Charge transfer based "turn-on" chemosensor for Zn2 + ion recognition using new triaryl pyrazoline derivative

Article abstract of DOI:10.1016/j.saa.2016.01.027

The fluoroionophore PY serves as a selective and fluorimetric chemosensor for Zn^{2 +} based on charge transfer (CT). A mechanism for the binding mode was proposed based on fluorescence changes, NMR experiments and theoretical calculations. The 1:

Full text of DOI:10.1016/j.saa.2016.01.027

Charge transfer based “turn-on” chemosensor for Zn2 + ion recognition using
new triaryl pyrazoline derivative
Dharmaraj Jeyanthi, Murugan Iniya, Karuppiah Krishnaveni, Duraisamy
Chellappa
PII:
DOI:
Reference:
S1386-1425(16)30027-0
doi: 10.1016/j.saa.2016.01.027
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12 January 2016
19 January 2016
Please cite this article as: Dharmaraj Jeyanthi, Murugan Iniya, Karuppiah Krishnaveni,
Duraisamy Chellappa, Charge transfer based “turn-on” chemosensor for Zn2 + ion recog-
nition using new triaryl pyrazoline derivative, (2016), doi: 10.1016/j.saa.2016.01.027
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ACCEPTED MANUSCRIPT
Charge transfer based “turn-on” Chemosensor for Zn2+ ion recognition using
new Triarylpyrazoline derivative
Dharmaraj Jeyanthi a, Murugan Iniya a, Karuppiah Krishnaveni a and Duraisamy
Chellappa*a
aSchool of Chemistry, Madurai Kamaraj University, Madurai, TamilNadu-625021, India.
*Corresponding author. Tel: +91 452 2456614; Fax: +91 452 2459181.
E-mail address: dcmku123@gmail.com
Abstract
The fluoroionophore PY serves as a selective and fluorimetric chemosensor for Zn2+ based on charge
transfer(CT). A mechanism for the binding mode was proposed based on fluorescence changes, NMR
experiments and theoretical calculations. The 1:1 stoichiometry between Zn2+ and the sensor was deduced
from Job’s plot. The addition of EDTA quenches the fluorescence of PY.Zn2+ complex offers PY as a
reversible chemosensor.
Keywords: charge transfer; triaryl pyrazoline; fluorimetric changes; reversibility
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1. Introduction
Fluorescent chemosensors for metal ions are indispensable tool and have received extensive
attention in light of their diverse applications in areas like environment, biology, chemistry and medicine
[1,2]. Zinc, the second most abundant and essential trace divalent transition-metal after iron in the living
systems, is an essential cofactor of many biological processes such as catalytic centers, regulators of gene
expressions and enzymes[3-5]. Zinc is also a contributory factor in several neurodegenerative diseases
exemplified as epilepsy, ischemia, stroke, Alzheimer’s and Parkinson’s diseases [6].
Moreover, apart from its wide applications, a high level of zinc is highly toxic and leads to
diabetes, skin disease and prostatic adeno carcinoma [7] and reduce the soil microbial activity causing
phytotoxic effects [8,9]. For Zn2+ sensing, fluorescence techniques are used because of their high
sensitivity, accurate determination and spatiotemporal resolution than other detection methods [10, 11].
Thus development of simple, selective and accessible fluorescent sensors for Zn2+ is crucially important
and strongly required for environmental and biological analysis [12].
The fluorescent activities of sensor was controlled by a number of mechanisms such as Charge
transfer
transitions(CT)[13],
Photo
electron
transfer(PET)[14,15],
Chelation
enhanced
fluorescence(CHEF)[16], Fluorescence resonance energy transfer(FRET)[17], and Excimer
formation[18]. Pyrazoline derivatives have many interesting bioactivities such as antimicrobial [19],
antiamoebic[20], anticancer[21], antinociceptive[22], antidepressant[23] and anti-inflammatory[24]. Up
to now, only few fluorescent reports based on this class of compounds are available for Zn2+ detection
compared to other heavy metal ions [25-30].
In continuation of our on-going research in the development of metal ion sensors [31-33], herein
we have designed a relatively simple triaryl pyrazole derivative as a metal chelating moiety. It has
selective binding affinity towards Zn2+ and exhibits sensitive “turn-on” fluorescence operating through
charge transfer mechanism.
