A novel pyridine-pyrazole based selective “turn-off” fluorescent chemosensor for Fe(III) ions

Article abstract of DOI:10.1016/j.jphotochem.2018.11.033

A novel pyridine-pyrazole based “turn-off” fluorescent chemosensor namely, 5-N-(pyridine-2-yl)-3-(pyridine-4-yl)-1H-pyrazole-5-carboxamide (PPPC) was designed, synthesized and well characterized by NMR, ESI-MS and FT-IR spectroscopic techniques. UV–vis absorption and fluorescence spectroscopic studies show that PPPC exhibits high selectivity and sensitivity towards Fe³⁺ ion in DMSO/H₂O solution (9:1, v/v) over other metal ions. The binding constant (K) of PPPC with Fe³⁺ was calculated to be 5.1 × 10^?2 M and 6.1 × 10^?2 M from Benesi-Hildebrand plot using UV–vis and fluorescence spectrophotometer respectively. The detection limit of PPPC for Fe³⁺ was further determined as 57 nM and 88 nM by UV–vis and fluorescence titrations. Moreover, the binding mechanism of Fe³⁺ with PPPC was confirmed by DFT study.

Full text of DOI:10.1016/j.jphotochem.2018.11.033

Accepted Manuscript
Title: A novel pyridine-pyrazole based selective
¨
¨
turn-offfluorescent chemosensor for Fe(III) ions
Authors: P. Madhu, P. Sivakumar
PII:
S1010-6030(18)31306-6
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.11.033
JPC 11605
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
7 September 2018
23 November 2018
23 November 2018
Please cite this article as: Madhu P, Sivakumar P, A novel pyridine-
¨
¨
pyrazole based selective turn-offfluorescent chemosensor for Fe(III) ions,
Journal of Photochemistry and amp; Photobiology, A: Chemistry (2018),
https://doi.org/10.1016/j.jphotochem.2018.11.033
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1
A novel pyridine-pyrazole based selective "turn-off" fluorescent chemosensor
for Fe(III) ions
P. Madhua,b,* and P. Sivakumarc
aResearch and Development Centre, Bharathiar University, Coimbatore-641 046, TamilNadu, India
bDepartment of Chemistry, Thiruvalluvar Government Arts College, Rasipuram-637 401, TamilNadu, India
cDepartment of Chemistry, Arignar Anna Government Arts College, Namakkal-637 002, TamilNadu, India
*
Corresponding Author E-mail: madhup.chem@gmail.com
Tel: +91 9944502310
Graphical abstract
Highlights

The new pyridine-pyrazole based chemosensor was designed, synthesized and characterized for
the detection of Fe ions.
3+
3
+



The PPPC exhibits high selectivity and sensitivity toward Fe ions over other metal ions.
3
+
Fluorescence quenching mechanism is due to the binding interaction of the Fe ion with PPPC.
3
+
Fe ion inhibits the ICT process by forming a complex with PPPC.

