Recognition of Fe3+ by a new azine-based fluorescent “turn-off” chemosensor and its binding mode analysis using DFT

Article abstract of DOI:10.1016/j.molstruc.2020.127834

A highly responsible Fe³⁺, azine chemosensor (EBHMM) was successfully designed and synthesized. The structure of the synthesized azine was confirmed by FT-IR, UV–Vis, ¹H and ¹³C{¹H} NMR spectroscopic techniques

Full text of DOI:10.1016/j.molstruc.2020.127834

Journal Pre-proof
3+
Recognition of Fe by a new azine-based fluorescent “turn-off” chemosensor and its
binding mode analysis using DFT
Subramani Manigandan, Athianna Muthusamy, Raju Nandhakumar, Charles
Immanuel David
PII:
S0022-2860(20)30158-7
DOI:
https://doi.org/10.1016/j.molstruc.2020.127834
MOLSTR 127834
Reference:
To appear in: Journal of Molecular Structure
Received Date: 19 August 2019
Revised Date: 23 January 2020
Accepted Date: 31 January 2020
Please cite this article as: S. Manigandan, A. Muthusamy, R. Nandhakumar, C.I. David, Recognition
3+
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DFT, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.127834.
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Article reference: MOLSTR_MOLSTRUC-D-19-02735
Article title: Recognition of Fe3+ by a new azine-based fluorescent "turn-off"
chemosensor and its binding mode analysis using DFT,To be published in: Journal of
Molecular Structure
3
+
RECOGNITION OF Fe BY A NEW AZINE-BASED FLUORESCENT
"TURN-OFF" CHEMOSENSOR AND ITS BINDING MODE ANALYSIS
USING DFT
a
a
b
Subramani Manigandan , * Athianna Muthusamy, * RajuNandhakumar and
b
Charles Immanuel David
a
PG and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of
Arts and Science, Coimbatore-641 020, Tamil Nadu, India.
b
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore-641 114, Tamil Nadu, India.
a
*
E-mail: muthusamy@rmv.ac.in and muthusrkv@gmail.com,
b
*
E-mail:nandhakumar@karunya.edu

3
+
RECOGNITION OF Fe BY A NEW AZINE-BASED
FLUORESCENT "TURN-OFF" CHEMOSENSOR AND ITS
BINDING MODE ANALYSIS USING DFT
a
a
Subramani Manigandan and * Athianna Muthusamy
PG and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of
Arts and Science, Coimbatore-641 020, Tamil Nadu, India
*
E-mail: muthusamy@rmv.ac.in and muthusrkv@gmail.com
&
b
b
*
Raju Nandhakumar and Charles Immanuel David
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore-641 114, Tamil Nadu, India
*
E-mail: nandhakumar@karunya.edu
Graphical Abstract

3
+
RECOGNITION OF Fe BY A NEW AZINE-BASED
FLUORESCENT "TURN-OFF" CHEMOSENSOR AND ITS
BINDING MODE ANALYSIS USING DFT
a
a
Subramani Manigandan and * Athianna Muthusamy
PG and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of
Arts and Science, Coimbatore-641 020, Tamil Nadu, India
*
E-mail: muthusamy@rmv.ac.in and muthusrkv@gmail.com
&
b
b
*
Raju Nandhakumar and Charles Immanuel David
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore-641 114, Tamil Nadu, India
*
E-mail: nandhakumar@karunya.edu
Abstract
3
+
A highly responsible Fe , azine chemosensor (EBHMM) was successfully designed
1
and synthesized. The structure of the synthesized azine was confirmed by FT-IR, UV-Vis, H
1
3
1
and C{ H} NMR spectroscopic techniques and high resolution mass spectrometry. The azine
3
+
shows sensitive and selective fluorescence “turn-off” towards Fe ion over other metal ions in
3
+
1
:1 DMF-H O (5µM, pH=7.4) solution. The fluorescence quenching on the addition of Fe ion
2
at various pH clearly indicate that the azine can be used for the quantitative determination of
3
+
3+
Fe . The binding of Fe with chemosensor was analyzed by confirming the formation of
metal-ligand complex using HRMS-ESI spectrometry. The binding stoichiometry (1:1) was
-1
calculated using Stern-Volmer method and the binding constant 1.0034 M was calculated
using
3
+
Benesi-Hildebrand plot. The exact binding sites of the chemosensor with Fe ion is
theoretically explained with Fukui Function using DFT study in B3LYP function with
B3LYP/6-311++G (d,p) level of basis set. The “turn-off” fluorescence through photo induced
electron transfer (PET) process is discussed and confirmed with the results of Fukui Function
and Frontier molecular orbital analysis.
3
+
Keyword: Azine Chemosensor, Fluorescence, Fe , DFT, Fukui Function.
. Introduction
In recent years, researchers have focused on organic fluorescent chemical probes,
1
2
+
2+
2+
2+
sensitive and selective for the metal ion recognition such as Cu , Zn , Pb and Hg , among
others [1-9]. The improvement of fluorescent chemical probes for detecting the metal ion is an
encouraging field because of their essential role in environmental, medical and biological