2. Experimental Section
2.1. Materials and methods
All the chemicals were purchased from Sigma-Aldrich and used as received unless otherwise mentioned.
Metal chloride salts obtained from Sigma-Aldrich were used as sources for metal ions. Solvents used
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were of spectroscopic grade. UV-visible absorption spectra were recorded on JASCO V-550
spectrometer. All fluorescence measurements were made on an F-4500 Hitachi fluorescence
spectrophotometer with slit width 5nm used for both excitation and emission. The NMR spectra were
recorded on a Bruker (Avance) instrument operating at 300MHz in DMSO-d6 with tetramethylsilane as an
internal reference. Chemical shifts were expressed in ppm and coupling constants(J) in Hz. Electrospray
ionization mass spectrometry (ESI-MS) analysis was performed in the positive ion mode on a liquid
chromatography-ion trap mass spectrometer(LCQ fleet, Thermo Fisher Instruments Limited, US).
Elemental analysis was carried out in a Perkin-Elmer 4100 elemental analyzer. The stock solution of the
probe (10µM) was prepared in DMSO. The solutions of metal ions were prepared from their chloride
salts. Metal ion stock solutions were prepared in deionized water keeping concentration range from 0-
10µM. Doubly distilled deionized water was used throughout. All titrations were carried out at room
temperature. For the fluorescence spectra, the excitation wavelength was kept at 350nm.
2.2. General synthetic procedure for the preparation of (5-(4-chlorophenyl)-3-
(4-hydroxyphenyl)-1H-pyrazol-1-yl)(2-hydroxyphenyl)methanone(PY)
Starting materials chalcone and 2-hydroxybenzohydrazide were prepared according to literatures.
To a stirred solution of chalcone (0.500g, 1.0mmol) in ethanol was added 2-hydroxybenzohydrazide
(0.353g, 1.2mmol) and NaOH (0.233g, 3.0mmol). The reaction mixture was refluxed for 4h. The progress
of the reaction was monitored by TLC. After completion, the reaction mixture was cooled to room
temperature and diluted with chilled water, and then hydrochloric acid was added to neutralize it. The
crude product was obtained as yellow precipitates. The precipitate was filtered, washed with water and
1
ethanol and then subsequently recrystallized from ethanol to afford product (PY). H NMR(300MHz,
DMSO-d6, TMS): δ(ppm) = 11.20(s, 1H, OH), 10.50(s, 1H, OH), 8.66(s, 1H, pyrazole-1H), 8.06(d, 1H,
J=8.7Hz, Ar-H), 6.87-7.95(m, 12H, Ar-H). 13C NMR(75MHz, DMSO d6, TMS): δ(ppm)=187.35, 162.62,
141.40, 135.01, 134.14, 131.44, 130.54, 129.29, 129.13, 123.28, 115.7. IR (KBr, cm-1): ν(O-H), 3452;
ν(C-H), 3050; ν(C=O), 1706; ν(C=N), 1650; ν(M-O), 628;ν(M-N), 528. Anal. Calcd for C12H15ClN2O3: C
67.61, H 3.87,Cl 9.07, N 7.17, O 12.28; Found: C 68.0, H 3.85, Cl 8.80, N 7.13, O 12.22%; MS (ESI):
391(M+H)+.
2.3. Computational details
Density functional theory (DFT) calculations were carried out with B3LYP-6-31G and
B3LYP/LanL2DZ basis set using Gaussian 03 program package to confirm the UV-visible and
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fluorescence changes upon the addition of Zn2+ ion. The TD-DFT calculations on the optimized
geometries of PY & PY.Zn2+ were performed using the above basis set in order to obtain the electronic
behavior and oscillator strength for the electronic transitions.
3. Results and discussion
Scheme 1
The receptor was synthesized by a simple and straightforward method with good yield by
condensation of chalcone with salicyloyl hydrazide in methanol as depicted in scheme 1. The receptor
1
was systematically and fully characterized by H, 13C-NMR, FT-IR, ESI-MS and elemental analysis (Fig
S1-S5, ESI†). To explore the practical utility of the probe in bio-environment, the signal response of
probe PY upon subjecting to interaction with different metal ions was monitored using both absorption
and emission spectrometers in aqueous DMSO [DMSO: H2O=8:2] at pH=7.4 (HEPES buffer). The uv-vis
spectral response of PY (10μM) towards Zn2+ and other metal ions are given in Fig. 1.
