A new Schiff-base as fluorescent chemosensor for selective detection of Cr3+: An experimental and theoretical study

Article abstract of DOI:10.1016/j.tet.2018.03.046

A novel Schiff base fluorescent sensor N,N′-bis(salicylidene)-2,6-bis(4-aminophenyl)-4-phenylpyridine (P3) was synthesized through condensation of 2,6-bis(4-aminophenyl)-4-phenylpyridine and 2-hydroxybenzaldehyde. The obtained results from fluorescence an

Full text of DOI:10.1016/j.tet.2018.03.046

Accepted Manuscript
3+
A new Schiff-base as fluorescent chemosensor for selective detection of Cr : An
experimental and theoretical study
Gholam Babaei Chalmardi, Mahmood Tajbakhsh, Nahid Hasani, Ahmadreza
Bekhradnia
PII:
S0040-4020(18)30311-9
10.1016/j.tet.2018.03.046
TET 29386
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 8 January 2018
Revised Date: 17 March 2018
Accepted Date: 21 March 2018
Please cite this article as: Chalmardi GB, Tajbakhsh M, Hasani N, Bekhradnia A, A new Schiff-base
3+
as fluorescent chemosensor for selective detection of Cr : An experimental and theoretical study,
Tetrahedron (2018), doi: 10.1016/j.tet.2018.03.046.
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ACCEPTED MANUSCRIPT
A new Schiff-base as fluorescent chemosensor for selective detection of Cr3+: An
experimental and theoretical study
Gholam Babaei Chalmardia, Mahmood Tajbakhsha*, Nahid Hasania, Ahmadreza Bekhradniab*
a
Department of Organic Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar 47416-95447, Iran
b
Pharmaceutical Sciences Research Center, Department of Medicinal Chemistry, Mazandaran University of
Medical Sciences, Sari, Iran
E-mail: Mahmood.Tajbakhsh32@gmail.com, abekhradnia@gmail.com
Graphical Abstract
N
..
N
..
N
N
N
CH
N
HC
Cr3+
CH
HC
Cr3+
OH
HO
OH
HO
Fluorescence
OFF
PET ON
Fluorescence
ON
PET OFF
Highlights
The Schiff base (P3) shows high selectivity towards Cr3+ ion compared with other competing
metal ions.
DFT calculations together with the experimental results obtained from Job’s plot method
showed a 1:1 binding ratio of Cr3+ with ligand P3.
The sensing mechanism are explained by photoinduced electron transfer (PET) and chelation -
enhanced fluorescence (CHEF).
It has high affinity (Ka =2.28 × 105 M-1) and selectivity for Cr3+.
The detection limit of the sensor towards Cr3+ is 1.3 × 10-7 M.
1