3
+
applications [10]. Among the transition metal ions, Fe ion has dynamic roles in the
environment as well as in many biological processes [11]. Over sufficiency of iron is causing
several diseases such as, kidney failure, liver damage, cancer, anemia, Huntington's,
Alzheimer’s and Parkinson's diseases [12, 13]. Furthermore, the development of chemosensor
3
+
that can detect Fe is very important to prevent and solve health and environmental problems
3
+
3+
induced by Fe . The literature survey revealed that there are few Fe selective fluorescent
-3
-7
probes with detection limit ranges between 3.38 x 10 and 1.63 x 10 M [14-19]. Among
them, fluorescent probes of azines are having very attractive ligating ability with metal ions.
Recently, azine derivatives have shown wide range of applications in various areas such as
material and analytical, environmental, biomedical, catalysis and supramolecular chemistry
[
20-25]. The N-N linked diimines of azine moieties are very tolerant to hydrolysis and are
having good ligating ability. The –C=N- moiety of azine shows potential detection of transition
and post transition metal ions in the chemosensor study [26-28]. There are several methods
available for detecting iron in a particular system [29-31]. Comparing with other methods,
fluorescence technique is most versatile one to detect metal ions due its sensitivity, simplicity,
instantaneous response, low-cost and capable of specific recognition of particular ions [32]. We
reported the synthesis and characterization of an azine and its application as chemosensor for
3
+
detecting Fe ion. The results obtained from experimental and DFT studies provide
information on the structural and photophysical properties of fluorescent compound and helps
in elucidating the fundamental aspects concerning the sensing mechanisms.
2
2
. Experimental
.1. Materials
Hydrazine, 4-hydroxy-3-methoxybenzaldehyde and glyoxal were purchased from
Merck chemical Co. and used without any further purification. Dimethylsulfoxide (DMSO),
tetrahydrofuran (THF), dimethylformamide (DMF), ethanol, methanol, acetone, acetonitrile,
ethyl acetate, heptane, toluene, n-hexane and chloroform were obtained from Merck, India and
used after suitable distillations [33].
2
.2. Synthesis of 4-(hydrazonomethyl)-2-methoxyphenol (PHM)
Equimolar quantities of 4-hydroxy-3-methoxybenzaldehyde and hydrazine were
dissolved separately in minimum volume of ethanol. The reaction mixture was stirred at room
temperature for 4 h (Scheme 1). After checking the completion of the reaction with TLC, the
reaction mixture was poured into ice cold water. The yellowish white coloured precipitate

0
1
formed was filtered and dried. (Yield:82%, M.P: 96 C). H NMR (DMSO-d6): δ ppm 9.17 (s,-
OH), 8.57 (s, -CH=N-), 7.62 (s, Ar-CH), 7.45(d, Ar-CH), 7.10(d, Ar-CH), 6.41 (s, -NH ), 3.83
2
(s,-OCH3).
1
3
C NMR (DMSO-d6): δ ppm 161.17 (C7), 150.31(C1), 140.20(C2), 123.99(C4), 115.94(C5),
1
10.43(C6), 108.37 (C3) and 55.96 (-OCH ). m/z (Found): 166.0806.
3
2
.3. Synthesisof 4, 4-((ethane-1, 2-diylidene)bis(hydrazine-2,1-diylidene))bis
(
methanylylidene)bis(methanylylidene))bis(2-methoxyphenol) (EBHMM)
In to the ethanolic solution of 4-(hydrazonomethyl)-2-methoxyphenol (0.02 mol), 0.01
ole of glyoxal was added slowly with constant stirring. After refluxing the reaction mixture for
h (Scheme 2), it was poured in to ice cold water. The deep yellow coloured product obtained
was filtered, washed thoroughly with distilled water, and recrystallized with ethanol. (Yield:
6
0
1
7
8%, M.P: 184 C). H NMR (DMSO-d6): δ ppm 9.86 (s, -OH), 8.59 (s, Ar-CH=N-), 8.36
(-CH=N), 7.49 (s, Ar-CH), 7.35 (d, Ar-CH), 6.91 (d, Ar-CH), 3.84(s, -OCH3).
1
3
C NMR (DMSO-d6): δ ppm 164.12(C7), 158.66(C1), 152.58(C8), 148.32(C2), 125.27(C4),
1
16.17(C5), 111.32(C6), 110.71(C3) and 56.11(-OCH ). m/z ( Found): 354.9497.
3
2
.4. Characterization Techniques
-1
FT-IR spectrum of EBHMM was recorded in the region of 4000-400 cm using KBr
1
13
1
pellet with Perkin Elmer 8000 FT-IR spectrophotometer. The H and C{ H} NMR spectra
were recorded with Bruker AV400 MHZ spectrometer by using DMSO-d6 as a deuterated
solvent. The UV absorption spectra were recorded using Shimadzu double beam UV-240
spectrophotometer. The mass spectra were recorded with PerkinElmer GC-MS spectrometer.
2
.5. Theoretical study
The quantum theoretical Fukui Function calculations of EBHMM was carried out using
DFT, B3LYP/6-311+ +G (d,p) level of basis set in the Gaussian 09 package. The exact active
sites of EBHMM for chemosensor activity was well identified using Fukui Function
calculations.
2
.6. Optical properties
The UV-visible spectra of various concentrated solutions of EBHMM in DMF were
recorded between of 200 and 800nm. Fluorescence measurements were carried out with Jasco
FP-8200 spectrofluorimeter using quartz cuvettes of 1cm path length. The absorption and

