A simple blue fluorescent probe to detect Hg2+ in semiaqueous environment by intramolecular charge transfer mechanism

Article abstract of DOI:10.1016/j.tetlet.2013.05.014

An efficient intramolecular charge transfer fluorescent probe bridging benzhydryl moiety and dansyl fluorophore through piperazine unit has been synthesized and characterized. The photophysical behavior of synthesized probe has been analyzed in the presence of different cations in aqueous acetonitrile solution. The probe has shown sensitivity to detect Hg²⁺ ion selectively over other tested cations at 20 nM level. The probable mode of binding of Hg²⁺ through the nitrogen and oxygen atoms of piperazine and dansyl has been established by spectral data analysis.

Full text of DOI:10.1016/j.tetlet.2013.05.014

Accepted Manuscript
A simple blue fluorescent probe to detect Hg2+ in semiaqueous environment by
intramolecular charge transfer mechanism
Priyanka Srivastava, Rashid Ali, Syed S. Razi, Mohammad Shahid, Satyakam
Patnaik, Arvind Misra
PII:
S0040-4039(13)00762-4
DOI:
http://dx.doi.org/10.1016/j.tetlet.2013.05.014
TETL 42914
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
19 February 2013
30 April 2013
3 May 2013
Please cite this article as: Srivastava, P., Ali, R., Razi, S.S., Shahid, M., Patnaik, S., Misra, A., A simple blue
fluorescent probe to detect Hg2+ in semiaqueous environment by intramolecular charge transfer mechanism,
Tetrahedron Letters (2013), doi: http://dx.doi.org/10.1016/j.tetlet.2013.05.014
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Graphical Abstract
4+Hg2+
N
N
4
O
S
O
Hg(L)2
O
S
O
N
N
Hg2+
N
N
1

A simple blue fluorescent probe to detect Hg2+ in semiaqueous environment
by intramolecular charge transfer mechanism
Priyanka Srivastava1, Rashid Ali1, Syed S. Razi1, Mohammad Shahid1, Satyakam Patnaik2 and
Arvind Misra1*
1Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi – 221 005,
INDIA
2Indian Institute of Toxicology and Research (IITR), Lucknow-226001, INDIA
Corresponding author: e-mail: arvindmisra2003@yahoo.com; amisra@bhu.ac.in Tel: +91-542-
6702503; Fax: +91-0542-2368127, 2368175.
An efficient intramolecular charge transfer fluorescent probe bridging benzhydryl moiety and
dansyl fluorophore through piperazine unit has been synthesized and characterized. The
photophysical behavior of synthesized probe has been analyzed in the presence of different
cations in aqueous acetonitrile solution. The probe has shown sensitivity to detect Hg2+ ion
selectively over other tested cations at 20 nM level. The probable mode of binding of Hg2+
through the nitrogen and oxygen atoms of piperazine and dansyl has been established by spectral
data analysis.
Keywords: Fluorescence, Hg2+, ICT
2

Over the last few decades, among the heavy and transition metal (HTM) ions Hg2+ is considered
as most toxic heavy metal ion and common pollutant that possesses severe risks for human
health and environment.1,2 Mercury is commonly spread in environment by anthropogenic and
industrial releases and is converted to toxic methylmercury by the bacterial and chemical actions.
The bioaccumulation of such toxic stuff in living tissues of human and animal bodies via a food
chain causes mercury poisoning and lethal diseases.1,2 Environmental Protection Agency (EPA)
has set maximum allowable level of inorganic mercury in drinking water 2 ppb.3
Considerable efforts have been devoted to develop chemosensors for Hg2+ and among several
methods fluorescence based detection methods have been found more reliable, inexpensive,
sensitive, easy to perform, and are suitable for real time monitoring of anions and metal ions.4
However, poor aqueous medium compatibility5 and nonspecific fluorescence quenching induced
by HTMs due to enhanced spin–orbit coupling6a and electron transfer mechanism6b,c are kind of
limitations for such sensing systems to become a good analytical tool. Therefore, a sensitive
fluoroionophore compatible with aqueous or partial aqueous medium and which can show
characteristic optical behavior upon interaction with metal ions particularly, Hg2+ is highly
demanding.7
Currently, fluorescent probes has attracted considerable interest because of their intrinsic
sensitivity and selectivity.8 In this direction several efforts have been made to design efficient
fluorescent molecular sensors9 to detect Hg2+ either by fluorescence enhancement or quenching
2
and color changes.10,11 The major challenges in the development of a sensitive Hg sensor are
+
due to silent optical spectroscopic signature of Hg2+ because of closed-shell d10 configuration, a
background of competing analytes12 and compatibility in an aqueous or partial aqueous media.
Many reports dealing with fluorescent probes for instance, azacrown ethers,13a dithioaza
compounds,13b calixarene-based ionophores,13c-d DNA,14a MerR family proteins,15a fluorescent
dye doped crystalline complexes,15b and so on15c have appeared in literature to detect Hg2+ ions
however, only a few of them are found competitive14 in terms of sensitivity, selectivity, and
measurements in aqueous media. Therefore, for Hg2+ design and synthesis of new systems based
on small organic molecular systems and/or scaffolds that are cost-effective, rapid, facile and
applicable to the environmental and biological milieus is an important goal to provide instant
optical feedback without involving sophisticated instrumentation.
3

