The structural properties of 5-methyl-2-phenyl-2H-1,2,3-triazole-4- carboxylic acid and chromogenic mechanism on its rhodamine B derivatives to Hg2+ ions

Article abstract of DOI:10.1016/j.saa.2018.04.009

5-Methyl-2-phenyl-2H-1,2,3-triazole-4-carboxylic acid (MPTC), a newly synthesized compound, was explored to study the structural properties and theoretical spectra by using GaussView5.0 program package and the time dependent density functional theory (TD

Full text of DOI:10.1016/j.saa.2018.04.009

Accepted Manuscript
The
structural
properties
of
5-methyl-2-phenyl-2H-1,2,3-triazole-4- carboxylic acid and
chromogenic mechanism on its rhodamine B derivatives to Hg2+
ions
Jianling Li, Guohua Ding, Yanyan Niu, Luyong Wu, Huajie Feng,
Wenying He
PII:
DOI:
Reference:
S1386-1425(18)30308-1
doi:10.1016/j.saa.2018.04.009
SAA 15966
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 5 December 2017
Revised date:
Accepted
date:
3 April 2018
5 April 2018
Please cite this article as: Jianling Li, Guohua Ding, Yanyan Niu, Luyong Wu, Huajie
Feng, Wenying He , The structural properties of 5-methyl-2-phenyl-2H-1,2,3-triazole-4-
carboxylic acid and chromogenic mechanism on its rhodamine B derivatives to Hg2+ ions.
The address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Saa(2017), doi:10.1016/j.saa.2018.04.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
The structural properties of 5-methyl-2-phenyl-2H-1,2,3-triazole-4-
carboxylic acid and chromogenic mechanism on its rhodamine B
derivatives to Hg2+ ions
Jianling Lia, Guohua Dinga, Yanyan Niua, Luyong Wub, Huajie Fenga*, Wenying Heb*
a College of Chemistry and Chemical Engineering,Hainan Normal University, Haikou,571158, China;
b Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Haikou, 571158, China
ABSTRACT
5-Methyl-2-phenyl-2H-1,2,3-triazole-4-carboxylic acid (MPTC), a newly
synthesized compound, was explored to study the structural properties and theoretical
spectra by using GaussView5.0 program package and the time dependent density
functional theory (TD DFT). The calculated quantum chemical values suggested that
it is easy for MPTC to lose electron with weak electron accepting ability. And the
results of experimental measurements on fluorescence and absorption spectra were
consistent with that of the calculated spectra in great degree. In addition, MPTC was
successfully used and synthesized a novel rhodamine B derivative RMPTC containing
1,2,3-triazole unit. It is found that there is special chromogenic response of RMPTC
to Hg2+ ions in N, N-dimethylformamide (DMF)-H2O (v/v=1/1, Tris-HCl, pH 7.4)
with the triazole appended colorless chemosensor turned to pink and enabled
naked-eye detection. The fluorescence signal for RMPTC-Hg2+ system was not
affected by other coexisting metal ions. The 1:2 stoichiometric structure of RMPTC
and Hg2+ is confirmed using a Job’s plot estimation and TD DFT calculations. The
corresponding “off-on” fluorescence mechanism of RMPTC binding to Hg2+ which
were ascribed to Hg2+ inducing the ring-opened rhodamine B moiety were proposed.
This study was an advancement for the application of 1,2,3-triazole compound in
photophysical chemistry field and provides guidance for exploring simple and
high-selectivity Hg2+ probes in aqueous solutions under physiological conditions.
Key words: 5-Methyl-2-phenyl-2H-1,2,3-triazole-4-carboxylic acid, structural
properties, rhodamine B, synthesis, chromogenic mechanism, Hg2+ ion.
1. Introduction
Since the landmark research on the synthesis of 1,2,3-triazoles in 2002[1], these
*Corresponding author at: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University,
Hainan Normal University, Haikou 571158, China.
E-mail address: hewenying@hainnu.edu.cn; 1370525899@qq.com