0
emission spectra of the compound in 1:1 DMF/H O solution were recorded at 25 ± 1 C in at
2
2
+
2+
3+
+
3+
+
3+
2+
2+
pH = 7.4. The solutions of Cu , Fe , Fe , K , La as chlorides and Ag , Al , Ba ,Bi
2
+
3+
3+
2+
2+
+
2+
2+
2+
2+
2+
2+
2+
+
Pb , Cr , Ce , Cd , Hg , Li Zn , Zr , Co , Ni , Mn , Mg , Ca. , Na as nitrate were
prepared using 1:1 DMF/H O mixture. The binding studies of the compound was carried out
2
by incorporating these metal ion solutions into 5µM 1:1 DMF/H O solution of EBHMM at pH
2
7
3
3
.4.
. Results and discussion
.1. Solubility
The solubility of EBHMM was tested by dissolving 0.1 mg of it in 1ml of a series of
solvents such as, THF, DMSO, DMF, ethanol, methanol, CHCl , CH Cl and ethyl acetate,
3
2
2
heptane, toluene, n-hexane and acetone at room temperature. The compound is soluble in all
the above mentioned solvents except in n-hexane, toluene and acetone.
3
.2 Spectral Characterization
The structures of PHM and EBHMM are confirmed by IR, H, C{ H}NMR, and mass
1
13
1
spectrometry techniques and their spectra are given in SF1 to SF7. The IR spectrum of
EBHMM showing –OH stretching vibration at 3469.31. PHM showing a broad band at
3
483.59
-1
cm due to the presence of one of the –NH stretching vibration along with –OH stretching
2
-1
vibration. The other –NH stretching vibration band of PHM appears at 3362.68 cm and the
2
-1
bands are absent in EBHMM. The bands at 2941.56-2933.20 cm is attributed to the –CH
stretching vibrations of aromatic hydrogens. The –C=N– stretching vibration bands are
-1
appearing at around 1589-1892 cm [34-37]
1
.
In H NMR spectra, the methoxy protons of both the compounds are appearing as a
singlet in the up field region of around 3.84ppm and the phenolic –OH proton signals are
appearing as a broad singlet at 9.87-9.17ppm. The two set of signals of azomethine protons of
EBHMM (Hd & He) are appearing as singlet at 8.59 and 8.36ppm respectively and the shifting
of Hd proton to downfield is due to the deshielding effects of benzene ring [38]. Similarly, the
aromatic protons of both compounds shows two doublet and one singlet signal between
7
.45 - 6.91ppm and 7.62 - 7.49ppm respectively. The shift at 6.41ppm is attributed to –NH2
proton of PHM and the absence of such shift in EBHMM confirms the formation of expected
structure.

1
3
1
In the C{ H} NMR spectra, the carbon atoms of different chemical environments are
appearing at their respective chemical shift values. The appearance of an additional signal at
1
58.80ppm indicates the formation of azine moiety. The high resolution mass spectra of PHM
and EBHMM respectively are giving their m/z values at 166.0806 and 354.9479, confirming
the formation of expected structures.
3
.3. UV-Vis spectrum of EBHMM
The absorption spectrum of EBHMM was recorded with various concentrated solutions
(
2, 4, 20 and 40µl in 1:1 DMF/H O) and is shown in SF. 8. The λmax appeared at 377nm is
2
attributed to n-π* transition of imine moiety of sensor [39]. The intensity of the spectrum
increases gradually with increase in concentration of EBHMM.
By observing the intensity of the spectra at various concentrations, the suitable
concentration for the fluorescence study was explored. From the spectrum, the optical band gap
(SF. 9) of the compound was calculated by Tauc method and is found to be 2.9 eV. This band
gap value was compared with the theoretical band gap value and used for fukui function
calculation to find the exact nucleophilic attack site in the molecule. For the fluorescence study
the excitation wavelength was fixed as λmax 377nm and the emission was observed at a higher
wavelength of 423nm. The combined spectra of absorption and emission are shown in the SF.
1
0.
3
.4. Selectivity studies of EBHMM to the metal ions
The fluorescence emission of EBHMM with and without metal ion was recorded to find
the selective and sensitive detection of metal ions by observing fluorescence emission intensity
changes. The selectivity of the probe EBHMM was evaluated in DMF/H O (1:1,
2
+
3+
2+
3+
2+
EBHMM=5µM, pH=7.4) solution with different metal ions such as Ag , Al , Ba ,Bi , Pb ,
2
+
3+
3+
2+
2+
3+
+
2+
2+
2+
2+
2+
2+
2+
2+
3+
+
Cu , Cr , Ce , Cd , Hg , La ,Li Zn , Zr Co , Ni , Mn , Mg , Ca , Fe , Fe , Na
+
3+
3+
2+
and K . In the selectivity study, Fe , La and Fe have shown significant change in the
fluorescence emission, while the other metal ions under same concentrations (100 equiv.) and
3
+
identical conditions does not. Among these three metal ions, Fe has shown greater intensity
variation in the selectivity fluorescence spectrum. It shows that there is an effective quenching
3
+
“
On Off” behavior with Fe ion through energy or electron transfer processes which
eventually leads to reverse photo induced electron transfer process. Additionally, the quenching
3
+
behavior may be due to the unfilled d subshell and paramagnetic property of Fe ion [40]. The
3
+
hydrated Fe ion in the solution is highly feasible to form Fe(OH) at pH above 7 [41]. The
3