Keeping these perspective in mind our ongoing research is currently paying attention toward
development of some efficient fluorescent organic scaffolds/motifs to detect cations, anions, and
biomolecules sensitively in different media.16 Through this contribution we present design and
synthesis of a new class of benzhydryl piperazine dansyl conjugate, 4 and its potential
application to detect Hg2+ in a partial aqueous medium. While synthesizing such typical motif we
considered that soft metal ions like, Hg2+ has affinity for ligands containing nitrogen as a donor
side. Simple heterocycles like, piperazine, a strong base (pKa, 9.8) than pyridine (pKa, 5.2) and
aniline (pKa, 4.6) will favor a stable 1:1 complexation with mercuric salts in aqueous
medium.17Also, introduction of benzhydryl moiety in conjugation to piperazine and dansyl
would generate a stable configuration by acquiring an appropriate amount of energy and become
an efficient electron rich charge transfer probe to work on push-pull mechanism.2,8
Scheme 1 illustrates the synthesis and structure of probe, 4. Benzophenone upon reduction with
sodium borohydride in methanol gave biphenylmethanol, 1 which was converted to
biphenylmethylchloride, 2 in good yield by reacting with thionyl chloride. Piperazine
dihydrochloride and compound 2 in the presence of potassium carbonate were reacted under
refluxing condition in THF to afford 1-benzhydrylpiperazine, 3 in 80% yield. Subsequently, 3
treated with dansyl chloride in THF under anhydrous condition to yield benzhydryl piperazine
dansyl conjugate, probe 4 as a light yellow color powder in ~75% yield. All products were
1
characterized by HNMR, FTIR, and ESI-MS spectral data analysis (See supplementary
information, Figure S1-S6).
Dansyl is sensitive to variation in external environment and a slight variation in the medium
would result a dramatic change in its typical fluorescence behavior attributed to intramolecular
charge transfer (ICT) reaction from N,N’-dimethylamine (-N(CH3)2) to sulfonamide function.18-
20
Also, dansyl appended probes show solvatochromic nature (sensitivity to polarity of the
medium due to a change in fluorophore’s dipole moment on optical excitation in solvents of
different dielectric constants and refractive indexes) owing to the presence of locally excited
(LE) and twisted intramolecular charge transfer (TICT) excited state.21a,22a So, upon excitation,
first a less polar semi-planar state, similar in structure to ground state is formed to exhibit
structured emission (LE). This LE state undergoes ICT from N,N’-dimethylamino (the donor) to
the sulfonamide fragment (the acceptor) with a twist along the bond connecting the donor and
acceptor units. That creates a relatively more polar TICT state in which donor and acceptor units
4