2
Abstract
A novel pyridine-pyrazole based “turn-off” fluorescent chemosensor namely, 5-N-
pyridine-2-yl)-3-(pyridine-4-yl)-1H-pyrazole-5-carboxamide (PPPC) was designed, synthesized
(
and well characterized by NMR, ESI-MS and FT-IR spectroscopic techniques. UV-Vis
absorption and fluorescence spectroscopic studies show that PPPC exhibits high selectivity and
sensitivity towards Fe3+ ion in DMSO/H
O solution (9:1, v/v) over other metal ions. The
2
3
+
-2
-2
binding constant (K) of PPPC with Fe was calculated to be 5.1 x 10 M and 6.1 x 10 M from
Benesi-Hildebrand plot using UV-vis and fluorescence spectrophotometer respectively. The
3
+
detection limit of PPPC for Fe was further determined as 57 nM and 88 nM by UV-vis and
3
+
fluorescence titrations. Moreover, the binding mechanism of Fe with PPPC was confirmed by
DFT study.
Keywords: Chemosensor; Fluorescent sensor, Iron ion; Pyridine-pyrazole derivative; DFT
studies.
1
. Introduction
The design and synthesis of chemosensors with high selectivity and sensitivity for heavy and
transition metal ions have considerable research attention over the years because of its important
applications in biological, industrial and environmental processes [1-3].
3
+
Among various metal ions, iron ion (Fe ) is an essential element within the human body
which plays an important role in the growth and development of living systems. Also, iron
provides the oxygen carrying capacity of hemoglobin and acts as a cofactor in many enzymatic
reactions viz. metabolism, electron transfer and nucleic acids (DNA and RNA) synthesis [4].
Presence of trace amount in the human body can cause an imbalance in the iron concentration
and can have a dangerous effect on human health. The deficiency of Fe3+ causes various
biological disorders in living systems such as low blood pressure, anemia [5] and decreased
3
+
immunity. Excess of Fe concentration may also equally harmful or even fatal [6], which could
cause damage the lipids, proteins and nucleic acids. Recently, researchers revealed that Fe3+
could involve some neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease [7-
1
1]. Thus, the development of new methods to detect iron has emerged and tremendous
attention in the field of research. Nowadays some of the instrumental techniques such as atomic

3
absorption spectroscopy (AAS), inductively coupled plasma atomic emission spectrometry (ICP-
AES), inductively coupled plasma mass spectrometry (ICP-MS) and electrochemical methods
[
12] have been used for the detection of iron ions, but these methods require highly expensive
instrument and professional operators. Compared with the traditional analytical methods,
colorimetric chemosensors are an excellent method because they have low cost, good resolution,
high sensitivity and selectivity and easy monitoring of the target ions. Although enormous
excellent fluorescent chemosensors were developed and reported to selective sensing ability for
2
+
2+
2+
2+
various transition metal ions such as Hg [13-24 ], Cu [25-29], Zn [30-33] and Ag [34-38],
there are very few fluorescent probes are reported for Fe3+ ions. Thus there is an immediate
requirement to develop some highly selective fluorescent chemosensor for Fe3+ to satisfy the
biological and environmental needs.
A number of organic molecules containing a wide range of donor sites were reported for
detection of heavy metals. Among them, pyrazole and its derivatives are one of the most
important classes of a chelating ligand for the heavy metal atoms. The combination with
pyridine moiety the pyrazole derivative strengthen in its complexing behavior and can act as a
good candidate for complexation with a wide range of metals due to its more nitrogen donor
sites. Thus, in this study, we designed and synthesized a novel pyridine-pyrazole based
fluorescent chemosensor for Fe3+ ion (scheme 1), which showed a selective and sensitive
3
+
3+
recognition towards the Fe ion over other metal ions. The sensing behaviour of Fe mostly
depends on the fluorescence quenching mechanism due to its high paramagnetic nature.
2
. Experimental
2
.1. Materials and Instrumentation
All chemicals were purchased from Sigma-Aldrich/Merck and used as received without
further purification. Solvents used were analytical grade and double distilled water was used
throughout the experiment. The progress of all reactions was monitored by TLC using silica gel
6
0F254 and visualized under UV 254-366 nm and iodine.
1
13
H NMR (300MHz) and C NMR (75MHz) were recorded on Bruker NMR instrument
in CDCl and DMSO-d as solvents and TMS as an internal standard. Electrospray ionization
mass spectra (ESI-MS) were recorded on LCQ Fleet mass spectrometer (Thermo Fisher
Instruments Ltd., US). Fluorescence measurements were recorded using fluorescence
3
6