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Abstract
A novel Schiff base fluorescent sensor N,N’-bis(salicylidene)-2,6-bis(4-aminophenyl)-4-
phenylpyridine (P3) was synthesized through condensation of 2,6-bis(4-aminophenyl)-4-
phenylpyridine and 2-hydroxybenzaldehyde. The obtained results from fluorescence analysis
revealed that by excess of Cr3+ to P3, a remarkable increase was observed in the fluorescent
intensity of the Schiff base at 663 nm with the ratio of CH3CN/H2O (95/5%), even though the
other cations would likely have no impact on the fluorescence intensity. The cause of this trend
might be ascribed to the formation of a 1:1 stoichiometric P3-Cr3+ complex, confirmed by Job’s
plot, which is resulted in preventing the photo-induced electron transfer (PET) process. From
fluorescence titration, the association constant Ka was gained 2.28 × 105 M-1 and the limit of
detection (LOD) was determined to be 1.3 × 10-7 M. Furthermore, the optimized structure
together with the electronic spectra of the proposed complex was determined by DFT and
TDDFT calculations.
Keywords: Fluorescence, Chemosensors, Schiff-base, PET, DFT, Chromium ion.
1. Introduction
The synthesis and development of new fluorescent chemosensors with the goal of
detecting the heavy and transition metal ions are attractive and promising, whereas their use is
growing fast in several research areas such as supramolecular chemistry, organic chemistry, drug
delivery, biological chemistry and environmental chemistry 1–2. Among these compounds, N-
heterocycles are considerable attention toward chemosensors due to their high selectivity and
sensitivity and also most important moieties in medicinal chemistry 3–5. To tackle the
environmental and medical challenges, a significant effort has been devoted in designing small-
molecule florescent organic issues for chromium (Cr3+) ion sensing. This vital ion, as an essential
nutrient, plays a major role in the metabolism of carbohydrates, lipids, proteins, and nucleic acids
6
in human body . Another most important reason for enhancing the risk factors connected with
diabetes and cardiovascular disease is concerned to insufficient dietary intake of chromium ion 7.
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On the other hand, chromium is recognized as a noxious pollutant, omitted through
various industrial and agricultural activities, that it can definitely be detrimental for both
environment and human health. 8. Despite the biological and environmental benefits of Cr3+, few
reports regarding fluorescent detection of Cr3+ are available 9-14. A diversity of electron transition
mechanisms, for example, chelation enhanced fluorescence (CHEF), intermolecular charge
transfer (ICT), fluorescence resonance energy transfer (FRET), metal to ligand charge transfer
(MLCT), photoinduced electron transfer (PET), photoinduced proton transfer (PPT), excited
state intermolecular proton transfer (ESIPT), chelation-enhanced fluorescence (CHEF) effect,
15–20
and C=N isomerization has been considered for developing fluorescent chemosensors
.
Among the mentioned mechanisms, PET process has been used as a sensing tool in which the
electron transfer directly occurs between the fluorophore (signaling unit) and the receptor
(“switch” of the fluorescence intensity) 21–23. The receptor unit can be connected chemically to
fluorophore with absorption of shorter wavelength, where the PET efficiently happens between
donor-acceptor probes 24–26. In this regard, the electron-donating Schiff base ligand, can be