ACCEPTED MANUSCRIPT
compounds have been receiving permanent interest due to their unique chemical
structure and physicochemical properties. 1,2,3-Triazoles are five member
N-heterocyclic compounds and are easy to conjugate or combine with other
substituent groups to form multiple types of derivatives. Furthermore, nitrogen
heterocycles with triazole ring have been widely applied in different fields of
chemical, biological, macromolecular materials and pharmaceutical sciences because
of their less toxic effects and good biological activity[2-3]. Some studies have also
shown that 1,2,3-triazoles or their derivatives can act as ligands capable of interaction
with various metal ions and have exhibited specific pharmacological action[4-6].
However, it is well known that the lack or overdose of metal ions will lead to the
physiologic disturbance of organisms. So it is especially practical to design and
synthetize a novel compound containing 1,2,3-triazole as a metal ion probe. For
instance, as one of the toxic metal ions, mercury ion (Hg2+) is one of the most
poisonous species to human beings due to the strong interaction of Hg2+ with
sulfhydryl groups of proteins in a wide variety of organs including brain, kidneys,
central nervous system and immune system. After sinking in water, mercury ions can
accelerate accumulation by the aquatic food chain. The contamination of mercury ions
has been becoming one of the most serious problems in the environment because of
the increasing industrial and agricultural activities as well as the improper release of
metal ions from waste waters and domestic effluents[7-8]. How to detect mercury
ions efficiently and sensitively has aroused the more and more concern of researchers.
And enormous studies have been carried out to develop a wide variety of analytical
techniques for Hg2+ detection [9-11]. Based on the broad chemical versatility and
biological activity of 1,2,3-triazoles, there is still an unmet demand to develop and
make full use of new 1,2,3-triazoles for highly sensitive and selective detection of
Hg2+ in environmental and biological samples.
The rhodamine-based fluorophores have attracted considerable interest due to
their excellent spectroscopic properties such as high fluorescence quantum yield,
broad absorption, large extinction coefficient, broad emission wavelength, and high
light stability [12]. Generally, the rhodamine B derivatives could show significant
fluorescence enhancement and color variations from the closed loop state turned to be
open loop state when binding to the ligands. So it has been widely applied to design
as “off-on” fluorescent probes for detecting metal ions[13]. Recent studies on the
fluorescence probes obtained from rhodamine-triazoles systems have shown that these

ACCEPTED MANUSCRIPT
complexes could response specifically to some metal ions such as Zn2+、Hg2+、Cu2+、
Al3+ etc [14-16]. Among different research techniques for detection of metal ions, the
more common methods include inductively coupled plasma mass spectrometry
(ICP-MS)[17], atomic absorption spectroscopy(AAS) [18], fluorescence spectroscope
[19] and electrochemistry [20] etc. Especially the color change is a simple but
extremely practical and economic tool to recognize and detect analytical species.
Compared with fluorescence channels, it worked in the natural light conditions
without excitation of extraneous light. Therefore the fluorescence analysis method
with the colorimetric sensors have attracted great attention due to the advantages of
easy operation, signal visibility (instrument-free), fast response, applicable for on site
and high throughput analysis[21-22]. In present work, 5-methyl-2-phenyl
-2H-1,2,3-triazole-4-carboxylic acid (MPTC, Scheme 1) was a new compound
synthesized under copper-catalyzed oxidative conditions[23]. Molecular properties for
MPTC like ionization potential, electron affinity, molecular orbitals and
theoretical spectra have been calculated by using TD-DFT method with HF/6-31G(d)
and B3LYP/6-31G(d) basis set. Then MPTC was designed and introduced to
rhodamine B to synthesize a novel derivative named RMPTC, which exhibited
excellent selectivity toward Hg2+ with chromogenic reaction over other metal ions and
can be applied to detect Hg2+ ions qualitatively under physiological conditions.
2. Experimental
2.1. Materials and Instrumentations
MPTC was provided by Organic Chemistry Lab of Hainan Normal University,
China, and the stock solution (5.0×10−4mol/L) was prepared in methanol. Tris-HCl
buffer was selected to keep the pH of the solution at 7.4. Sodium hydroxide (NaOH)
and chloride salts including Pb2+ , Mn2+, K+, Na+, Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe3+,
Zn2+, Ni2+, Hg2+, Li+ and Mg2+ were acquired from Sinopharm Chemical Reagent Co.,
Ltd (Shanghai, China). All other reagents were of analytical reagent grade and
obtained commercially without further purification. Double distilled water was used
throughout the experiment.
NMR spectra were measured at room temperature on a Bruker Avance 400MHz
spectrometer at 400 MHz in CDCl3. Fluorometric experiments were carried out on a
RF-5301PC spectrofluorophotometer (Shimadzu, Japan) and
a
F-7000
spectrofluorophotometer (Hitachi, Japan) equipped with a xenon lamp source and 1.0