4
spectrophotometer (Agilent Technologies, US). Shimadzu UV-1800 UV-vis spectrophotometer
was used to record UV absorption spectra.
2
2
.2. Synthesis and Characterization
.2.1. Synthesis of ethyl 2,4-dioxo-4-(pyridin-4-yl)butanoate (1) [39]
To a sodium hydride (60 % in paraffin oil) (1.98 g, 0.049 mol), 10 mL of Dimethyl
°
formamide was added dropwise at 5 C under nitrogen atmosphere. To this mixture, 4-
acetylpyridine (3.0 g, 0.024 mmol) was added dropwise and stirred for 30 min at ambient
temperature. Then diethyl oxalate (4.03 mL, 0.029 mol) was added slowly and the reaction
mixture was again stirred for 3 h at room temperature. The completion of the reaction was
monitored by TLC and quenched with crushed ice. Then the reaction mixture was extracted with
ethyl acetate (1 x 100 mL), washed with water (100 mL) and brine solution (100mL). The
organic layer was separated, dried over anhydrous sodium sulfate and evaporation of solvent
affords the expected intermediate 1. (White solid, Yield 78%). 1H NMR (300 MHz, CDCl
) δ
3
8
.78 (d, J = 5.5 Hz, 2H), 7.85 (d, J = 5.5 Hz, 2H), 4.42 (q, J = 14.0, 7.1 Hz, 2H), 1.42 (t, J = 7.0
Hz, 3H). 13C NMR (75 MHz, CDCl
) δ 192.89, 173.26, 165.10, 150.57, 137.70, 122.79, 61.75,
3
2
2
9.62, 14.15.
.2.2. Synthesis of ethyl 3-(pyridin-4-yl)-1H-pyrazole-5-carboxylate (2) [39]
To acetic acid (10 mL) solution of intermediate 1 (2.0g, 9.04 mmol), hydrazine hydrate
(2.26 mL, 45.2 mmol) was added dropwise over 5 min at 0°C. The reaction mixture was stirred
for 15 h at room temperature and quenched with crushed ice, basified with sodium bicarbonate
solution. Then the reaction mixture was extracted with ethyl acetate (150 mL) washed with
water (150 mL) and brine solution (150 mL). The organic layer was separated, dried over
anhydrous sodium sulfate and evaporated under reduced pressure yielded intermediate 2. (White
1
solid. Yield 87 %). H NMR (300 MHz, DMSO-D
6
) δ 14.36 (s, 1H), 8.64 (d, J = 5.7 Hz, 2H),
7
.85 (d, J = 5.7 Hz, 2H), 7.51 (s, 1H), 4.36 (q, J = 14.0, 7.0 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).
13C NMR (75 MHz, DMSO-D
) δ 163.78, 150.81, 135.88, 127.73, 120.08, 107.35, 61.33, 14.68.
6
+
ESI-LC/MS calculated m/z 217.09, found 218.08 (M+1) . IR: 3124, 2978, 1725, 1608, 1571,
8
2
35, 765.
.2.3. Synthesis of 4-(5-carboxy-1H-pyrazol-3-yl)pyridin-1-ium (3) [39]