considered as a receptor unit to absorb short wavelength light through forming chelate complex
with the metal ions. Due to paramagnetic property of Cr3+ ion and the deficiency of a selective
multi-chelating ligand, the appropriate fluorescent turn-on chemosensors to monitor intracellular
Cr3+ are still under developed 27. With this approach, we report a new chemosensor, N,N’-
bis(salicylidene)-2,6-bis(4-aminophenyl)-4-phenylpyridine (P3) for Cr3+ that was prepared by
condensing 2,6-bis(4-aminophenyl)-4-phenylpyridine with 2-hydroxybenzaldehyde (Scheme 1).
Upon addition of Cr3+, P3 shows a large fluorescence enhancement because of the formation of a
1:1 P3-Cr3+ complex that inhibits photo-induced electron transfer (PET) process. The other
metal ions including Na+, K+, Bi2+, Hg2+, Co2+, Cu2+, Zn2+, Cd2+, Fe3+ and Al3+ have almost no
influence on the fluorescence.
To get more insight related to the electronic structures of P3 and P3-Cr3+ complex, density
functional theory (DFT) calculations were carried out on the plausible structures of the
molecules. Moreover, time dependent density functional theory (TDDFT) was used
as an accurate method for finding the excited state energies and UV-Vis absorption spectra of P3
and P3-Cr3+ complex.
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2. Experimental
2.1. General and reagents
Melting points were measured on an Electrothermal 9100 apparatus. Mass spectra were
1
obtained on an Agilent 5975c spectrometer operating at 70 eV. H and 13C spectra were
measured with Bruker DRX-400 AVANCE spectrometer at 400.1and 100.6 MHz, respectively.
The UV-Vis spectra were recorded using a Perkin-Elmer lambda-EZ 201 and a Jasco FP-200
spectra fluorometer was used to obtain fluorescence emission spectra. Fluorescence intensity
measurements were performed in CH3CN/H2O (95/5%) at room temperature.
2.2. Synthesis
2.2.1. Synthesis of 2,6-bis(4-nitrophenyl)-4-phenylpyridine ( P1)
A mixture of benzaldehyde (6.4 g, 0.06 mol), p-nitroacetophenone (20 g, 0.12 mol),
28
ammonium acetate (60 g), in glacial acetic acid (150 ml) was refluxed for 2 h . After cooling
the reaction mixture to room temperature a solid residue was obtained which then filtered and
washed with acetic acid (50%) and cold ethanol, respectively. The crude 4-phenyl-2,6-bis(4-
nitrophenyl) pyridine (P1) was recrystallized from absolute ethanol, and dried under vacuum
◦
1
(Scheme1). Yield: 66%, mp: 319-321 C, H NMR (400 MHz, CDCl3), δ, ppm: 8.43-8.38 (8H,
13
m), 8.07 (2H, S), 7.80-7.77 (m, 2H), 7.63-7.56 (3H, m). C NMR (100 MHz, CDCl3), δ, ppm:
155.52, 151.39, 148.44, 144.83, 137.90, 129.73, 129.42, 127.94, 127.19, 124.11 and 119.12 (Fig.
S1).
2.2.2. Synthesis of 2,6-bis(4-aminophenyl)-4-phenylpyridine (P2)
A mixture of 4-phenyl-2,6-bis(4-nitrophenyl) pyridine (13.75 g, 48mmol) and palladium
on carbon 5% (5 g) in ethanol (500 mL) as solvent were prepared in a two-necked round-
bottomed flask (500 ml) equipped a dropping funnel. The mixture was warmed up to 50 ºC and
then hydrazine hydrate 85% (35 ml) in ethanol (50 ml) was added dropwise over a 1.5 h period
through the dropping funnel while maintaining the temperature at about 50 ºC. The reaction
mixture was then refluxed for 2 h and filtered. Upon cooling, the filtrate gave white colored
crystals of the 2,6-bis(4-aminophenyl)-4-phenylpyridine (P2), which were recrystallized from
◦
1
ethanol and vacuum dried. (Scheme1). Yield: 64%. mp: 207-209 C. H NMR (400 MHz,
CDCl3), δ, ppm: 8.068 (4H, d, J=8.4 Hz), 7.77-7.72 (4H, m), 7.54 (2H, T, J=7.6 Hz), 7.48(1H, d,
J=7.2 Hz), 6.82 (4H, d, J=8.4 Hz), 3.85 (4H, S, NH2). 13C NMR (100 MHz, CDCl3), δ, ppm:
4