1
interaction of Fe(OH) with the probe is confirmed by H NMR titration and mass
3
+
spectrometry. The HRMS-ESI m/z value of the probe on binding is:[EBHMM+H ]= 462.3699.
1
The spectra of H NMR titration and mass analysis are shown in SF.11 and SF. 12. The
selectivity of the probe with a series metal ions is shown in Figure 1.
3
.5. Interference studies
The investigation of possible interferences of other metal ions (100 equiv.) with the
3
+
sensor EBHMM-Fe ion complex were carried out with the help of competitive complexation
3
+
analysis. The results show that there is no significant change in the intensity of azine-Fe ion
on addition of other metal ions in to the solution. The extent of intensity variation due to the
2
+
2+
3+
+
3+
+
interference of other metal ions (Chloride of Cu , Fe , Fe , K , La and nitrate of Ag ,
3
+
2+
2+
2+
3+
3+
2+
2+
+
2+
2+
2+
2+
2+
2+
2+
+
Al , Ba ,Bi Pb , Cr , Ce , Cd , Hg , Li Zn , Zr , Co , Ni , Mn , Mg , Ca. , Na )
is shown in Figure 2. It is concluded from the results that the sensor EBHMM can specifically
3
+
detect Fe ion without the interference of potentially competing metal ions.
3
.6. Stern–Volmer and Benesi–Hildebrand plot
In the fluorescence study, the sensor exhibits strong fluorescence at 423nm in
DMF/H O (1:1, EBHMM=5µM, pH=7.4) solution. A fluorescence titration was carried with
2
3
+
different concentrations Fe ion of FeCl from 0 to 100 equiv. to inspect the sensing ability of
3
EBHMM. The fluorescence intensity gradually decreases with the increase in concentration up
3
+
to 80 equiv. of Fe , after that there is no much change in fluorescence intensity. The
3
+
quenching of fluorescence intensity with various concentration of Fe is shown in Figure 3.
3
+
Further, the stoichiometry of EBHMM with Fe is confirmed by the Stern–Volmer and
Benesi–Hildebrand plot. The fluorescence quenching of EBHMM with different concentration
3
+
of Fe ion was analyzed with Stern-Volmer plot (Figure 4). The Stern-Volmer equation is,
I /I = 1 + K [Q]
0
SV
Where, I and I are the fluorescence intensities of the probe in absence and presence of the
0
3
+
quencher (Fe ) respectively. [Q] is the concentration of the quencher and K is Stern-Volmer
sv
quenching constant. The Stern-Volmer plot between I /I and [Q] gives a linear line indicating
0
3
+
that there is a dynamic and dominant diffusion process taken place upon the addition of Fe
ion in to the sensor solution. The correlation coefficient value calculated from Stern-Volmer
2
linear plot is (R =0.97532) shows that there is a good linearity and it can be used for the

3
+
qualitative determination of Fe ion both in environmental and biological systems. The limit
of detection (LOD) was calculated using the formula,
LOD = 3.3σ/k
Where, σ is the y-intercept of the regression line and k is the slope of the calibration
-8
graph [42]. The estimated value of LOD is 7.7 × 10 M.
3
+
The Benesi–Hildebrand plot (Figure 5) is used to determine the binding constant of Fe
ion with the sensor EBHMM. Binding constant (Ka) calculated from the slope of the plot is
-1
1
.0034 M .
3
.7. Effect of Time
3
+
The effect of time during the detection of Fe ion utilizing the sensor EBHMM was
analyzed with DMF/H O solution (1:1, EBHMM=5µM, pH=7.4). The sensor shows high
2
3
+
fluorescence intensity at 423nm in the absence of Fe ion in the solution. Upon the addition of
3
+
Fe ion into the sensor solution, the fluorescence intensity was gradually decreased and
attained saturation in 4 minutes. This instantaneous quenching of intensity is a spot response of
3
+
3+
EBHMM towards the detection of Fe ion. Therefore, it can be used for the detection of Fe
3
+
ion real time analysis [43]. The time response of azine with Fe is shown in the SF. 13.
3
.8. Effect of pH
The fluorescence emission of EBHMM at different pH (from 1 to 12) both in presence
3
+
and absence of Fe ion was investigated in (DMF/H O1:1, EBHMM=5µM) solution to get the
2
3
+
response of EBHMM in the detection of Fe . When the pH of the solution increased the
equilibrium between phenolic and phenoxide is shifted to phenoxide formation. The formation
of phenoxide ion reduces the energy gap between HOMO and LUMO by increasing the
conjugation of π electrons. Therefore, less energy is required for the electron transition and the
intensity of fluorescence emission is gradually increasing [44].The quenching and enhancing of
fluorescence at different pH indicates the capability of EBHMM to choose a suitable pH for the
3
+
detection of Fe ion. The effect of pH on fluorescence emission is illustrated in the SF. 14.
3
.9. Reversibility of the chemosensor
The reversible capability is an important requirement of a chemosensor for the
3
+
detection of ions for several cycles. The reversibility of EBHMM with Fe ion was
investigated in 1:1 DMF/H O solution (EBHMM=5µM, pH=7.4) by the addition of ethylene
2
diamine tetraacetate (EDTA). The fluorescence intensity was increasing gradually by the