5
Intermediate 2 (1.5 g, 6.9 mmol) was dissolved in 6N hydrochloric acid (7.5 mL) and
heated to reflux for 3 h. The completion of the reaction was monitored by TLC. Then the
reaction mixture was stirred with tetrahydrofuran (15 mL) then filtered and washed with
tetrahydrofuran (15 mL). The filtered intermediate 3 was taken to the next step. (White solid,
1
Yield 95%). H NMR (300 MHz, DMSO-D
6
) δ 8.93 (bs, 2H), 8.47 (d, J = 5.3 Hz, 2H), 7.80 (s,
1
H). 13C NMR (75 MHz, DMSO-D
6
) δ 160.58, 148.04, 142.48, 122.51, 109.74. ESI-LC/MS
+
calculated m/z 189.05, found 190.11 (M+1) . IR: 3046, 2865, 1694, 1631, 1589, 830, 773.
2
.2.4. Synthesis of N-(pyridin-2-yl)-3-(pyridin-4-yl)-1H-pyrazole-5-carboxamide (PPPC)
The intermediate 3 (1.0g, 5.29 mmol) was dissolved in Acetonitrile (20 mL) and then
TBTU (2.03g, 6.33 mmol) and triethylamine (1.10 mL, 7.93 mmol) was added. The reaction
mixture was stirred for 30 min at room temperature. To the reaction mixture, 2-aminopyridine
(0.497 g, 5.29 mmol) was added at 0° C and again stirred for 24 h at room temperature. Then the
reaction mixture was quenched with crushed ice. The precipitate obtained was filtered and dried
in vacuum yielded the target compound PPPC and used for further studies. (White solid, Yield
1
9
2 %). H NMR (300 MHz, DMSO-D
6
) δ 14.30 (s, 1H), 10.97 (s, 1H), 8.68 – 8.63 (m, 2H), 8.42
(
bs, 1H), 8.23 (d, J = 7.3 Hz, 1H), 7.89 – 7.81 (m, 3H), 7.46 (s, 1H), 7.25 – 7.17 (m, 1H). 13C
NMR (75 MHz, DMSO-D
6
+CDCl
3
) δ 158.98, 148.43, 148.22, 146.16, 136.38, 118.01, 117.75,
+
1
3
12.10, 111.11, 104.61, 103.10. ESI-LC/MS calculated m/z 265.10, found 266.14 (M+1) . IR:
108, 2161, 1736, 1638, 1538, 830, 797.
2
.3. Stock solution preparation
-3
The stock solution of PPPC was prepared at a concentration of 1 x 10 M in DMSO/H
9:1, v/v). 20 µL of this stock solution was diluted to 2 mL with the respective solvent system to
cover the final concentration as in 10 µM level. The stock solution of metal ions was prepared in
2
O
(
-2
1
x 10 M concentration by adding the appropriate amount of metal cation using double distilled
water. For selectivity experiment 10 equiv. of the metal ions were used and the stock solution
-5
was diluted (10 times) for the sensitivity experiments. For Job’s plot analysis 1 x 10 M of
PPPC and metal solutions were used. For the purpose of spectral measurements, detection
solutions of PPPC and metal solutions were freshly prepared and used.

6
2
.4. UV-vis and fluorescence spectral studies
Using UV-vis spectrophotometer and fluorescence spectrophotometer, the absorption and
emission spectra were recorded respectively. 10 µM solution of PPPC in DMSO/H O solution
9:1, v/v) was prepared at room temperature and added with the metal ion solution to the quartz
2
(
optical cell. After the addition of metal ions, spectral data were recorded. In the fluorescence
measurements, the probe PPPC shows an admirable fluorescence band at 354 nm with the
excitation wavelength 310 nm.
2
.5. Calculation of binding constant
The binding constant (K) for the formation of complex, PPPC-Fe has been determined
3
+
by using both UV-vis and Fluorescence Spectrophotometer. First, the binding constant was
determined by UV-vis Spectral titration. The concentration of PPPC was kept constant
3
+
throughout the experiments and varying the concentration of the Fe ions. The constant value of
PPPC with the corresponding Fe3+ was determined by using Benesi-Hildebrand equation.
Binding constant K was calculated by following the equation stated below.
3
+
1
/(A-A
o
) = 1/{Ka(Amax–A
o
) [Fe ]} + 1/[Amax-A
o
]
3
+
Here, A
o
is the absorbance of the receptor, PPPC in the absence of guest Fe , A is the
3
+
absorbance recorded in the presence of added Fe ions, Amax is absorbance in presence of added
3
+
-1
[
Fe ]max and K is the binding constant (M ). The binding constant (K) could be determined
3
+
from the slope of the straight line obtained in the plot of 1/(A-A ) against 1/[ Fe ]. Similarly,
o
the binding constant value can also be determined by fluorescence spectrophotometer by using
modified Benesi-Hildebrand equation stated below,
3
+
1
/ I-Imin = 1/ Imax-Imin + (1/K[Fe ])(1/ Imax-Imin).
Where, Imin, I, and Imax are the emission intensities of PPPC, at an intermediate Fe3+
concentration with PPPC, and at a concentration of complete saturation respectively. K is the
3
+
3+
binding constant and [Fe ] is the concentration of Fe ions. From the plot of 1/(I-Imin) against
3
+ -1
3+
[
Fe ] for PPPC with Fe ion, the binding constant value (K) has been determined from the
slope.
3
. Results and discussion
3
.1. Synthesis and structural characterization PPPC

