with various keto heterocycles and aminoketones are taken for molecular

modeling studies for synthesized 2-(thiophen-2-yl)-1H-benzo[d]imidazole compounds.
Methionine aminopeptidases (MetAPs) are ubiquitous enzymes found in both eukaryotic and
prokaryotic cells and play a critical role in the maturation of proteins for proper function,
targeting, and degradation (Giglione).[51] Based on the above said biological factor
Staphylococcus aureus methionine amino peptidase I, L1-Pd2+ ion is targeted protein, we have
retrieved from the Protein Data Bank (PDB ID: 1QXY) (Douangamath ).[52] The study reveals
that the molecules have good binding capability with the active site amino acids of
Staphylococcus receptor. In all these complexes, the non-bonded interactions limit is varied from
2.5Å to 3.5Å which reveals that the ligands may result in a strong inhibition. Therefore, it can be
concluded that both the size and flexibility play a prominent role in binding with the
Staphylococcus enzyme.
The three-dimensional representations of complexes of the Staphylococcus aureus
methionine aminopeptidase I complex are shown in (Figure S15&S16). Here two different types
of ligands are docked with Staphylococcus aureus methionine amino peptidase I receptor using
the program Docking Server. The Ligplot depicts that the compounds (Figure S18 &S19) have
similar mode of binding interactions with Staphylococcus protein. The Est. Free Energy of
Binding, vdW+Hbond+desolv Energy and Total Inter molec. Energy is calculated for each
compound. Both compounds form the best interaction with the active site residues of His 151,
which is involved in the key interactions with compounds are strictly conserved residues in
targeted protein. Compared to L1, L1-Pd2+ ion have more hydrogen bond interaction and Est.
Free Energy of Binding, vdW+Hbond+desolv Energy and Total Inter molec. Energy is more
active in the targeted protein. The hydrogen bond interaction of the best compounds with the
receptor L1 molecule is given in (Figure S17) and the energy values were tabulated in Table 2.
Table 2
Hydrogen bond Interaction and docking score of best compounds With the Staphylococcus
Targeted Protein
Sl.
No
Ligand
Name
Est. Free
Energy of
Binding
vdW+Hbond
+desolv
Total
Intermolec.
Energy
Interactions (dista
nce in
Ǻ)
Energy
1
Probe L1
-5.34kcal/mol -5.58kcal/mol
-5.64kcal/mol
N2-HIS151
3.86

2
Probe L1- -6.57kcal/mol -6.71kcal/mol
-6.75kcal/mol
N1-HIS151
N2-HIS151
3.85
3.85
Pd2+
3.11 Bio imaging:
Bio probing assessment of probe L1 on Artemia salina showed clear incidents of cellular
uptake. Epifluorescence image of Artemia salina showed an excitation in Ex 250-350 nm. Both
the intracellular and pheriphral components of Artemia salina was observed with complete
fluorescence at 370 nm (Figure 12). Specific internal cellular component of fluorescence in
Artemia salina was reported by Murugase [53]. No fluorescence was observed in the palladium,
probe L1-Pd2+ ion complex and solvents confirms inhibition of fluorescence by palladium. These
observations support the interaction of probe L1 with Pd2+ ion in the solvent medium during
photometry assessment.

Figure 12. Bioimaging of Artemia Salina (a, c, e, g, i) green microscopic images, Artemia Salina
(b, d, f, h, j) red fluorescence microscopic images. (a) green image of Artemia Salina (b) red
image of Artemia Salina, (c) green field image of probe L1(20µM) accumulated to Artemia
Salina, (d) red field image of probe L1(20µM) accumulated to Artemia Salina (20µM), (e) green
field image of probe L1(20µM) and Pd2+ (20µM) accumulated to Artemia Salina (20µM), (f) red
field image of probe L1(20µM) and Pd2+ (20µM) accumulated to Artemia Salina (20µM), (g)
green field image of Pd2+ (20µM) accumulated to Artemia Salina (20µM), (h) red field image of
Pd2+ (20µM) accumulated to Artemia Salina (20µM), (i) green field image of CH3CN (20µM)
accumulated to Artemia Salina (20µM), (j) red field image of CH3CN (20µM) accumulated to
Artemia Salina (20µM),
4. Conclusion
In summary, a simple reversible chemosensor L1 for selective detection Pd2+ with
absorption “off-on” and emission “on-off” signals was achieved compared to other potentially
competing meta ions. Both the emission and absorbance spectroscopy delivers information for
the creation of 1:2 stoichiometry complexes between Pd2+ and L1. The association constants and
the limit of detection by both the modes have been calculated. The DFT calculations further
supports the experimental evidences. Biological applications like antibacterial properties and in
vitro bio imaging studies on Artemia salina has been studied and discussed. Currently, other
imidazole based chemosensors and their varied environmental and biological applications are
underway in our laboratory.
5. Acknowledgement
This work is supported by the SERB-EMR grant by the DST (Sanction NO.SERB-
EMR/2016/005692) to RN. Center for research in Nanosciences, KITS, Panjab University –
SAIF and The Gandhigram Rural Institute (Deemed to be University) supported the microscopic
and spectral details for this work.
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