Fig. 1
It is obvious from the spectra that only Zn2+ ion induced an observable change in the absorption
spectrum of probe PY. However, other metal ions cause least variation in the absorption spectra (Fig. 1).
The absorption intensity at 308nm decreased linearly with increasing concentration of Zn2+ (0-10μM).
Simultaneously a new absorption peak appeared and gradually increased at 372nm upon addition of Zn2+
ion with distinct isobestic point at 350nm. The new peak at 372nm increased up to the addition of
1equivalent of Zn2+ and leveled off thereafter, indicating a molar ratio (PY/Zn2+) of 1:1 (Fig. S6). This
observation in absorption spectra is attributable to an interaction between Zn2+ and the receptor PY in
ground state.
Fig. 2
To gain further insight into the sensing properties and mechanism, emission profile of sensor
molecule PY upon interaction with different metal ions were examined (Fig. S7). Only Zn2+ induced
sizable enhancement in the fluorescence spectra of PY[34]. Negligible change in emission spectra was
observed for other tested metal ions such as Na+, K+, Li+, Ag+, Ca2+, Mg2+, Cu2+, Co2+, Ni2+, Mn2+, Zn2+,
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Cd2+, Hg2+, Pb2+,Cr3+, Al3+, Fe3+ even when present in excess (Fig. S8). The fluorescence titration of
ligand PY was performed with increasing concentration of Zn2+ (0-10μM) in DMSO/H2O system
[DMSO: H2O=8:2] at pH = 7.4 (HEPES buffer).
Fig. 3
Fig. 4
PY shows a weak fluorescent emission band at 446nm when excited at 350nm (φf=0.03). Upon
sequential addition of Zn2+ the emission band at 446nm undergoes a red shift by 12nm with
hyperchromism to 458nm (quantum yield φf=0.31) (ESI†) at room temperature (Fig. 2). The emission
intensity reached maximum when the amount of Zn2+ reached 1equivalent thereby suggests 1:1
stoichiometry between PY and Zn2+. This was further confirmed by Job’s plot. The fluorogenic changes
of PY with Zn2+ is shown in Fig. 3. The observed dramatic red shift in the fluorescence emission could be
a result of charge transfer mechanism (CT). A proposed binding mode is given in Fig. 4
Fig. 5
In order to determine the stoichiometry of complex formed between PY and Zn2+, the method of
continous variation (Job’s method)[35,36] using the emission changes was applied by varying the mole
fraction of Zn2+ from 0.1 to 0.9 and by keeping the sum of the concentration of Zn2+ and PY as constant
(Fig. 5). The emission maximum was observed when the mole fraction of Zn2+ reached 0.5, in accordance
with the proposed host-guest binding stoichiometry.
Fig. 6
The composition of complex PY.Zn2+ (1:1) has also been proved by ESI-MS analysis. The
receptor PY shows m/z at 391 corresponding to [M+H]+ ion, where M = PY; for PY.Zn2+ complex the
observed m/z peak at 514 corresponding to [M-H+Na]+, where M=[PY+Zn2++Cl-], confirms 1:1
binding stoichiometry. From the fluorescence titration profile of PY with Zn2+, the association constant ka
was found to be 2.4x104M-1(ESI†) (Fig. 6)[37]. In addition, the detection limit of PY for the analysis of
Zn2+ was calculated to be 1.02μM (ESI†); this shows that PY is a more effective and excellent fluorescent
sensor for Zn2+ over other cations[38]. The calculated association constant and detection limit are
tabulated with that reported in the literature respectively in (Table 1) and (Table 2). On comparison it is
clear that the present probe resembles many of the reported probes in its binding constant. Also obvious is
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that the present probe is having higher efficiency in detection of zinc relative to benzimidazole based
receptor but lower efficiency with reference to probes like MCB dye.