7
H
N
O
O
O
O
O
N
O
(i)
(ii)
N
O
N
N
2
1
(iii)
N
H
N
N
N
3
OH
NH
NH
(iv)
O
N
O
N
PPPC
Reagents and conditions: (i) NaH, Diethyloxalate, DMF, 1h, RT; (ii) NH NH .H O, ethanol, 24h, RT; (iii) 6N HCl,
2
2
2
o
6
h, reflux; (iv) TBTU, 2-aminopyridine, TEA, 3h, 0 C-RT
Scheme 1: Synthetic route of PPPC
As illustrated in scheme 1, the targeted probe PPPC was synthesized in a multi step
process. Intermediate 1 was prepared from 4-acetylpyridine and diethyl oxalate in the presence
of NaH. Treatment of intermediate 1 with hydrazine hydrate promotes cyclisation and gives the
intermediate 2. The resulting intermediate 2 was hydrolyzed using HCl gives intermediate 3.
Finally, the intermediate 3 reacts with 2-aminopyridine gives the probe PPPC via a condensation
1
13
reaction. All the compounds were satisfactorily characterised using H NMR, C NMR, FT-IR
1
and Mass spectral measurements. The characterization of PPPC can be well explained by H
NMR spectral studies. The singlets at 14.30 ppm and 10.97 ppm clearly indicates the presence
of pyrazole NH and carboxamide NH respectively. The doublets for 2 protons and singlet for 1
proton at 8.65 ppm, 8.63 ppm appears for pyridyl protons (2 protons from the 4-substituted ring
and 1 proton from the 2-substituted ring). The singlet appeared at 7.19 ppm indicates the
1
3
presence of pyrazole CH. Similarly, the C NMR spectrum also confirms the product formation.
The appearance of a peak at 158.97 ppm indicates the presence of amide carbonyl carbon. The 3
peaks appeared around 148 ppm indicates the presence of pyridyl carbons (2 carbon from the 4-
substituted ring and 1 carbon from the 2-substituted ring). The peak appeared around 103.1 ppm
indicates the presence of pyrazolyl carbon. In addition, the PPPC was characterized by using the
ESI-Mass spectroscopic technique. The Mass spectrum clearly showed positive mode signal at

8
2
66.14. The FT-IR spectrum also gives some additional information about the probe PPPC. The
-1
band appeared around 1638 cm indicates the presence of carboxamide unit. Similarly, the band
-1
appeared around 1578 cm indicates the presence of C=N unit.
3
+
3
.2. Selectivity of PPPC to Fe using UV-vis and fluorescence spectra
The selectivity of PPPC in DMSO/H O solution (9:1, v/v) was investigated with various
2
2
+
2+
2+
2+
3+
2+
2+
2+
2+
2+
2+
2+
metal ions such as Mg , Ca , Mn , Fe , Fe , Co , Ni , Cu , Zn , Cd , Ba and Hg .
Initially, the synthesized probe PPPC (10 µM) in DMSO/H O solution (9:1, v/v) shows a single
band at 255 nm. On the addition of 10 equiv. of various metal ions, no significant changes
2
3
+
occurred except iron ions. As shown in Fig. 1, the addition of Fe ion a new absorption band
2
+
was observed at 340 nm and Fe also produced some small interaction with the probe PPPC,
3
+
but it is negligible compared to Fe ions.
2
1
1
0
0
0
.0
.6
.2
.8
.4
.0
PPPC +other metal ions
2+
PPPC + Fe ions
3
+
PPPC + Fe ions
3
00
350
400
450
500
Wavelength (nm)
Fig. 1: Selectivity spectrum of the PPPC with 10 equiv. of the metal ions in UV-vis spectrum
To acquire further strong evidence of the selectivity of PPPC for metal ions, the
2
+
2+
2+
fluorescence spectra were recorded between PPPC and various metal ions (Mg , Ca , Mn ,
2
+
3+
2+
2+
2+
2+
2+
2+
2+
Fe , Fe , Co , Ni , Cu , Zn , Cd , Ba and Hg ) under the same conditions. Actually, the
probe PPPC in DMSO/H O solution (9:1, v/v) shows an excellent fluorescence band at 354 nm
2
in fluorescence spectrometer with the excitation wavelength of 310 nm. Upon addition of Fe3+
ions, the fluorescence intensity was quenched significantly (Fig. 2). The quantum yield (Φ) of