ACCEPTED MANUSCRIPT
cm quartz cells. A thermostatic water bath was used to keep the reaction systems at
a constant temperature. The UV-vis absorbance spectra were recorded on a Hitachi
U3900/3900H spectrophotometer (Hitachi, Japan) equipped with 1.0 cm quartz cells.
2.2 Synthesis
According to the reported procedure [24], the first step is to synthesize
B hydrazide 1 as shown in Scheme 1. Rhodamine B (1.0 g, 2.1 mmol) was dissolved
in 20 mL ethanol in a 50mL flask, 80% hydrazine hydrate (2 mL) was added with
stirring at room temperature and the stirred mixture was heated to reflux for 3 h. Then
50 mL water was added to the mixture and extracted with 50 mL ethyl acetate three
times. The combined organic layer was dried over anhydrous sodium sulfate and then
1
filtered, affording intermediate 1 as a yellow solid (0.52 g, 54.74%); H NMR (400
MHz, CDCl3):δ (ppm) = 1.16 (t, J = 6.8 Hz, 12H),3.34 (q, J = 7.2 Hz, 8H), 3.61 (s,
2H), 6.30 (m, 2H), 6.45(m, 4H), 7.1 (m, J = 2.64 Hz, 1H), 7.44 (m, 2H), 7.94(m, 1H);
13CNMR (400 MHz, CDCl3) δ (ppm) = 166.03, 153.75, 151.45, 148,78,132.39,
129.91, 128.00, 127.95, 123.71, 122.83, 107.94, 104.47, 97.89, 77.32, 77.00, 76.68,
65.82, 44.25, 12.50.
The second step is the synthesis of RMPTC as follows: A mixture of MPTC
(100mg, 0.49 mmol) and a solid of 2.3mL SOCl2 in a 10 mL round-bottomed flask
was heated to reflux for 3 h. Then the reflux device was substituted by the distillation
device and the residuary SOCl2 was distilled out to give the product 2, after which,
the product 2, the solution of intermediate 1 (234mg, 0.51mmol), 10mL
dichloromethane and 1mL ethylenediamine were mixed and stirred at room
temperature for 12h then extracted with ethyl acetate and H2O. When the reaction was
completed the organic phase was washed three times by deionized water and dried
over Na2SO4. The solvent was evaporated and the obtained solid was dried and
purified by a silica column chromatography (elute: petroleum ether: ethyl acetate=4:1).
The target product RMPTC was obtained as pale yellow solid in 37.01% yield and
1
116.3mg. H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.92 (s, 1H), 7.90 (s, 1H),
7.54-7.46 (m, 2H), 7.40 (t, J = 7.7 Hz, 2H), 7.32 (d, J = 7.2 Hz, 1H), 7.16 (d, J = 6.9
Hz, 1H), 6.77 (s, 1H), 6.75 (s, 1H), 6.38 (s, 1H), 6.36 (s, 3H), 3.33 (dd, J = 13.7, 6.7
Hz, 8H), 2.48 (s, 3H), 1.15 (t, J = 7.0 Hz, 12H);13C NMR (100 MHz, CDCl3) δ 165.23,
158.65, 153.73, 151.64, 148.96, 148.15, 139.15, 138.06, 133.16, 129.27, 129.16,
128.95, 128.26, 127.91, 124.08, 123.51, 119.06, 108.11, 104.52, 97.80, 66.24, 44.32,
12.54, 11.08. HRMS (ESI) (m/z): [M+H]+ calcd. for C38H40N7O3, 642.3193; found,