Fig. 7
To check out the potential utility and efficiency of PY for Zn2+, the competitive experiments were
conducted in the presence of other biologically relevant cations under the same conditions (Fig. 7). The
results showed insignificant effect on Zn2+-induced fluorescence enhancement. Thus, this probe should
have good selectivity and remarkable sensitivity for Zn2+ in the biological studies. To gain further insight
into the reversible behavior of the receptor PY towards Zn2+, the effect of addition of chelator EDTA was
examined (Fig. S9). This feature is highly desirable for practical applications and to rule out the
possibility that the fluorescent enhancement observed is not due to a chemical reaction [(i.e)
chemodosimeter][39]. It revealed that addition of 1 equivalent of EDTA to the PY.Zn2+ complex results
in recovery of original fluorescence spectra of PY. The same extent of fluorescence enhancement was
again recovered by the addition of another 1equivalent of free Zn2+ ions. It shows the reversible nature of
binding interaction between PY and Zn2+.
Fig. 8
1
To get insight into cation binding interaction of PY with Zn2+, H NMR titrations were also
carried out in d6-DMSO at room temperature. The changes in the 1H NMR spectra of PY before and after
the addition of Zn2+ (0.5, 1.0equivalent) are shown in Fig. 8. All aromatic protons in the receptor PY,
showed a downfield shift upon chelation to Zn2+. When PY interact with 0.5equivalent of Zn2+, the –OH
proton of salicyloyl hydrazide moiety underwent downfield shift with broadening and disappeared on
addition of 1equivalent Zn2+ indicating the involvement of the phenolic oxygen atom in complexing the
metal ion via deprotonation. Moreover, Zn2+ coordination also triggers a clear downfield shift of the
pyrazole protons. All these findings indicated a proposed binding mode of PY with Zn2+ and the dynamic
nature of the zinc sensing process. In addition, almost same fluorescent enhancement of PY was noted
after the addition of different counter anionic zinc salts such as Zn(NO3)2, ZnCl2, ZnSO4 and Zn(OAc)2
(Fig. 9). The result reveals the influence of counter anions on the detection of Zn2+ with PY is rather
small. In otherwords, the anion effect of different zinc salts is insignificant.
Fig. 9
Fig. 10
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3.1. DFT Calculations
The bathochromic shift of the emission of probe upon binding with Zn2+ and plausible reason for
that was elucidated using quantum calculations. The geometries of receptor PY and PY.Zn2+ optimized in
the ground state using density functional theory (DFT) with Becke’s three parameter hybrid exchange
functional (B3LYP) but using basis set 6-31G(d,p) and LANL2D(d) levels respectively, are shown in
Fig. 10. To further gauge the electronic behavior of the probe in the presence and absence of Zn2+, time
dependent density functional theory (TD-DFT) calculations were carried out using the same functional
and basis set. All above computations were performed with Gaussian 03 program package [40].
Fig. 11
The zinc is bound by one N-atom (not adjacent to >C=O group) from pyrazole ring, carbonyl
oxygen, phenolic oxygen and one chloride ion in a distorted tetrahedral arrangement. Fig. 11 shows
calculated HOMO and LUMO of PY and PY.Zn2+. In PY HOMO resides on molecule bearing
chlorophenyl moiety while LUMO spans only pyrazole and salicyloyl moieties. In PY.Zn2+, HOMO
almost resembles that of pure probe PY, while LUMO resides intensively on Zn2+ ion. This indicates
substantial charge transfer from donor moiety to Zn2+ when the molecule is excited. Furthermore, the
lowering of HOMO-LUMO energy gap after Zn2+ binding corroborates the experimentally observed red
shift of emission band [41] (Fig. S10). Therefore, based on the above analysis, the proposed mechanism is
rational and reasonable.
4. Conclusion
In conclusion, we have designed and successfully synthesized a new triaryl pyrazole derivative
(PY) as a good selective and sensitive “turn-on” fluorescent sensor for Zn2+ over other cations. The
structure was confirmed by 1H, 13C-NMR, mass spectrometric and elemental analysis. Based on DFT/TD-
DFT calculations it is proposed that ligand to metal charge transfer is responsible for fluorescence
enhancement. These findings clearly demonstrate that PY could be applied as a strong sensor for Zn2+
with biological applications.
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Acknowledgement
The authors D.J., M.I., K.K., are grateful to UGC-BSR for research fellowship and also thankful to DST-
IRHPA, FIST and PURSE for funding and instrumental facilities.