become perpendicular to each other. The emission corresponding to TICT state is structureless
and appears at higher wavelength in comparison to LE state. The TICT state is sensitive to
solvent polarities which help to mediate CT. Therefore, high energy fluorescence is associated
with LE while low energy fluorescence is through TICT state. The polarity of the solvent up to a
certain extent also governs the height of the energy barrier for the conversion of LE to ICT as
well as stabilize the ICT state. Therefore, when the ICT behavior of 4 has been studied in
solvents of different dielectric constants such as, hexane, chloroform, benzene, acetonitrile,
methanol and dimethylformamide the emission maxima appeared at different wavelengths. The
red shift observed in emission band along with increase in Stoke’s shift were found in the order
as; Hexane < benzene < chloroform < acetonitrile < dimethylformamide < methanol (Figure S7
and Table S1).
Further, the optical behavior of present fluorescence motifs 4 has been examined at different pH
by absorption and emission spectroscopy (Figure 1). In the alkaline medium (pH 7 to 14)
absorption spectra showed a slight enhancement in the absorption band centered at 342 nm (ε =
5.8 x 104 M-1 cm-1) while, in acidic medium (pH 6 – 1) band centered at 342 nm decreased
gradually and a new blue shifted band of relatively high extinction coefficient (ε = 1.0 x 105 M-1
cm-1) appeared at 286 nm (Figure 1a). Similarly, emission spectra of 4, in alkaline medium (pH 7
to 14) showed insignificant change in fluorescence intensity at 540 nm. However, in acidic pH 6
– 1 fluorescence intensity at 540 nm decreased gradually (80%) and a new emission band
appeared at 400 nm (Figure 1b). The variation in the optical behavior of 4 at different pHs may
˜
be attributed to TICT processes18 wherein under the acidic conditions decrease in emission
intensity with a large blue shift is probably due to the protonation of -N(CH3)2 function and of
piperazine unit (high pKa, 9.8), ultimately restricting typical ICT reaction.18 The pH studies also
suggested about the applicability of 4 to work around physiological pH 7. Moreover, when the
pH studies have been performed in HEPES buffer almost similar optical pattern was observed
(Figure S8). Thus, the above experimental observations clearly suggest about the ICT process in
the studied probe 4.
The photophysical behavior of 4 in the absence and presence of metal ions has been examined in
aqueous-acetonitrile (H2O-MeCN; 50%) through absorption and emission spectrometry. It is
expected that MeCN or MeCN-H2O gradient system would facilitate the TICT process. The
absorption spectrum of 4 (10 µM) show characteristic intramolecular charge transfer band at 342
5

nm (ε = 58000 M-1 cm-1) (Figure 2, S9). Upon excitation at 342 nm 4 displayed a weak emission
band centered at 540 nm with Stoke’s shift of 10721 cm-1. The ability of 4 to interact with
different cations has been examined by the addition of different metal ion (5 equiv) namely, Na+,
K+, Ca2+, Zn2+, Pb2+, Ag+, Cd2+, Co2+, Ni2+, Cu2+ and Hg2+ to a solution of 4. The absorption
spectra in the presence of metal ions do not show any significant modulation except, a marginal
change with Hg2+ ion in which, absorption band centered, at 342 nm enhanced marginally (ε =
0.03%). Further, the fluorogenic affinity of 4 (1 µM) toward tested metal ions (5 equiv) has been
analyzed through emission spectra, at 342 nm excitation. The emission spectra revealed local and
TICT excited states at ~ 400 and 540 nm respectively. The emission band centered at 540 nm
showed fluorescence quenching selectively with Hg2+ in which the relative fluorescence intensity
of 4 decreased, ~30% (Figure 3a). The competitive metal ions interference experiment when
performed by the addition of excess of tested cations (20 equiv) to a solution of complex, 4-Hg2+
revealed insignificant change in the absorption and emission spectra of 4-Hg2+ thus, suggesting
high sensitivity of 4 toward Hg2+ ion selectively (Figure 2b, 3b, S9). To ascertain that the process
of complexation between 4 and Hg2+ is reversible a strong chelating reagent, EDTA was added,
in excess to a solution of 4+Hg2+. The fluorescence intensity revived and was found almost close
to the intensity of 4 (Figure S10a) thus, suggested about the reversible mode of complexation
between 4 and Hg2+ions. In contrast, when Hg2+ ions were added to a solution of 4 containing
EDTA in excess, emission spectra show insignificant change probably due to the formation of a
strong EDTA-Hg2+ complex in the medium (Figure S10b).
The binding affinity of 4 with Hg2+ has been determined by absorption titration studies (Figure
S11a). Upon a gradual addition of Hg2+ (0-15 equiv.) absorption band centered, at 342 nm
enhanced marginally (ε = 0.03 %). However, excess of Hg2+ ions (20-50 equiv.) was added to the
same solution ICT band decreased almost completely (ε = 0.82 %) and a new band appeared at
288 nm (ε = 1.8 x 105 M-1 cm-1). That may be attributed to either protonation at -N(CH3)2
16b
function22b and/or interaction of Hg2+ with -N(CH3)2 function of dansyl
as well as nitrogen
atoms of piperazine unit.17 To confirm the possibilities of interactions absorption titration
experiment was performed at pH 7 in HEPES buffer (10 mM; H2O-MeCN; 50%). Upon a
gradual addition of Hg2+ (0-15 equiv) ICT band centered at 342 nm increased marginally (ε = 6 x
104 M-1 cm-1) with a blue shift of 4 nm. However, upon increasing concentration of Hg2+ ions
(20-50 equiv.) absorption spectra showed insignificant change (Figure S11b). This clearly
6