ACCEPTED MANUSCRIPT
642.3199.
2.3 Quantum chemical calculation of MPTC
GaussView5.0 program package was used to fully optimize the molecular
structure of MPTC in the ground state at Hartree-Fock Restricted algorithm level. The
molecular orbital was analyzed by the data obtained from HF/6-31G (d) method. The
electronic properties of MPTC including frontier molecular orbital LUMO, HOMO
energies, and their band gap were determined to show the effect of electron density
transfer within the molecule. Then the values of ionization potential and electron
affinity of MPTC were obtained by Gaussian 09 software at the DFT//B3LYP/6-31G*
algorithm level. The theoretical absorption spectra of MPTC were performed by using
the time dependent density functional theory
(TD DFT).
The
Configuration
Interaction with Single excitations (CIS) method was used to fully optimize the
configurations of lowest excited state. Based on the above, the theoretical
fluorescence emission spectra of MPTC was obtained from TD DFT [25].
Additionally, based on Gaussian 09 program package, B3LYP method was applied to
fully optimize the molecular structure of RMPTC and calculate the structural energy
in terms of LANL2TZ basis set for Hg and 6-311G(d) basis set for H, C, N, O and F
respectively[26].
2.4 Ions detection experiments of RMPTC
The different concentration solutions of RMPTC and Hg2+ ions were prepared by
stepwise dilution of stock solutions (0.5 mM for RMPTC and 5mM for Hg2+) in
acetonitrile and water, respectively. The test solution of RMPTC was diluted with
DMF-water (v/v=1:1, Tris–HCl，pH=7.40) buffer solution to give a concentration of
1×10-5 mol/L. The fluorescence and UV-vis absorption spectra titrations were
performed by adding the corresponding volume of metal ions to the test solution of
RMPTC. The excitation wavelength of fluorescence measurement is fixed at 563 nm
and that of emission wavelength at 584nm respectively.
To evaluate the selectivity of RMPTC for Hg2+ detection, a series of competitive
metal ions, including Pb2+ , Mn2+, K+, Na+, Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe3+, Zn2+,
Ni2+, Hg2+, Li+ and Mg2+, at a final concentration of 500 nM, were tested as following
the same experimental procedure. All spectroscopic titration measurements were
repeated at least three times until consistent values were obtained.
3. Results and discussion

ACCEPTED MANUSCRIPT
3.1 Molecular Geometry of MPTC
Quantum chemical calculation method such as the time dependent density
functional theory (TD DFT) has become a powerful tool in the investigation of the
molecular structure and optical properties and are finding increasing use in
application related to biological systems[27]. The calculation of ground-state
geometry optimization of MPTC obtained from GaussView5.0 software at the
Hatree-Fock Restricted algorithm level is shown in Fig. 1A, which indicated that the
structure of MPTC is non planar with various groups. The compound MPTC consists
of 24 atoms and the geometrical parameters of MPTC including bond length and bond
angle are listed in Table S1 by using DFT/B3LYP method with HF/6-31G(d),
CIS/6-31G(d), and B3LYP/6-31G(d) as the different basis sets respectively. It is
found that the values of calculated bond lengths and bond angles are similar at all
levels of calculations. The bond length varied from 0.95 A to 1.49A. The bond angle
varied from 104.09 to 130.56°. But the dihedrals show relatively small differences
due to using various computing methods. It is noteworthy that the values of the C-C
bond length in benzene ring (marked 7-12) were between 1.35 and 1.44 Å for three
different basis sets, which are much shorter than the typical C-C single bond (1.54 Å),
longer than the C=C double bond (1.34 Å) and shorter than that of normal C-N bond
(1.47 Å)[28]. The results on the bond lengths and angles indicated that the molecule
of MPTC is no planar.
3.2 Frontier Molecular Orbital Analysis
The highest occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO) known as Frontier Molecular Orbitals (FMO's) are two of
the most important tools used in quantum chemistry. According to FMO's theory, the
formation of a transition state is due to the interaction between HOMO and LUMO
levels of the reacting species. In general, LUMO can be considered as the innermost
orbital which containing free places representing the ability to accept an electron,
while HOMO can be thought as the outermost orbital which contains electrons
representing the ability to donate an electron. The lower the LUMO energy, the easier
the acceptance of electrons from electron-donating substitutes. Energy of HOMO is
directly related to the ionization potential and LUMO energy is directly related to the
electron affinity[29-30]. Fig. B1 and Fig. B2 displayed the lowest unoccupied
molecular orbital (LUMO, 0.08028ev) and highest occupied molecular orbital
(HOMO, -0.3214ev) of MPTC respectively. The other information on molecular