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Figure Captions
Scheme 1. The synthetic route of receptor PY
Fig 1. UV-vis absorption spectra of PY(10μM) in the presence of various metal ions(1x10-5M) like Na+,
K+, Li+, Ag+, Ca2+, Mg2+, Cu2+, Co2+, Ni2+, Mn2+, Zn2+, Cd2+, Hg2+, Pb2+, Cr3+, Al3+, Fe3+ in aqueous
DMSO[DMSO:H2O=8:2] at pH = 7.4 (HEPES buffer).
Fig 2. Changes in fluorescence intensity of PY(10μM) in aqueous DMSO[DMSO:H2O=8:2] at pH = 7.4
(HEPES buffer) upon successive addition of Zn2+(0-1 equivalent). Excitation wavelength = 350nm
Fig 3. Fluorogenic changes of a 10μM solution of PY in aqueous DMSO[DMSO:H2O=8:2] at pH = 7.4
(HEPES buffer) in the presence of 1 equivalent Zn2+ (irradiated by 365nm light)
Fig 4. A proposed binding mode of PY for Zn2+
Fig 5. Job’s plot for the complex formed between receptor PY and Zn2+
Fig 6. Plot of fluorescence intensity variation at 458nm upon varying equivalents of Zn2+ ions
Fig 7. The fluorescence intensity of PY(10μM) in the presence of 20μM for Na+, K+, Li+, Ag+, Ca2+,
Mg2+, Cu2+, Co2+, Ni2+, Mn2+, Zn2+, Cd2+, Hg2+, Pb2+, Cr3+, Al3+, Fe3+ (violet bars) followed by addition of
10μM of Zn2+ (pink bars) in aqueous DMSO[DMSO:H2O=8:2] at pH = 7.4 (HEPES buffer). Excitation at
350nm (slit width=5nm)
1
Fig 8. Plot of H NMR spectra of receptor PY(10μM) upon addition of Zn2+ in DMSO-d6 at room
tempertature
Fig 9. Fluorescent emission spectra of free receptor PY(1x10-5M) in aqueous DMSO[DMSO: H2O=8:2]
at pH = 7.4 (HEPES buffer) upon addition of 1.0 equivalent of different zinc salts
Fig 10. Conformations of PY(left) and PY.Zn2+(right) optimized by density functional theory calculations
Fig 11. HOMO and LUMO plot of PY & PY/Zn2+ calculated at B3LYP/LANL2DZ(d) level of theory
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Figures
Scheme 1. The synthetic route of receptor PY
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Fig 1. UV-vis absorption spectra of PY(10μM) in the presence of various metal ions(1x10-5M) like
Na+, K+, Li+, Ag+, Ca2+, Mg2+, Cu2+, Co2+, Ni2+, Mn2+, Zn2+, Cd2+, Hg2+, Pb2+, Cr3+, Al3+, Fe3+ in
aqueous DMSO[DMSO:H2O=8:2] at pH = 7.4 (HEPES buffer).
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Fig 2. Changes in fluorescence intensity of PY(10μM) in aqueous DMSO[DMSO:H2O=8:2] at pH =
7.4 (HEPES buffer) upon successive addition of Zn2+(0-1 equivalent). Excitation wavelength =
350nm
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Fig 3. Fluorogenic changes of a 10μM solution of PY in aqueous DMSO[DMSO:H2O=8:2] at pH =
7.4 (HEPES buffer) in the presence of 1 equivalent Zn2+ (irradiated by 365nm light)
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Fig 4. A proposed binding mode of PY for Zn2+
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Fig 5. Job’s plot for the complex formed between receptor PY and Zn2+
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Fig 6. Plot of fluorescence intensity variation at 458nm upon varying equivalents of Zn2+ ions
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Fig 7. The fluorescence intensity of PY(10μM) in the presence of 20μM for Na+, K+, Li+, Ag+, Ca2+,
Mg2+, Cu2+, Co2+, Ni2+, Mn2+, Zn2+, Cd2+, Hg2+, Pb2+, Cr3+, Al3+, Fe3+ (violet bars) followed by
addition of 10μM of Zn2+ (pink bars) in aqueous DMSO[DMSO:H2O=8:2] at pH = 7.4 (HEPES
buffer).