3
+
addition of EDTA (10 to 100 equiv.) into the solution of EBHMM-Fe . The entire quenching
3
+
was nullified by the addition of EDTA in the EBHMM -Fe solution. The reversibility of
3
+
EBHMM with Fe ion on the addition of EDTA is clearly shown in Figure 6.
4
4
. Computational Method
.1. Optimized molecular Structure
3
+
The exact binding mode of EBHMM with Fe ion was found with the help of DFT
study. The structure of EBHMM was theoretically studied with DFT. The optimized structure
is in trans form due to steric effect and is depicted in the SF. 15.
4
.2. Frontier molecular orbital analysis
The analysis of wave function ascertains that the electron absorption corresponding to
transition from the ground state to the first excited state via one-electron excitation from
HOMO to LUMO. So, both HOMO and LUMO are taking part in the chemical reaction.
HOMO energy characterizes the capability of electron donating and LUMO characterizes the
capability of electron accepting [45]. The energies of HOMO and LUMO were calculated with
B3LYP/6-311++G (d, p) level and is shown in SF. 16, which shows the distribution and energy
levels of the HOMO and LUMO orbitals. In the HOMO and LUMO of EBHMM the electron
densities are localized on all the atoms except the methoxy group (EHOMO=-5.8097eV &
ELUMO=-2.5598eV). The value of energy separation between the HOMO and LUMO is
3
.2499eV. In the contour map, the lines with red colors are regarded as the negative phase and
the lines with green colors are the positive phase. The contour of HOMO-LUMO diagram is in
accordance with the frontier orbital. The frontier orbital gap helps to characterize the optical
polarizability chemical hardness-softness and chemical reactivity of a molecule [46]. Small
HOMO-LUMO energy gap means, the compound has low excitation energy with low chemical
hardness and good stability. The molecules having small energy gap are known as soft, and
molecules having large energy gap are known as hard molecules. The soft molecules are more
polarizable than the hard ones, because they need small energy for excitation. The hardness
value of a molecule can be determined by the formula [47],
ɳ= 1/2(I - A)
Where, EHOMO and ELUMO are the energies of HOMO and LUMO molecular orbitals.
The value of η of azine is -1.62495 (a.u), indicates that the compound has low chemical

hardness [48] and less resistance to charge transfer. The result clearly illustrated that the metal
ions can easily make bond with azine molecule.
Mechanism of sensing
3
+
To study the mechanism of “Turn off” fluorescence on binding Fe with azine probe is
proved by carrying out DFT studies of azine probe and Fe(OH) . The HOMO-LUMO energy
3
diagram of the probe and metal ion (Fe(OH) are depicted in SF.17. In general, the quencher
3
(metal ion) will act as electron acceptor in photoinduced electron transfer (PET) process when
+
3
its LUMO is lower than the probe. Since the LUMO of Fe is lower than the azine probe they
involve in PET and is responsible for Turn off fluorescence [49-52]. The values HOMO-
LUMO are given in the Table 3.
4
.3. Molecular electrostatic potential [MEP] and Contour map
The different values of the electrostatic potential are symbolized by different colors at
the MEP surface. The red and blue colours represent the regions of most negative (electrophilic
reactivity) and most positive (nucleophilic reactivity) sites. Other colours in the map indicate
zero electrostatic potential sites of the molecule and the contour map shows that the electron
flow is maximum around the azine moiety than the other atoms. Due to high electron density,
the nitrogen atom is more prone to electrophilic attack. The three dimension charge
distributions of the azine MEP and the contour map are clearly shown in the SF.18.
Furthermore, the exact active site for electrophilic and nucleophilic attack on the molecule can
be explained using global and local descriptor.
4
.4. Global descriptors
The global descriptors were calculated from the energy gap values of HOMO-LUMO.
By using this energy gap value, Koopmans’s theorem can be used to determine the global
descriptors of electronegativity (χ), electrophilicity index (ω), global softness (S), chemical
potential (ꢀ) and global hardness (η) of the molecule [53]. Here, A and I indicates the HOMO
and LUMO energy values.
χ =-(I+A)/2
µ = - χ = (I+A)/2
ɳ= 1/2(I - A)
2