suggested that Hg2+ is not interacting with -N(CH3)2 function of dansyl at pH 7 while in aqueous-
ACN (50%) it could happened probably because of acidic pH of medium generated by the
addition of excess of mercury as its nitrate salt.
The binding affinity of 4 with Hg2+ ions has been determined by emission titration studies. Upon
a gradual addition of Hg2+ (0 - 15 equiv) to a solution of 4 the emission spectra displayed, ~ 81%
fluorescence quenching, in which relative fluorescence intensity of emission band centered at
540 nm decreased gradually with a bathochromic shift of 20 nm and the naked-eye sensitive
color of solution changed from a bright fluorescent blue to a bluish-green (Figure 4). The
estimated quantum yield21a of 4 (Φ4 = 0.053, in MeCN/H2O) upon interaction with Hg2+ ions
decreased to 0.012 (∆Φ4 + Hg2+ = 0.041). The resultant Job’s plot revealed a 1:1 stoichiometry for
4 - Hg2+ interaction (Figure 5a). The binding constant for a 1:1 stoichiometry calculated from
Benesi-Hildebrand (B-H) method21b utilizing emission titration experiment data, gave Kass = 3.3
x 105 M-1 (Figure 5b). The extent of fluorescence quenching was estimated quantitatively by
obtaining Stern-Volmer (S-V) plot between concentration of Hg2+ ions and change in relative
fluorescence intensities (∆I). An almost straight line S-V plot with quenching constant, Ks-v = 2 x
105 M-1 (Figure 5c) suggested fluorescence quenching in the present sensing event. The
sensitivity (LOD) of 4 to detect Hg2+ in H2O-MeCN solution has been estimated by our
previously reported method16a,b and was found to be 20 nM (4 ppb) which is low, (Figure 6) but
comparable to other reported methods.16a,k
Moreover, to confirm the interaction of Hg2+ emission titration experiment was repeated at pH 7
in HEPES buffer (Figure S12). The emission spectra revealed local and TICT excited states and
upon addition of Hg2+ fluorescence quenching occurred in which intensity of CT band centered
at 540 nm decreased with a bathochromic shift of 12 nm without generation of any additional
emission band corresponding to the protonation of -N(CH3)2 function. Furthermore, the pH-
emission spectra of 4 showed that from pH 7 to 5 the CT emission decrease with a marginal rise
in intensity of band centered at 400 nm in HEPES buffer (Figure S13a). However at pH 4 the
low energy band decreased abruptly and high energy band become prominent. This is attributed
to protonation of -N(CH3)2 function of dansyl as well as piperazine unit. Upon addition of Hg2+
at pH 4.0, insignificant change in the emission spectra of 4 was observed (Figure S13b) which
clearly suggested about the interaction of Hg2+ with nitrogen atoms of piperazine unit rather than
interaction with dansyl fluorophore.
7