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157.13, 149.72, 147.32, 139.67, 130.30, 128.98, 128.65, 128.26, 127.16, 115.05 and 114.86 (Fig.
S2).
2.2.3. N,N’-bis(salicylidene)-2,6-bis(4-aminophenyl)-4-phenylpyridine (P3)
A solution included 2-(6-(2-aminophenyl)-4-phenylpyridin-2-yl)benzenamine (1 mmol)
in absolute ethanol was added to an ethanolic solution of 2-hydroxybenzaldehyde (2 mmol). The
mixture was refluxed for 6 h and then cooled to room temperature. The solvent was evaporated
to afford yellow product which was recrystallized from ethanol (Scheme1). Yield: 87%. mp:
189-193 ◦C. 1H NMR (400 MHz, CDCl3), δ, ppm: 13.29 (2H, S), 8.74 (2H, S), 8.32 (4H, d, J 8),
7.94 (2H, S), 7.79 (2H, d, J=7.2 Hz), 7.59-7.52 (3H, m), 7.47-7.41 (8H, m), 7.07 (2H, d, J=8 Hz),
7.01(2H, t, J=7.2 Hz). 13C NMR (100 MHz, CDCl3), δ, ppm: 162.75, 161.25, 156.69, 150.43,
149.15, 138.92, 138.08, 133.34, 132.41, 129.19, 129.13, 128.21, 127.21, 121.59, 119.25, 119.16,
117.33, and 117.03. The mass spectrum showed a peak at m/z = 545.2 corresponding to
compound P3 (Figs. S3 and S4).
O
CHO
acetic acid
NH4OA
c
O2N
N
reflux
O2N
NO2
P1
Pd/C, NH2NH2
EtOH
N
N
H2N
NH2
O2N
NO2
P2
CHO
OH
EtOH
reflux
N
2
N
N
N
H2N
NH2
CH
HC
OH
HO
P3
Scheme 1. The synthetic route of compound P3.
5