9
3
+
PPPC and PPPC-Fe were found to be 0.044 and 0.010 respectively. The dramatic changes in
3
+
fluorescence can be attributed to the complex formed between the chemosensor PPPC and Fe .
This “turn-off” fluorescence mechanism could be imputed to the paramagnetic quenching
3
+
3+
behaviour of Fe . Further to confirm the binding mode of PPPC with Fe , the phenyl analogue
PA) of PPPC (replacing 2-pyridyl moiety by phenyl) was synthesized and characterized (Fig.
S14 and S15) and the sensing effect in emission spectroscopy was studied. In the presence of
(
3
+
Fe the fluorescent of the PA was quenched (Fig. S16), but not like the present probe, PPPC.
3
+
The quenching efficiency of the Fe was very less compare to PPPC. This is due to the PA
have two binding sites and PPPC has three sites along with 2-pyridyl moiety which interact
3
+
strongly with Fe than PA. Thus, the nitrogen atom in 2-pyridyl moiety plays a crucial role in
3
+
the sensing system. Therefore the PPPC has better selectivity towards Fe ions and could be
3
+
used as a selective chemosensor for Fe ions.
1
00
8
6
4
2
0
0
0
0
0
PPPC+ other metal ions
3+
PPPC+Fe ions
3
50
400
450
500
Wavelength (nm)
Fig. 2: Selectivity spectrum of PPPC with 10 equiv. of the metal ions in fluorescence
spectroscopy
3
+
3
.3. Sensitivity of PPPC to Fe using UV-vis and fluorescence spectra
3
+
The sensing behaviour of PPPC toward Fe was examined by the absorption spectral
3
+
O solution (9:1, v/v). Upon gradual addition of Fe3+
titration of PPPC with Fe in DMSO/H
2
ions (0-22 µM) to the probe PPPC, good linearity was observed with the detection limit of 57

1
0
3
+
2
-1
nM (Fig. 3 & 4). The binding constant for PPPC with Fe was determined to be 5.1 x 10 M
using Benesi-Hildebrand plot (Fig. 5) [40,41]. The complex stoichiometry of PPPC with Fe3+
also examined using Job’s plot [42-44]. Fig. 6 confirmed the 1:1 stoichiometry between PPPC
3
+
3+
and Fe where the absorbance at 340 nm got maximum when the mole fraction of Fe was 0.5.
3
+
Fig. 3: Sensitivity spectrum of PPPC with increasing concentration Fe ions in UV-vis
spectrum

1
1
Fig. 4: Linear plot derived from sensitivity titrations in UV-vis spectroscopy
3
+
Fig. 5: B-H plot analysis of Fe with PPPC from UV-vis. spectroscopy