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Excitation at 350nm (slit width=5nm)
1
Fig 8. Plot of H NMR spectra of receptor PY(10μM) upon addition of Zn2+ in DMSO-d6 at room
tempertature
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Fig 9. Fluorescent emission spectra of free receptor PY(1x10-5M) in aqueous DMSO[DMSO:
H2O=8:2] at pH = 7.4 (HEPES buffer) upon addition of 1.0 equivalent of different zinc salts
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Fig 10. Conformations of PY(left) and PY.Zn2+(right) optimized by density functional theory
calculations
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Fig 11. HOMO and LUMO plot of PY & PY/Zn2+ calculated at B3LYP/LANL2DZ(d) level of theory
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Table 1: The binding constant comparison of our probe with sensors available in the
literature
Probe
Binding constants
References
Receptor PY
2.4x104M-1
Present study
Carboxamidoquinoline
based macrocycle
6.7x106M-1
2.4x104M-1
1
2
Hydrazone-pyrene
derivative
Tryptophan based Schiff
base
3.0x104M-1
1.1x104M-1
1.8x106M-1
3
4
5
Aminoacid based Schiff
base
Pyrene derived molecule
Benzimidazole based
receptor
6
7
1.4x104M-1
7.4x103M-1
Benzoimidazole dye
References
1. Y. Zhang, X. Guo, W. Si, L. Jia, X. Qian, Org. Lett., 10 (2008) 473-476.
2. Y. Zhou, H.N. Kim, J. Yoon, Bioorg. Med. Chem. Lett., 20 (2010) 125-128.
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3. L. Li, Y.Q. Dang, H.W. Li, B. Wang, Y. Wu, Tetrahedron Lett., 51 (2010) 618-621.
4. L. Li, F. Liu, H.W. Li, Spectrochim. Acta A, Mol. Biomol. Spectrosc., 79 (2011) 1688-1692.
5. E. Manandhar, J.H. Broome, J. Myrick, W. Lagrone, P.J. Cragg, K.J. Wallace, Chem. Comm., 47
(2011) 8796-8798.
6. M.J. Kim, K. Kaur, N. Singh, D.O. Jang, Tetrahedron, 68 (2012) 5429-5433.
7. X. Wang, Z. Liu, F. Qian, W. He, Inorg. Chem. Comm., 15 (2012) 176-179.
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Table 2: The detection limit comparison of our probe with sensors available in the
literature
Probe
Detection limits
References
Receptor PY
Pyrazole
1.02μM
Present study
0.12µM
2.4µM
1
2
Napthyl imino conjugate
MCB dye
2.5x10-8M
3
Self-assembled monolayer
receptor
1.7x10-5M
1.0x10-7M
4
5
Napthaldehyde based
Schiff base
Benzimidazole based
receptor
3.0µM
6
7
6.1x10-7M
Pyrazoline derivative
References
1. Z.L. Gong, F. Ge, B.X. Zhao, Sens. Actuators, B. 159 (2011) 148-153.
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2. A. Mitra, V.K. Hinge, A. Mittal, S. Bhakta, P. Guionneau, C.P. Rao, Chem. Eur. J., 17 (2011)
8044-8047.
3. N. Aksuner, E. Henden, B. Yenigul, I. Yilmax, A. Cukurovali, Spectrochim. Acta A, Mol.
Biomol. Spectrosc., 78 (2011) 1133-1138.
4. D.W. Kim, K.K. Kim, E.B. Lee, J.S. Kim, J. Photochem. Photobiol. A: Chem., 250 (2012) 33-39.
5. R. Azadbakht, H. Keypour, Spectrochim. Acta A, Mol. Biomol. Spectrosc., 85 (2012) 293-297.
6. M.J. Kim, K. Kaur, N. Singh, D.O. Jang, Tetrahedron, 68 (2012) 5429-5433.
7. Z. Zhang, F.W. Wang, S.Q. Wang, F. Ge, B.X. Zhao, J.Y. Miao, Org. Biomol. Chem., 10 (2012)
8640-8644.
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Graphical abstract
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Highlights
 The receptor PY was synthesized by a simple method with high yield
 The detection limit of Zn2+ by PY was very low (1.02μM)
 The reversible behavior of the receptor PY towards Zn2+ was studied with EDTA

The turn-on response with remarkable red shift in the fluorescence spectra was further
supported by quantum(DFT) calculations
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