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2.3. Spectrometric procedure
Stock solutions (10 µM) of nitrate salts of Na+, K+, Bi2+, Hg2+, Co2+, Cu2+, Zn2+, Cr3+,
Cd2+, Fe3+, Al3+ in CH3CN/H2O (95/5%) were prepared and compound P3 was dissolved in
CH3CN/H2O (95/5%), (10 µM). Test solutions were prepared by placing 2 mL of the probe stock
solution into cuvettes, adding 2mL of each metal ion stock. For all measurements, excitation
wavelength was at 360 nm.
2.4. DFT procedure
The electronic structures of the titled compounds were optimized by density functional
29
calculations (DFT) without any symmetry constraints, employing the Gaussian09 package
.
Frequency calculations were performed on all the optimized geometries to ensure the correct
local minima. Al the calculations were carried out in the framework of the Becke three-
parameter hybrid exchange and Lee–Yang–Parr correlation functional (B3LYP) together with a
split-valence Pople basis set plus polarization and diffuse functions, 6-31+G (d,p) for H, C, N,
and O atoms and a double-ζ quality LANL2DZ basis set for Cr atom 30. All the “inner electrons”
of metal ions were replaced and described with a scalar relativistic electron core potential (ECP).
Time-dependent density functional theory (TD-DFT) calculations were performed to attain the
UV-Vis spectra and characterization of the frontier orbitals, at B3LYP/6-31+G (d,p) level with
the polarized continuum model (IEFPCM) in acetonitrile solvent 31.
3. Results and discussion
3.1. Spectral studies
The outcomes obtained from absorption and fluorescence spectral analysis, upon addition
of a variety of metal ions to P3 solution with the ratio of CH3CN/H2O (95/5%) were shown in
Fig.1 and Fig. S5. The absorption spectra of P3 (10 µM) in CH3CN/H2O (95/5%) solution
display three bands at 236 nm, 316 nm and 360 nm. After addition of various metal ions such as
Cr3+, Na+, K+, Bi2+, Hg2+, Co2+, Cu2+, Zn2+, Cd2+, Fe3+and Al3+, the absorption intensity of the
peaks were decreased which can be attributed to the hypochromic effects in the UV-Vis spectra
of P3 (Fig. S5). It seems the UV-Vis spectra cannot be likely an alternative to demonstrate the
specific sensitivity of P3 to the cations due to the ambiguous absorption spectra within mixture
species 32. To this reason, the selectivity of P3 towards Cr3+ among other metal ion solutions (10
6

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µM) was investigated by fluorescence spectroscopy in CH3CN/H2O (95/5%) solution. As shown
in Fig 1, the sensor P3 without Cr3+ exhibits the weak fluorescence intensity with excitation at
363 nm. Upon addition of various metal ions, the Cr3+ has only displayed a considerably
enhancement of fluorescence, which is indeed demonstrated the high selectivity of P3 for Cr3+.
This could be assigned to the formation of a 1:1 stoichiometric P3-Cr3+ complex associated with
the inhibiting of photo-induced electron transfer (PET) process 33-35
.
a)
b)
Fig. 1. (a) Fluorescence responses of compound P3 (10 µM) in CH3CN/H2O (95/5%) solution upon the addition of
various metal ions (λex =360 nm, 2 equiv). (b) Bar graph representing the change of the relative emission intensity
of P3 at 663 nm upon treatment with various metal ions (λex = 360 nm).
By considering the fact that the Schiff base P3 is quite sensitive to Cr3+ rather than other
metal ions, the absorption of ligand P3 in a mixture of other metal ions in CH3CN/H2O (95/5%)
7

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solution both in absence and presence of Cr3+ have been measured which resulted in observing a
drastic enhancement in fluorescence intensity of (P3-other cations-Cr3+) mixture than the another
one (shown in Fig. 2). These observations can likely confirm the trend of P3 as an efficient and
selective fluorescence chemosensor for Cr3+ ion.
Fig. 2. Fluorescence intensity of P3 (10 µM) in the absence of metal ions (red curve), and presence of 2 equiv of all
kinds of competitive metal ions (blue curve) in CH3CN/H2O (95/5%, λex = 360 nm. The green curve represents the
addition of Cr3+ to the above mixture. Each spectrum was acquired 1 minute after cations addition at room
temperature.
In order to gain further insights concerning the chemosensing properties of receptor P3, a
fluorescence titration of receptor P3 with Cr3+ has been carried out. The results were in
accordance with our claim so that by raising the concentration of Cr3+, the sensitivity of P3
towards Cr3+ ions was obviously increased as it is shown in Fig. 3. The fluorescence spectrum of
P3 (
λ
= 663 nm) shows the rapid turn-on responses and the inset is indication of the relative
em
fluorescence intensity changes as a function of Cr3+ concentration.
8