=µ /2ɳ
S=1/2ɳ

N max=-µ/ɳ
The global descriptors of the molecule calculated using the above equations are depicted in
Table 1.
Electrophilicity index measures the energy stabilization when the system gains
additional electronic charge from surroundings [54]. The higher value of electrophilicity index
indicates the greater electrophilic behaviour of the system as well as lower chemical potential
[
55]. The low value of global electrophilicity index and higher value of chemical potential of
EBHMM, clearly indicate strong nucleophilic behavior of the azine sensor.
.5. Local descriptors
Fukui function is one of the widely used local reactivity descriptors for chemical
reactivity and site selectivity. Fukui functions (f , f , f ) were calculated using B3LYP/6-
4
k+ k- k0
3
11+ +G (d,p) and the following equations based on the research work of Yang and Mortier
[
56].
+
f = [q(N+1)-q(N)] – For nucleophilic attack
k
-
f = [q(N)-q(N-1)] – For electrophilic attack
k
0
f = 1/2[q(N+1)-q(N-1)] – For radical attack
k
Where N, N-1 and N+1 are total electrons present in neutral, cationic and anionic state
on the molecule respectively.
Local softness
+
-
0
+
-
0
Local softness (S , S and S ) and local electrophilic index (ωk
,
ωk and ωk ) are also
k
k
k
used to describe the reactivity of atoms in molecule. Here +, - and 0 indicate nucleophilic,
electrophilic and radical attacking sites and their values are presented in Table 2.
The atoms N17 and N18 are having high electrophilicity index value respectively
0
.9817 and 1.1524 than the other atoms. It is confirmed that N17 and N18 atoms have more
electronegativity and are more prone to electrophilic attack [57] in the chemosensor study. The
results of local descriptor confirmed the fluorescence quenching of probe due to PET by
3
+
involving N17 and N18 atoms. The possible mode of binding between azine and Fe ion is
1
shown in Figure 7. The non-involvement of -OH in binding is verified by H NMR titration.
The spectra don’t show any decrease in intensity of -OH signal during the titration. Therefore,
3
+
the binding of Fe with azine probe occur only with N17 and N18 atoms and not with phenolic
groups.
5
. Conclusion