ACCEPTED MANUSCRIPT
orbital were provided in Table S2 and Fig. S1 Supplemental Materials. From Table 2s,
it indicated that the values of LUMO is higher than that of HOMO, which means that
the acceptance of electrons of MPTC from other electron donating group is not easy
to obtain. The energy difference between the HOMO and LUMO orbitals is called the
energy gap which serves as an important stability factor for the compound [31]. The
value of HOMO-LUMO energy gap for MPTC is 0.4017ev, which means that the
molecule has stable structure. And more easily electrons are excited from the ground
state to excited state due to lower the energy gap, which is proved by the following
results obtained from the spectroscopic experiments. In addition, some
physicochemical parameters were calculated such as vertical ionization potential
(6.377 eV), adiabatic ionization potential (6.572 eV), vertical electron affinitie(1.804
eV) and adiabatic electron affinity (2.073 eV) by using the diffuse functions
B3LYP/6-311G+(d) at the DFT//B3LYP/6-31G* algorithm level. These values
suggested that it is easy for MPTC to lose electron or the electron accepting ability of
MPTC is very weak [32].
3.3 Spectra analysis of MPTC
Since the HOMO-LUMO gap is also a crucial factor related to the luminescent
properties[31, 33]. In the present study, the calculated results indicated that the
electron transition of MPTC mainly focused on HOMOLUMO transition, which
suggested that MPTC had luminescent properties and confirmed by the spectroscopic
measurements. Fig. 2A showed the predicted electronic absorption spectra of MPTC
by TD-DFT calculations. The inset in Fig. 2A is the absorption spectra of MPTC in
methanol. It can be seen that both of two graphs showed the similar shape with double
peaks, but the positions of their absorption maximum bands are different. In principle,
the absorption band observed at about 280 nm was more likely to attribute to the
formation of polyene π bonds or π-π* transition by a triazole ring and benzene ring of
MPTC. On the other hand, the increase of solvent polarity accounts for the shift of
absorption band towards the shorter wavelength direction due to n-π* transition. It is
possible that MPTC with the nitrogen atoms as lone electron pair coordinated to
methanol solvent via hydrogen bonds, which increased the energy gap between
ground state and excited state. So it needed greater increased energy supply for n-π*
transition and resulted in the blue-shift effect [34]. Therefore, it is noticed that
the maximum absorption peak is at 286 nm by TD DFT calculations, which is in line