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Cr3+
Fig.3. Fluorescence titration of P3 (10 µM) with various concentration of Cr3+ in CH3CN/H2O (95/5%) solution
(excitation = 360 nm) at room temperature. inset: changes of fluorescence upon addition of Cr3+ (0-2 equiv) at
emission=663 nm.
Furthermore, the association constant (Ka) of binding of P3 with metal ions was
determined by Benesi–Hildebrand equation 36 as follows:
F and F0 represent the fluorescent intensity of compound P3 in the presence and absence of metal
ions, respectively. Fmax is the saturated fluorescent intensity of compound P3 in the presence of
excess amount of metal ions and [M] is the concentration of the added metal ions. The
association constant of P3-Cr3+ complex was determined to be 2.28 × 105 M-1 using the results
obtained from fluorescence titration. Moreover, the detection limit of P3 was estimated based on
the fluorescence titration while to gain the S/N ratio, the emission intensity of the complex (P3-
Cr3+) was calculated along with the standard deviation of blank measurements. Then, the
detection limit for Cr3+, according to 3 × δb/m, was found to be 1.3 × 10-7 mol/L, where δb and m
are the standard deviation of blank solutions and the slope between intensity versus sample
concentration, respectively37.
9

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In alignment with our research studies, the fluorescence quantum yield for the P3-Cr3+ complex
was determined through comparison of both emission and absorption intensities of the bands
38
with those of a fluorescence standard (fluorescein in 0.1 N NaOH) which was obtained to be
13.8%. For all the fluorescence measurements, the region of excitation was in 360 nm with slit
widths of 3.0 nm. However, the negligible quantum yields calculated for combined receptor and
the other cations as well as free receptor are not reported.
3.2. Binding mode studies
To find out the stoichiometry of binding between the receptor P3 and Cr3+, the
Job's method of continuous variation was utilized. The total concentration of the complex P3-
Cr3+was constant (10 µM), with a continuous variable molar fraction of guest ([P3]/[ Cr3+] +
[P3]). Fig. 4 shows the Job's plot of ligand P3 with Cr3+ (at 663 nm) in which the fluorescence
emission intensity indicates a maximum, whereas the molar fraction of P3 is 0.5. As a result,
a stoichiometry of 1:1 was confirmed for the complex Cr3+-P3.
Fig. 4. Job’s plot for determining the stoichiometric complexation of P3 with Cr3+ in CH3CN/H2O (95/5%) at room
temperature. The total concentration of P3 and Cr3+ is 10 µM.
1
In addition, the H NMR titration experiments were applied to support the binding of
receptor P3 to Cr3+ to form a metal complex. The experiment was conducted by addition of
increasing concentrations of Cr3+ to the P3 solution, so that the phenolic OH signals of P3,
centered at 13.29 ppm, was disappeared after addition of 2 equivalent of Cr3+. This implies that
10

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the phenolic OH groups are vital to make a stable complex between the P3 and Cr3+. The binding
mode of P3 and Cr3+ is meaningful for the formation of novel Cr3+ fluorescent chemosensor 39, 40
.
3+2 eq Cr3+
3+1.5 eq Cr3+
3+1 eq Cr3+
3+0.5 eq Cr3+
3+0.25 eq Cr3+
OH
1
Fig. 5. Partial H NMR spectra of P3 (10 µM) in CD3CN at room temperature and the corresponding changes after
the addition of increasing amounts of Chromium nitrate (0-2 equiv). Each spectrum was acquired 1 minute after Cr3+
addition at room temperature.
3.4. Solvent effect
It is found that the character of optical sensing in the chemosensors is connected to the
polarity of solvents. Hence in this study, the response of ligand P3 to Cr3+ ion in various media
such as DMSO, DMF, methanol, acetonitrile and acetonitrile/H2O was investigated at maxima of
emission intensity. The diagram (shown in Fig. 6) shows the optimum fluorescence emission
occurs both in acetonitrile and acetonitrile/H2O (95/5%) solvents. On the other hand, the aprotic
(DMSO, DMF) and protic (methanol, H2O) media results in a significantly reduction in
fluorescent intensity These observations can be due to hydrogen bonding formation between
11

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solvents and hydroxyl groups, rather than general solvent effects 41. Nevertheless, the highest
fluorescent intensity was observed in acetonitrile and acetonitrile/H2O (95/5%), and hence the
later was chosen as solvent 42.
Fig. 6. Solvent effect on fluorescent intensity of P3 (10 µM) with Cr3+ (2 equiv) in different solvent DMSO, DMF,
methanol, acetonitrile, acetonitrile/H2O (50/50%), acetonitrile/H2O (80/20%), acetonitrile/H2O (95/5%).
3.5. Effect of pH on the binding affinity of Cr3+ to P3
Due to the critical role of the medium pH in the fluorescence detection of the
chemosensors, in this study the effect of pH (in the range of 2 to13) on the fluorescence response
of P3 was investigated in CH3CN/H2O (95/5%) mixture. The changes in the fluorescence
intensity at 663 nm of P3 in both absence and presence of Cr3+ ion were plotted as a function of
pH (Fig. 7). The overall intensity of P3–Cr3+ remained higher than the free receptor P3.
Fluorescent intensity was found to be the highest at pH 9. For measurements outside the pH 9,
Cr3+ might be appeared as Cr(OH)3 species, removed from P3, thus the outcomes can be
impacted on emission intensity as decreasing. In the same manner, below pH 9, P3 might be
combined with H+ and show no tendency to bind Cr3+ resulting a decrease in the fluorescence
intensity. So, P3 can tolerate the detection of Cr3+ in the pH 9 43.
12