Fluorescently spot chemosensor responded azine EBHMM was synthesized and
1
13
1
confirmed by using FT-IR, UV-Vis, H, C{ H}NMR spectroscopic techniques and mass
spectrometry. In chemosensor study, EBHMM selectively gives very spot response towards
3
+
Fe ion and is confirmed by HRMS-ESI spectrum. A good linearity was observed in Stern-
3
+
Volmer plot of EBHMM with Fe between 15 and 85µM concentration. The variation of
3
+
fluorescence intensities indicate the capability of azine towards the detection of Fe ion in
different pH.
The concentration depended quenching and reversibility of the probe with EDTA clearly
3
+
indicating the strong binding between azine and Fe ion. No change in the intensity of
1
3+
phenolic proton in H NMR titration indicate that it is not coordinated with Fe ion. The
binding sites of molecule with metal ion was theoretically calculated by using DFT study in
B3LYP functional with B3LYP/6-311+ +G (d,p) level of basis set. ESP and Contour analysis
map clearly indicates the electron rich and electron flow mostly around the azine in the
molecule. Global and Local descriptors are strongly gives the evidence that molecule has high
nucleophilic behavior and the atoms N17 and N18 atoms are more prone to electrophilic attack
in the chemosensor study due to high electrophilicity index. The results of Fukui Function and
the Frontier molecular orbital analysis strongly supported the quenching mechanism occurred
by the electron transition from the probe to metal through the N17 and N18 atoms.
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3
+
RECOGNITION OF Fe BY A NEW AZINE-BASED
FLUORESCENT "TURN-OFF" CHEMOSENSOR AND ITS
BINDING MODE ANALYSIS USING DFT
a
a
Subramani Manigandan and * Athianna Muthusamy
PG and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of
Arts and Science, Coimbatore-641 020, Tamil Nadu, India
*
E-mail: muthusamy@rmv.ac.in and muthusrkv@gmail.com
&
b
b
*
Raju Nandhakumar and Charles Immanuel David
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore-641 114, Tamil Nadu, India
*
E-mail: nandhakumar@karunya.edu
Table 1: Global descriptors values of EBHMM
2
I= - HOMO
5.8097
A= -LUMO
-2.5598
ɳ= 1/2(I - A)
S=1/2ɳ
ꙍ=µ /2ɳ
Nmax=-µ/ɳ
χ
= -(I+A)/2
4.1847
µ= -
χ
-
-4.1847
-1.6249
-0.8123
-14.2281
-2.5753
Table 2: Local descriptors values of EBHMM
Natural atomic charges (eV)
Fukui functions(eV)
Local softness(eV)
Local electrophilicity index(eV)
Atoms
qN+1
-0.263
0.293
0.014
-1.101
0.876
0.728
-0.872
0.201
1.083
-0.223
0.139
0.047
-0.627
-0.535
0.116
0.085
-0.217
-0.195
0.002
0.003
-0.491
-0.364
0.184
-0.449
-0.34
qN0
-0.251
0.314
-0.021
-1.125
0.832
0.656
-0.881
0.161
1.048
-0.213
0.086
0.006
-0.637
-0.521
0.060
0.021
-0.251
-0.22
qN-1
-0.239
0.311
-0.05
fk+
fk0
fk-
Sk+
Sk0
Sk-
ꙍ+
ꙍ0
ꙍ-
1
2
3
4
5
6
7
8
9
C
C
C
C
C
C
C
C
C
-0.012
-0.021
0.035
0.024
0.044
0.072
0.009
0.04
-0.012
-0.009
0.032
0.029
0.040
0.061
0.038
0.019
-0.002
-0.034
0.05
-0.012
0.003
0.029
0.034
0.036
0.05
0.0097
0.0170
0.0097
0.0073
0.0097
-0.0024
-0.0235
-0.0276
-0.0292
-0.0406
-0.0544
0.0016
0.0324
0.0479
-0.0381
-0.0211
-0.0568
-0.1178
0.0113
0.0219
-0.0560
-0.0658
-0.0333
-0.0390
-0.0203
-0.0032
0.0048
-0.0268
-0.0097
-0.0048
0.1707
0.1707
0.17073
-0.0426
-0.4126
-0.4837
-0.5122
-0.7114
-0.9532
0.0284
0.5691
0.8394
-0.6687
-0.3699
-0.9959
-2.0630
0.1991
0.3841
0.9817
1.1524
-0.5833
-0.6829
0.3557
0.0569
0.0853
-0.4695
-0.1707
-0.0853
0.2987
0.1280
-0.0284
-0.0195
-0.0357
-0.0585
-0.0073
-0.0325
-0.0284
0.0081
-0.0260
-0.0235
-0.0325
-0.0495
-0.0308
-0.0154
0.0020
-0.4979
-0.3414
-0.6260
-1.0244
-0.1280
-0.5691
-0.4979
0.1422
-0.4553
-0.4126
-0.5691
-0.8679
-0.5406
-0.2703
0.0355
-1.159
0.796
0.606
-0.948
0.163
1.088
-0.154
0.039
-0.02
0.067
-0.002
-0.040
-0.059
0.047
0.026
0.07
0.035
-0.01
0.053
0.041
0.01
10C
11C
12C
13C
14C
15N
16N
17N
18N
19C
20C
21O
22O
23C
24O
25O
26C
0.0280
0.4908
-0.0430
-0.0333
-0.0081
0.0113
-0.0406
-0.0272
-0.0325
-0.0532
-0.0170
-0.0150
-0.0418
-0.0430
-0.0174
-0.0215
-0.0280
-0.0044
-0.1438
-0.0373
-0.0130
-0.0036
-0.7540
-0.5833
-0.1422
0.1991
-0.7114
-0.4766
-0.5691
-0.9319
-0.2987
-0.2632
-0.7327
-0.7540
-0.3059
-0.37705
-0.49087
-0.07825
-2.51839
-0.6545
-0.22765
-0.06403
0.033
0.04
-0.707
-0.666
0.074
0.048
-0.32
-0.014
0.056
0.064
0.034
0.025
0.002
0.005
0.044
0.007
0.36
0.065
0.021
0.018
0.051
0.053
0.021
0.026
0.034
0.005
0.177
0.046
0.016
0.004
0.145
-0.014
-0.027
0.069
0.081
0.041
0.048
0.025
0.004
-0.006
0.033
0.012
0.006
-0.0455
-0.0520
-0.0276
-0.0203
-0.0016
-0.0040
-0.0357
-0.0056
-0.2924
-0.0479
-0.0162
-0.0020
-0.7967
-0.9106
-0.4837
-0.3557
-0.0284
-0.07114
-0.62604
-0.0996
-5.12214
-0.83946
-0.28456
-0.04268
-0.301
-0.041
-0.05
0
-0.002
-0.535
-0.371
-0.176
-0.508
-0.360
-0.111
-0.56
-0.375
-0.17
-0.541
-0.372
-0.117
0.059
0.02
-0.108
0.003

Table 3: HOMO LUMO and band gap value of ligand and metal
Compound
EBHMM
Fe(OH)3
I=-Homo
-5.8097
-6.8395
A=-Lumo
-2.5598
-6.6582
Band Gap(eV) MO.Number
3.2499 93(U),94(O)
0.1812 27(O),28(U)

3
+
RECOGNITION OF Fe BY A NEW AZINE-BASED
FLUORESCENT "TURN-OFF" CHEMOSENSOR AND ITS
BINDING MODE ANALYSIS USING DFT
a
a
Subramani Manigandan and * Athianna Muthusamy
PG and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts
and Science, Coimbatore-641 020, Tamil Nadu, India
*
E-mail: muthusamy@rmv.ac.in and muthusrkv@gmail.com
&
b
b
*
Raju Nandhakumar and Charles Immanuel David
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore-641 114, Tamil Nadu, India
*
E-mail: nandhakumar@karunya.edu
2
Figure 1. Selective responses of EBHMM in DMF-H O solution (1:1, EBHMM=5µM,
pH=7.4) in the presence of various metal ions (100 equiv. λex=423 nm)