Additionally, the mechanism of fluorescence quenching either by charge/electron transfer or
energy transfer can be determined thermodynamically by performing spectrofluorometric
investigations on a frozen solution at liquid nitrogen temperature.19b,23 In a frozen matrix,
immobilization of the solvent molecule prevents solvent reorganization surrounding the
generated charge separated molecules and raises the energy of the ion-pair species and prevent
electron transfer. Consequently, if the electron transfer quenching mechanism prevails at room
temperature, freezing of the solution at 100K would partially or fully restore the fluorescence
emission. To observe that when the emission spectra of the probable complex, 4+Hg2+ was
acquired in EtOH/MeOH solvent mixture (9:1, v/v) and fluorescence quenching (~73%) was
observed with a bathochromic shift of ~ 16 nm (Figure 7). In contrast on a frozen solution (rigid
glass matrix, at ~100K), the emission of complex restored partially (49%). Thus, the
experimental observations clearly suggesting about quenching mechanism through a
charge/electron transfer process.19b,23
Further insight about actual mechanism of interaction between 4 and Hg2+ were followed by 1H
NMR titration studies. The 1HNMR spectrum of 4 in DMSO-d6 (Figure 8) showed three doublets
at δ 8.53-8.08 ppm assigned to of H2, H8 and H4 protons while multiplet at δ 7.68-7.54 ppm to
H3 and H7 proton resonances of naphthyl ring while H6 and benzhydryl ring protons resonates
at δ 7.39-7.11 ppm. The resonances appeared at δ 4.24, 3.02 and 2.29 ppm can be assigned to
methyl protons, –CH (1’’) and -CH2 (2’ and 1’) of benzhydryl and piperazine rings respectively,
and signal at δ 2.82 ppm is attributable to protons of -N(CH3)2 function. Upon a gradual addition
of Hg2+ ions (0 – 2 equiv) to a solution of 4 –CH (1’’) resonances shifted downfield (∆δ = 1.1
ppm) and emerged at δ 5.34. The -CH2 (1’) proton of piperazine ring showed significant
downfield shift, ∆δ = 0.71 ppm to appear at δ 3.01 ppm and while -CH2 (2’) shifted downfield,
∆δ = ~ 0.26 ppm and merged with signal of DMSO-d6. The resonances attributed to –N(CH3)2
protons showed almost negligible change whereas the benzhydryl and naphthalene ring protons
shifted downfield narrowly in the range of δ 0.03-0.12 ppm. Thus, the change in the spectrum of
the 4 upon addition of Hg2+ ions suggested about the possibility of interaction of Hg2+ with
piperazine ring nitrogen atoms well stabilized with oxygen atom of sulfonamide function.
Moreover, it is reasonable to assume that upon interaction with Hg2+ ions the orientation of
piperazine unit undergo some structural changes, in which the chair form is flipped to a boat
form. A 1:1 binding equilibrium between 4 and Hg2+ ions and insignificant change in the
8