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Fig. 7. Fluorescence response for P3 in the absence and presence of Chromium as a function of pH in CH3CN/H2O
(95/5%) at 663 nm.
3.6. Comparison
To find reliable results, the chemosensor P3 was compared to those previously reported
chemosensors for Cr3+ and the data are tabulated in Table 2. In comparing manner, the detection
limit, binding constant, sensing response, and pH range exhibited that P3 was efficient sensor in
compare with other synthetic ionospheres 44-48 in table 2.
Table 2: Comparative analysis of chemosensor P3 with previously reported sensors.
Ionophores
mechanism
Methods of
detections
Medium of
detection
Detection
limit
Binding
Constant
M-1
Sensing
pH
response
range
(M)
8
7
Ref. 40
PET and
CHEF
-
fluorescent
fluorescent
CH3CN/H2O
(95/5%)
2.2×10-7
8.77×104
Cr3+ (Turn on)
7.94 × 10-5
1.21 × 10-6
1.6 × 104
1.01× 104
Cr3+ (Turn on)
Cr3+ (Turn on)
CH3CN/H2O
(8/2)
Ref. 44
Ref. 45
2-7
PET
-
colorimetric
and
MeOH
MeOH
CH3CN
CH3CN
fluorescent
colorimetric
and
-
Ref. 46
Ref. 47
Ref. 48
1.1 × 10-5
2.75 × 10-6
9.86 × 10-6
1.4 × 104
5.97 × 104
2.1× 103
Cr3+ (Turn on)
Cr3+ (Turn on)
Cr3+ (Turn on)
fluorescent
colorimetric
and
-
C=N
isomerization
and ESIPT
-
fluorescent
fluorescent
6-8
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1.3 × 10-7
2.28 ×105
Cr3+ (Turn on)
9
PET and
CHEF
fluorescent
CH3CN/H2O
(95/5%)
P3
3.7. Theoretical study
3.7.1. The optimization calculations
The experimental results revealed that the Schiff base ligand (P3) is quite sensitive to Cr (III) ion
compared to the other metal ions, nevertheless, the coordination geometry of the ligand to bind
Cr(III) ion is still ambiguous. Since the crystal structures of P3 ligand and P3–Cr3+ complex
could not be determined by X-ray spectroscopy due to taken fine powders of the compounds. To
this reason, plausible tetrahedron geometry of P3–Cr3+ complex was considered to the titled
compound. The assuming structures of both of both P3 and P3–Cr3+ compounds were optimized
using DFT calculations at B3LYP/6-31G (d,p) levels of theory. The Optimized structures of P3
and also P3–Cr3+ complex together with the selected calculated bond distances and angles are
shown in Fig. 8 and Fig. 9, respectively. As shown in Fig. 8, the Schiff base (P3) behaves as a
tridentate ligand that was coordinated through N atom of phenylpyridine (N1) and two oxygen
atoms of salicylidene (O1 and O2) groups. So, the best possible structure of P3–Cr3+ complex
results in a distorted tetrahedral coordination geometry in which one oxygen atom (O3) of nitrate
group and also O1, O2 and N1 atoms of P3 ligand could bind to Cr3+ ion, as is supported by the
NMR spectra in experimental section (vide infra 3.2).
1
In order to investigate of binding mode of P3 with Cr3+ ion, the H NMR spectra of P3 in the
absence and presence of Cr3+ were investigated in CD3CN (Fig. 5). The proton shift of P3 at 8.74
ppm may be assigned to the proton of imine, which is fairly intact with addition of chromium ion
to the P3 solution. In spite of our previous study, N,N’-bis(salicylidene)-2,6-bis(2-aminophenyl)-
40
4-phenylpyridine (3)
( Table 2), the results indicate that the proton of imine group is not
involved in the formation of complex between the P3 with Cr3+ ion. To attain a deeper insight,
the evaluation was performed with the comparable studies. The comparison between P3 and 3 40
implied both fluorescent probes exhibit a fluorescence enhancement in the presence of Cr3+
through inhibition of photo-induced electron transfer (PET) mechanism.
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In addition of Cr+3 ion, the fluorescent chemosensor of ligand 3 showed a single emission band
at 537 nm upon excitation at 237 nm, whereas ligand P3 revealed an outstanding increase in the
fluorescent intensity at 663 nm upon excitation at 360 nm according to fluorescence emission
studies. Also, the association constant (Ka) of binding of P3-Cr+3 was calculated around 10 times
greater than 3- Cr+3 by Benesi–Hildebrand equation.
A bond length increase of 0.029 Å for C2-C3 and 0.015 Å for C5-C6, from ligand to complex,
lead to easier rotation of C-C groups. On the other hand, by decreasing the bond lengths of N2-
C4 and N3-C1, the free rotation around of the complex is restricted which results in a tetrahedral
geometry around the metal ion.
Fig. 8. Optimized geometry of the Schiff base ligand (P3) with B3LYP/6-31G(d, p) level.
15