1
2
3
+
-5
Fig. 6: Job’s plot analysis of the PPPC with Fe ions (10 M) in UV-vis spectroscopy.
3
+
3+
To promote further insight into the binding ability of PPPC with Fe , titration of Fe
against PPPC in DMSO/H O solution (9:1, v/v) was monitored using fluorescence spectra. In
the absence of Fe , the free PPPC showed an excellent fluorescence band at 354 nm. Upon
2
3
+
3
+
gradual addition of Fe ions (0-34 µM) to PPPC, a good linearity was observed from 6 to 30
µM with a strong fluorescence quenching. Furthermore, the lower detection limit of PPPC for
3
+
Fe was calculated as 88 nM using the formula 3σ/slope (Fig. 7 & 8), which is much lower than
3
+
the maximum level of Fe (5.4 µM) permitted by the US Environmental protection agency [45,
3
+
4
1
6] in drinking water. The association constant for PPPC with Fe was calculated to be 6.1 x
2
-1
0 M using a Benesi-Hildebrand plot (Fig. 9).

1
3
3
+
Fig. 7: Sensitivity spectrum of PPPC with increasing concentration Fe ions in fluorescence
spectroscopy
Fig. 8: Linear plot derived from sensitivity titrations in fluorescence spectroscopy

1
4
3
+
Fig. 9: B-H plot analysis of Fe with PPPC from fluorescence spectroscopy
3
+
3
.4. Competitive experiments of PPPC for Fe with other metal ions
The selectivity of PPPC for Fe3+ over other metal ions was further investigated by
performing competitive experiments in DMSO/H
2
O solution (9:1, v/v). Chemosensor PPPC
3
+
2+
was mixed with 10 equiv. of Fe ions in the presence of 15 equiv. of other metal ions (Fe ,
2
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
Zn , Cu , Ni , Co , Mn , Mg , Ba , Ca , Cd and Hg ) prepared separately. The
resulting individual solution was analyzed by UV-vis and fluorescence spectrophotometers. As
3
+
illustrated in Fig. 10 & 11, the probe PPPC shows remarkable sensing ability towards Fe ions
with a higher concentration of other metal ions, which reveals that the probe PPPC has higher
3
+
affinity with Fe than other metal ions.

1
5
PPPC with other metal ions
PPPC+Fe(III) with other metal ions
0.5
0.4
0.3
0.2
0.1
0.0
PPPC Fe(III) Fe(II) Zn(II) Cu(II) Ni(II) Co(II) Mn(II) Mg(II) Ba(II) Ca(II) Cd(II) Hg(II)
3
+
Fig. 10: Competitive analysis of the probe PPPC towards 10 equiv. Fe ions in the presence of
1
5 equiv. of other metal ions
PPPC with other metal ions
90
80
70
60
50
40
30
20
10
0
PPPC+Fe(III) with other metal ions
PPPC Ba(II) Ca(II) Cd(II) Co(II) Cu(II) Fe(II) Fe(III) Hg(II) Mg(II) Mn(II) Ni(II) Pb(II)
3
+
Fig. 11: Competitive analysis of the probe PPPC towards 10 equiv. Fe ions in the presence of
5 equiv. of other metal ions
1
3
.5. Proposed mechanism
To determine the binding mechanism of PPPC with Fe , the H NMR titration was
3
+
1
3
+
3+
carried out both in the absence and presence of Fe (Fig. 12). Gradual formation of PPPC- Fe
3
+
complex with the addition of Fe to PPPC caused the N-H proton signal of pyrazole ring and N-