resonances of –N(CH3)2 function of dansyl fluorophore ruled out the possibility of interaction
through –N(CH3)2 unit as demonstrated previously.16a,24 Therefore, the observed fluorescence
quenching upon interaction with Hg2+ is probably due to the transfer of electron21 from the
excited state of the fluorophore to the available d – orbital of Hg2+ precisely, due to strong spin-
orbit coupling25 enforced by Hg2+ on the probe.
Further, the probable complexation between 4 and Hg2+ ion was analyzed through FT-IR spectra
measured in range 4000–400 cm−1 (Figure S14). The FTIR spectrum 4 showed characteristic
bands at 2801, 1660 1492, 1447 1340 846 and 1281 1137 cm-1 probably due to C-H, C=C, C-N
and S=O stretching vibrations. Similarly, bands appeared at 1000, 744, 705 and 805 cm-1 may be
assigned to C-S, C-H (aromatic) and S-O (sulfonamide) stretching vibrations, respectively.16e,l
Upon interaction with Hg2+ ions, C-N and S=O stretching vibrations shifted to appear at lower
frequencies at 1434, 1329, 794 and 1143, 1110, 1087 cm-1. The C-S stretching vibrations also
showed a shift in lower frequency region, at 916 and 707 cm-1. The stretching vibrations
appeared at 626, 569 and at 482 cm-1 are possibly due to the M–O and M–N (Hg-O/N)
interactions.16e,l Therefore, supporting about the involvement of nitrogen and oxygen atoms of
piperazine and sulfonamide units, as a potential donor site to form a complex, 4-Hg2+ as
predicted in Scheme 1. The ESI-MS spectral data (Figure S6 and 15) analysis of 4 before and
after interaction with Hg2+ [(HgClO4)2] revealed m/z at 486 (4 +H)+ and at 787.8 [4 + Hg + ClO4
+ H]+ supported the mode of binding between 4 and Hg2+ involving the N and O atoms of
piperazine and sulfonamide oxygen of dansyl.26
In summary, through the present contribution we have demonstrated the application of a new
charge transfer blue fluorescent probe to detect Hg2+ selectively in a partial aqueous medium
with sensitivity of detection up to 20 nM. The optical behavior of probe and NMR spectral data
analysis suggested about the interaction of probe with Hg2+ ion through the nitrogen atoms of
piperazine unit well stabilized by oxygen atom of sulfonamide function.
Acknowledgement: We gratefully acknowledge financial support and fellowships (SRF to PS,
RA, SSR and RA to MS) from the Council of Scientific and Industrial Research (CSIR), New
Delhi and The Head, Department of Chemistry for extending necessary basic facilities.
1 13
Supplementary data: Synthesis, HNMR, CNMR, FT-IR, ESI-MS, UV-Vis and fluorescence
spectra.
9

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Balzani, V.; Barigelletti, F.; Cola, L. D.; Flamigni, L. Chem. Rev. 1994, 94, 993-1019.
24 (a) Rotkiewicz, K.; Grellmann, K. H.; Grabowski, Z. R. Chem. Phys.Lett. 1973, 19, 315-318;
(b) Grabowski, Z. R.; Rotkiewicz, K.; Siemiarczuk, A.; Cowley, D. J.; Baumann, W. Nouv. J.
Chim. 1979, 3, 443-454.
25 (a) Kasha, M. J. Chem. Phys. 1952, 20, 71-74; (b) El-Sayed, M. A. Acc. Chem. Res. 1968, 1,
8-16; (c) Koziar, J. C.; Cowan, D. O. Acc. Chem. Res. 1978, 11, 334-341; (d) Svejda, P.; Maki,
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12

Figure Captions
Scheme 1: Synthesis of fluorescent probe 4 and plausible mode of complexation with Hg2+ ion.
(i) NaBH4/ Ethanol/ rt (ii) SOCl2 / DCM / rt (iii) Piperazine / K2CO3/ DMF/ reflux / 8h (iv)
Dansyl chloride / Acetone / rt
Figure 1. Change in (a) absorption and (b) emission spectra of 4 at different pH in aqueous ACN
(50%).
Figure 2. (a) Change in absorption spectra of 4 (10 µM) upon interaction with different metal
ions. (b) Interference study by the addition of different cations to a solution of 4-Hg2+ in HEPES
buffer (10 mM; aqueous ACN (50%) pH 7).
Figure 3. Change in emission spectra of 4 (10 µM) (a) upon interaction with different metal ions
and (b) Interference study by the addition of different cations to a solution of 4-Hg2+ in aqueous
ACN (50%). Inset: Bar diagram of interaction study.
Figure 4. Emission titration spectra of 4 upon addition of Hg2+ (0- 15 equiv) in aqueous-
acetonitrile (50%). Inset: Change in color of 4 under UV light.
Figure 5. (a) Jobs plot (b) B-H plot (c) Stern-Volmer plot of 4 and Hg2+ ions in aqueous ACN
(50%).
Figure 6. (a) Calibration curve for 4 and (b) Calibration sensitivity of Hg2+ ions in aqueous
ACN (50%).
Figure 7. Emission spectra of 4 and 4+Hg2+ at normal temperature and revival of fluorescence at
liquid nitrogen temperature.
Figure 8. 1H NMR titration spectra of probe 4 upon addition of Hg2+ ions (0-2 equiv.) in DMSO-
d6.
13