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Fig. 9. Optimized geometries of P3–Cr3+ complex with B3LYP/6-31G(d, p)/LANL2Z level.
3.7.2. The TDDFT calculations
The electronic spectra of both P3 (Schiff base) and P3–Cr3+ complex, optimized with
inclusion of acetonitrile solvent, were ascertained by TDDFT calculations to determine the
energies and compositions of the molecular orbitals (MOs). The assignment of the most
important occupied (HOMOs) and unoccupied (LUMOs) orbitals involved in electronic
transitions in α and β spin states, with the fragment Mulliken contributions have been expressed
in terms of the compositions from the central metal (Cr), (PhN) and (PhO) parts and also (Py)
fragment (shown in Fig S6). The energy gap between HOMO and LUMO can be related to
chemical activity of the molecules and tendency to intra. The TDDFT calculation revealed that
upon coordination of ligand P3 to Cr3+ ion, the energy levels of both the HOMO and LUMO
slightly decreased relative to those of free P3 (as shown in Fig. 10). Whereas, a small HOMO–
LUMO energy gap favors electron hopping between both occupied and unoccupied molecular
orbitals, the charge transfer in P3–Cr3+ complex would enhance, significantly than that for P3
ligand 49-50. The decrease of energy levels of LUMOs is more significant than those for the
HOMOs ones, indicating that the LUMOs were considerably stabilized relative to the HOMOs.
On the other hand, the conversion of P3 to P3–Cr3+ complex becomes simple by narrowing the
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energy gap between the HOMO and LUMO orbitals. So, it provides a reasonable explanation
about that decrease of the energy gap of HOMO-LUMO from P3 to its chromium complex (Fig.
10), which is in agreement with the subsequent generation of the new red shifted absorbance
peak on the addition of Cr3+ to P3 50-51. The formation of highly stable tetrahedron complex of
P3 with Cr3+ central metal, are resulted through the expansion of the conjugation of the aromatic
groups and also an increase of charge transfer possibility, that caused to initiate this unusual
fluorescence response. In addition, the theoretical calculations indicate that, in the P3–Cr3+
complex, the electron contributions in HOMO and HOMO-1 orbitals mainly were distributed
over the donor part, namely PhN and PhO fragments, whereas the electron density of the LUMO
and LUMO+1 were contributed significantly over the acceptor moiety of d orbitals of Cr3+ and
also NO3 group (Fig. S6). Additionally, the mapping of the frontier orbitals regarding the titled
compounds associated with their energies are depicted in Fig. 11. As seen in Fig 11, in free
ligand P3 the electron contributions are localized over the almost whole molecule, except the
phenyl ring attached to pyridine part. This result leads us to this fact that the coordination of Cr3+
with the P3 and NO3 moiety can be reinforced through electron donation of the coordinating
oxygen atoms of ligand P3 and moiety of the nitrate group and electron accepting of the nitrogen
atom of phenylpyridine fragment.
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Fig. 10. The molecular orbital and some characters of HOMO and LUMO molecular orbitals of the (a) P3, (b)
P3–Cr3+ (α-spin) and (c) P3–Cr3+ (β-spin) compounds.
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Fig. 11. Calculated the frontier molecular orbitals of the titled compounds with B3LYP/6-31G(d, p) level. Positive
values of the HOMOs and LUMOs contour are represented in blue and purple and the negative values in yellow
and green, respectively.
Figure 12 shows the UV-Vis spectra of both free ligand P3 and its plausible tetrahedron complex
P3–Cr3+. The obtained UV-Vis spectra by means of TDDFT calculations are in good agreement
with the experimental UV-Vis spectroscopy (Fig. S5). From the TDDFT calculations the
exhibited band lines for P3 showed at 311.7 nm (f=0.120), 318.2 nm (f=0.171), 355.5 nm
(f=0.530), 367.9 nm (f=0.843), 385.0 nm (f=1.180) and for P3–Cr3+ complex revealed at 355.70
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nm (f=0.010), 356.3 nm (f=0.011), 359.3 nm (f=0.014), 367.2 nm (f=0.007), 370.3 nm (f=0.008),
372.8 nm (f=0.006), 378.7 nm (f=0.008) and 380.4 nm (f=0.017), that are in agreement with the
experimental spectrum. By complexation of the P3 ligand to Cr3+ ion the intensity of the bands
were decreased because of spin-forbidden transitions in 3d transition metals. The major
transitions of the ligand P3 at 367.9 nm and 385.0 nm are due the electron transition of (HOMO)
→ (LUMO) with dominant excitation characters of π-PhN (52%), π-PhO (27%) and π-Py (20%)
to π*-PhN (59%), π*-PhO (23%) and π*-Py (18%). The contributions on P3 show that nitrogen
lone pair electrons of Py fragment as belonging to HOMO is responsible for the fluorescence
quenching. P3. On the other hand, the major lines for P3–Cr3+ at 367.2 nm are assigned to
2
2
(HOMO-2) → (LUMO+2) associated with π-PhN (64%), π-PhO (25%) and π-Py (7%), dx -y -Cr
(3%) to π*-NO3 (59%), dxy -Cr (7%). As a result, the strong interaction between N atom of Py
fragment and Cr3+ d-orbital causes to block the PET process from the nitrogen lone pair electron
to the benzene, hence block the fluorescence quenching. The possibility of the restricted
chelation-enhanced fluorescence (CHEF) process in P3 coordinated to Cr3+ can be contributed
the fluorescence enhancement along with the PET process 40.
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Fig. 12. The calculated UV-Vis spectra of the (a) P3 and (b) P3–Cr3+ compounds.
3.8. Investigation of Binding reversibility
The reversibility P3 binding with Cr3+ was evaluated by adding EDTA to P3–Cr3+ solution. It is
assumed that the addition of EDTA will liberate Cr3+ from the P3–Cr3+ complex, releasing the
53
P3 ligand, because of the EDTA–Cr3+ complex (stability constant log KEDTA–Cr = 23.4)
.
Therefore, 1 equiv. of EDTA (10 µM) was added to the Cr3+ (10 µM) complex of P3 (10 µM) in
CH3CN/H2O (95/5%) solution, exhibiting significant decrease of fluorescence signal at 663 nm.
The addition of excess Cr3+ to the previous solution regenerates the fluorescence by participation
of P3 with another Cr3+ binding process (Fig. 13). The regenerated free P3 can then participate in
another Cr3+ binding process 54.
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Fig. 13. Reversibility of Cr3+–complex (10 µM) coordination to probe P3 (10 µM) by EDTA (10 µM).
4. Conclusions
In summary, we have successfully developed a novel chemosensor P3 which exhibited the
high selectivity for Cr3+ in the presence of other metal ions. The sensor shows a large fluorescence
enhancement in the presence of Cr3+ which is attributed to the interaction of P3 and Cr3+ resulting in
the efficient inhibition for the PET process. The results of Job’s plot method together with
theoretical study indicate that P3 and Cr3+ form a 1:1 complex. The association constant Ka was
determined to be 2.28 × 105 M-1 and the limit of detection (LOD) was calculated to be 1.3×10-7
M exhibiting more efficient than many other reported Cr3+ sensors. Besides, DFT calculations
were carried out to get a comprehensive insight towards the plausible structure of P3–Cr3+ in the gas
phase. The results confirmed tetrahedron geometry around the Cr+3 ions. In order to investigate the
major excitation states and the absorption spectra of the compounds, TDDFT calculations were
employed that were in agreement with the experimental results.
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