3
+
Figure 2. Fluorescence response of EBHMM with Fe over the
presence of other metal ions

Figure 3. Fluorescence titration spectrum of EBHMM (1:1, EBHMM=5µM, pH=7.4) in the
3
+
presence of different concentrations of Fe (0–100 equiv.) excited at 423 nm

3
+
Figure 4. Stern–Volmer linearity plot of EBHMM with [Fe ]
3
+
Figure 5. Benesi–Hildebrand plot of EBHMM -Fe complex
1:1) binding stoichiometry
(

3
+
Figure 6. Reversibility of EBHMM
-
Fe with EDTA
3
+
Figure 7. Proposed binding mode of Fe with EBHMM

3
+
RECOGNITION OF Fe BY A NEW AZINE-BASED
FLUORESCENT "TURN-OFF" CHEMOSENSOR AND ITS
BINDING MODE ANALYSIS USING DFT
a
a
Subramani Manigandan and * Athianna Muthusamy
PG and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of
Arts and Science, Coimbatore-641 020, Tamil Nadu, India
*
E-mail: muthusamy@rmv.ac.in and muthusrkv@gmail.com
&
b
b
*
Raju Nandhakumar and Charles Immanuel David
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore-641 114, Tamil Nadu, India
*
E-mail: nandhakumar@karunya.edu
Scheme 1: Synthesis of 4-(hydrazonomethyl)-2-methoxyphenol (PHM)
Scheme 2: Synthesis of 4,4-((ethane-1,2-diylidene)bis(hydrazine-2,1-
diylidene))bis(methanylylidene) bis(methanylylidene)) bis(2-methoxyphenol) (EBHMM)

3
+
RECOGNITION OF Fe BY A NEW AZINE-BASED
FLUORESCENT "TURN-OFF" CHEMOSENSOR AND ITS
BINDING MODE ANALYSIS USING DFT
a
a
Subramani Manigandan and * Athianna Muthusamy
PG and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts
and Science, Coimbatore-641 020, Tamil Nadu, India
*E-mail: muthusamy@rmv.ac.in and muthusrkv@gmail.com
&
b
b
*
Raju Nandhakumar and Charles Immanuel David
Department of Chemistry, Karunya Institute of Technology and Sciences (Deemed-to-be
University), Karunya Nagar, Coimbatore-641 114, Tamil Nadu, India
*
E-mail: nandhakumar@karunya.edu
Highlights
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ꢀ
ꢀ
ꢀ
Azine based chemosensor was successfully designed and synthesized.
3
+
The sensor showed“turn-off” responses for Fe ion over other metal ions.
+
3
The sensor is sensitive and selective to Fe ion at different concentrations and various pH.
The binding sites of the molecule with metal ion was theoretically obtained using DFT study
in B3LYP functional with B3LYP/6-311+ +G (d,p) level of basis set.

AUTHORSHIP STATEMENT
AZINE BASED FLUORESCENT Fe3+ CHEMOSENSOR AND BINDING MODE
Manuscript title: _______________________________________________________________________
ANALYSIS USING DFT
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All persons who meet authorship criteria are listed as authors, and all authors certify that they have
participated sufficiently in the work to take public responsibility for the content, including participation in
the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that
this material or similar material has not been and will not be submitted to or published in any other
publication before its appearance in the Hong Kong Journal of Occupational Therapy.
Authorship contributions
Please indicate the specific contributions made by each author (list the authors’ initials followed by their
surnames, e.g., Y.L. Cheung). The name of each author must appear at least once in each of the three
categories below.
Category 1
Subramani Manigandan, Athianna Muthusamy
Conception and design of study: ____________, _____________, ____________, _____________;
Subramani Manigandan, Charles Immanuel David
acquisition of data: _____________, _____________, _____________, _____________;
Subramani Manigandan, Raju Nandhakumar
analysis and/or interpretation of data: ____________, ___________, ____________, ___________.
Category 2
Subramani Manigandan, Athianna Muthusamy
Drafting the manuscript: ______________, ______________, ______________, ______________;
Subramani Manigandan,
revising the manuscript critically for important intellectual content: ____________, ____________,
Athianna Muthusamy
____________, _____________.
_
Category 3
Approval of the version of the manuscript to be published (the names of all authors must be listed):
Subramani Manigandan, Athianna Muthusamy, Raju Nandhakumar, Charles Immanuel David
_
______________, _______________, _______________, _______________, _______________,
______________, _______________, _______________, _______________, _______________.
_

Acknowledgements
All persons who have made substantial contributions to the work reported in the manuscript (e.g., technical
help, writing and editing assistance, general support), but who do not meet the criteria for authorship, are
named in the Acknowledgements and have given us their written permission to be named. If we have not
included an Acknowledgements, then that indicates that we have not received substantial contributions from
non-authors.
This statement is signed by all the authors (a photocopy of this form may be used if there are more than 10 authors):
Author’s name (typed)
Author’s signature
Date
Subramani Manigandan
31-12-2019
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Athianna Muthusamy
31-12-2019
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Raju Nandhakumar
31-12-2019
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Charles Immanuel David
31-12-2019
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