1
6
1
H proton signal of amide moiety was disappear gradually in H NMR spectra, indicating that the
3
+
pyrazole nitrogen atom and amide nitrogen atom are the binding sites for Fe . In addition, to
3
+
assigning the sensing mechanism of PPPC with Fe , FT-IR spectral analyses were done for
3
+
-1
PPPC with and without Fe ions. IR spectrum of PPPC showed a strong band at 1578 cm ,
which is shifted in PPPC-Fe3+ (Fig. S17) indicating that the N-atom in 2-pyridyl moiety is
3
+
another binding site in the PPPC-Fe complex.
3
+
The plausible recognition of PPPC to Fe was further supported by the results of mass
spectral studies. ESI-Mass spectra gave a peak at m/z 320.02, which corresponds to
3
+
+
PPPC+Fe +H (Fig. S18). This clearly indicates the 1:1 binding stoichiometry between PPPC
3
+
1
and Fe . Research for the H NMR, FT-IR, and ESI-Mass spectra gave a remarkable evidence
for the proposed binding mechanism.
1
3+
Fig. 12. H NMR titration of PPPC in the presence of the varying amounts of Fe (in DMSO-d )
6
3
.6. Density functional theory (DFT) calculation

1
7
3
+
To get more evidence for the interaction between PPPC and Fe , the energy-optimized
structure of PPPC and its corresponding complex PPPC-Fe3+ were obtained by density
functional theory (DFT) calculations with the B3LYP/6.311++G(d,p) and LANL2DZ level of the
Gaussian 09W program. As shown in Fig. 12, the probe PPPC shows more electron density on
pyrazole part at HOMO. In LUMO, these electron densities were transferred to pyridine moiety.
This observation clearly indicates that the probe involved intra-molecular charge transfer (ICT),
3
+
which leads to fluorescence on in emission profile. In general, Fe ions have best quenching
3
+
behaviour due to its paramagnetic nature. Here, the Fe ion not only acts as a perfect quencher,
but it also inhibits the ICT process by forming a complex with PPPC. This was clearly observed
3
+
in Fig. 13. In the energy-optimized structure of PPPC-Fe , the electrons are highly localized on
pyridine ring at HOMO, whereas in LUMO, the electron cloud transferred into the metal
environment. In addition, the energy gap between HOMO and LUMO for PPPC (4.73 eV) is
3
+
higher than in the PPPC-Fe complex (3.31 eV). On the other hand, the energy gap between
3
+
HOMO and LUMO of PPPC-Fe complex has decreased to 1.42eV due to the formation of the
3
+
3+
PPPC-Fe complex. The results show that the probe PPPC was easily bind with Fe ions and
3
+
form stable PPPC- Fe complex as shown in scheme 2.
Fig. 13: DFT analysis of PPPC and iron complex (left to right).

1
8
3
+
Scheme 2: Proposed sensing mechanism of PPPC with Fe ion.
4
. Conclusion
In conclusion, we have designed and synthesized a novel pyridine-pyrazole based
fluorescence chemosensor PPPC by the multi-step process and satisfactorily characterized by
different spectroscopic techniques. UV-vis and fluorescence spectral studies reveal that the
chemosensor PPPC exhibits excellent selectivity and sensitivity toward Fe3+ ion over other
metal ions. In the presence of Fe3+ ions, the fluorescence intensity of PPPC was selectively
3
+
quenched due to the formation of the complex PPPC-Fe . Job’s plot analysis supports the
stoichiometry of the complex between PPPC and Fe3+ ion as 1:1. Furthermore, DFT
3
+
investigation confirms the complexation behaviour of PPPC with Fe from the HOMO-LUMO
3
+
orbitals of PPPC and PPPC- Fe complex.
Acknowledgement
The authors grateful to Biochemie Innovations Lab, Tindivanam-604001 for consultancy
service during this research work.
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5
Author Biographies
Mr.P. Madhu is an Assistant Professor in the Department of Chemistry, Thiruvalluvar
Government Arts College, Rasipuram, India. He is pursuing his Ph.D. degree in chemistry in
Bharathiar University, India, under the guidance of Dr P. Sivakumar. His current research
interest includes synthesis of Fluorescence Chemosensors and Biosensors.
Dr P. Sivakumar is an Assistant Professor in the Department of Chemistry, Arignar Anna Govt.
Arts College, Namakkal, India. He received his Ph.D. degree in chemistry from Anna
University, Chennai, India. His current research interest includes Nanomaterials, Photocatalysis
and Fluorescence Chemosensors.