A simple blue fluorescent probe to detect Hg2+ in semiaqueous environment
by intramolecular charge transfer mechanism
Priyanka Srivastava1, Rashid Ali1, Syed S. Razi1, Mohammad Shahid1, Satyakam Patnaik2 and
Arvind Misra1*
1Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi – 221 005,
INDIA
2Indian Institute of Toxicology and Research (IITR), Lucknow-226001, INDIA
Corresponding author: e-mail: arvindmisra2003@yahoo.com; amisra@bhu.ac.in Tel: +91-542-
6702503; Fax: +91-0542-2368127, 2368175.
14

(i)
(ii)
(iii)
N
OH
1
O
Cl
2
N
H
3
6
-
7
2
L
N
Hg2+
8
1
N
O
N S
O
5
4
Hg2+
(iv)
O
S
N
3
N
1''
N
EDTA
O
3
1'
4
2'
Scheme 1: Synthesis of fluorescent probe 4 and plausible mode of complexation with Hg2+ ion.
(i) NaBH4/ Ethanol/ rt (ii) SOCl2 / DCM / rt (iii) Piperazine / K2CO3/ DMF/ reflux / 8h (iv)
Dansyl chloride / Acetone / rt
15

pH 6 - 1
pH 14 - 7
120
)
1.0
0.5
0.0
pH 6 - 1
80
pH 14 - 7
(
pH 6 - 1
40
0
360 420 480 540 600
Wavelength nm
300
350
400
450
Wavelength nm
Figure 1. Change in (a) absorption and (b) emission spectra of 4 at different pH in aqueous ACN
(50%).
1.0
0.5
0.0
1.0
0.5
0.0
b
a
2+
2+
4+Hg
4+Hg +M ions
4+M ions
4
300
350
400
450
300
350
400
450
Wavelength nm
Wavelength nm
Figure 2. (a) Change in absorption spectra of 4 (10 µM) upon interaction with different metal
ions. (b) Interference study by the addition of different cations to a solution of 4-Hg2+ in HEPES
buffer (10 mM; aqueous ACN (50%) pH 7).
16

30
20
10
0
n+
120
120
4+ M
a
4
)
80
)
80
I
I
I
I
∆
∆
∆
∆
(
(
4+Hg2+
525
40
0
40
4+Hg2++M
n+
Na K Ca Zn Pb Ag Co Ni Cd Cu Hg
0
375
450
600
675
375
450
525
600
675
Wavelength nm
Wavelength nm
Figure 3. Change in emission spectra of 4 (10 µM) (a) upon interaction with different metal ions
and (b) Interference study by the addition of different cations to a solution of 4-Hg2+ in aqueous
ACN (50%). Inset: Bar diagram of interaction study.
120
Hg2+
)
80
(
40
0
400
500
600
Wavelength
nm
Figure 4. Emission titration spectra of 4 upon addition of Hg2+ (0- 15 equiv) in aqueous-
acetonitrile (50%). Inset: Change in color of 4 under UV light.
17

60
50
40
30
20
4.5
3.0
1.5
(b)
2.0
1.6
1.2
(a)
(c)
∆
∆
∆
∆
∆
∆
∆
∆
0.1
0.2
0.3
0.4
0
0.0 0.2 0.4 0.6 0.8 1.0
3
6
9
12 15
2+
1/Hg
2+
4/[4+Hg ]
2+
Hg (µM)
Figure 5. (a) Jobs plot (b) B-H plot and (c) Stern-Volmer plot for 4 upon interaction with Hg2+
ions in aqueous ACN (50%).
45
36
27
18
9
800
)
600
(
400
I
I
I
I
∆
∆
∆
∆
200
1
2
3
4
5
0
1
2
3
4
5
6
7
Hg2+
mM
Probe mM
Figure 6. (a) Calibration curve for 4 and (b) Calibration sensitivity of Hg2+ ions in aqueous
ACN (50%).
180
)
4
(
120
60
0
4 + Hg2+
560
400
480
640
Wavelength nm
Figure 7. Emission spectra of 4 and 4+Hg2+ at normal temperature and revival of fluorescence at
liquid nitrogen temperature.
18

Figure 8. 1H NMR titration spectra of probe 4 upon addition of Hg2+ ions (0-2 equiv.) in DMSO-
d6.
19