ACCEPTED MANUSCRIPT
with the strongest absorption peak at 277 nm obtained from the experimental
measurement for MPTC.
Fig.2 (B) showed the emission spectra by TD DFT methods and that of measured
in methanol (plot “a” in the inset of Fig. 2 (B)). The calculated spectra exhibited
the maximum fluorescence emission peak at 365nm, which has some difference
in comparison with the experimental data measured at 346nm. The reasonable
explanation is that the conjugated aromatic compound MPTC caused π→π* transition
from ground state to excited state. Since the polarity of excited state is stronger than
that of ground state and low polar solvent can have more influence on stabilizing the
ground state. So the position of maximum fluorescence emission shifted towards the
longer wavelength side [35]. However, methanol, a high polar molecule, was used as
a solvent of MPTC and had more effect on stabilizing the excited state. Therefore
produced the blue-shift effect with the change of the maximum fluorescence emission
peak from 365 nm to 346 nm.
Hence, the theoretical results on the UV-Vis absorption and fluorescence spectra
by using TD DFT methods are in agreement with the experimental results in great
degree. According to Lambert-Beer theory, the molar absorption coefficient of MPTC
(ε) was determined from the plot of absorbance vs concentration (Fig. S2 in
Supplemental Materials). The ε value is obtained to be 2.338×105 L/mol/cm. In
consideration of the particular structure of MPTC, it is speculated that MPTC should
have larger fluorescence quantum efficiency due to it’s fluorescent emission peak at
346 nm and is a prospective blue light emitting material in the organic light-emitting
diodes[36].
3.4 Synthesis of RMPTC
A number of researches have shown that using the triazole motifs as the
platform has potential application in the development of new fluorescence
chemosensors. A variety of highly selective chemosensors for the detection of
different metal ions (such as Fe3+, Zn2+ , Cu2+, Cd2+and Hg2+ etc ) based on click
generated triazole have been designd and applied [37-40]. Especially, it is feasible and
possible for MPTC to react with rhodamine B hydrazide in theory due to the triazole
structure containing ester group [41]. By optimizing the experimental conditions,
MPTC was designed and introduced to the rhodamine hydrazide to successfully
synthesize a new rhodamine B derivative RMPTC through a serious of reactions
13
(Scheme 1). The structure of RMPTC were characterized by 1HNMR, C NMR, and

ACCEPTED MANUSCRIPT
HRMS (Fig. S3, Fig. S4 and Fig. S5 in Supplemental Materials, respectively.). The
results achieving the expected goal can provide reasonable guidance for synthetic
reaction between similar 1,2,3-triazoles and rhodamine B.
3.5 Fluorescence spectra studies on response of RMPTC to metal ions
To determine the binding properties of RMPTC with metal ions, the
fluorescence spectroscopy of RMPTC (50μM) in the presence of 15 kinds of metal
ions such as Pb2+ , Mn2+, K+, Na+, Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe3+, Zn2+, Ni2+, Hg2+,
Li+ and Mg2+ (40μM) was investigated respectively, and the results were displayed in
Fig. 3A. It can be seen that RMPTC showed no obvious fluorescence emission with
excitation at 563 nm in DMF/H2O (v/v=1:1, Tris-HCl, pH=7.4) solutions, which is a
typically characteristic peak of rhodamine B [15, 24]. However, upon binding to
different metal ions, it was only observed that coordination of Hg2+ to sensor RMPTC
resulted in a distinct fluorescence enhancement at 584 nm, while other metal ions
showed no obvious fluorescence emission. Coupled with the fluorescent spectra
changes, the emission color change from colorless to pink was clearly detected by
naked eye (Fig. 3B inset), while other metal ions did not induce any apparent
emission color change under the same conditions. In general, rhodamine spirolactam
or spirolactone derivatives are nonfluorescent and colorless, whereas ring-opening of
the corresponding spirolactam/lactone gives rise to strong fluorescence emission and a
pink color. An appropriate ligand on a spirolactam ring can induce a color change as
well as a fluorescence change upon addition of metal ions, even though this process is
somewhat dependent on the solvent system[42]. Herein, it was attributed to ring
opening from the spirolactam (RMPTC) to ring-opened amide (RMPTC-Hg2+ system)
and RMPTC probably formed a novel compound with Hg2+. The result suggested that
RMPTC can act as a sensor to detect Hg2+ through the distinct fluorescence “off-on”
responses. Then the selectivity of RMPTC to Hg2+ over other metal ions were
investigated by compared experiments (Figure 3(B)). It displayed that the addition of
Hg2+ caused a 2900-fold increase in fluorescence intensity at 577 nm. The inset in Fig.
3(B) showed the photographs of the RMPTC in the presence of Hg2+ and other metal
ions. It is obvious that other metal ions, such as Pb2+, Mn2+, K+, Na+, Ca2+, Cd2+, Co2+,
Cu2+, Fe3+, Zn2+, Ni2+, Li+ and Mg2+ ions, did not induced any significant color and
spectral change under identical conditions. The result demonstrated that the developed
colorimetric sensor RMPTC possessed high selectivity for sensing Hg2+ due to the
possible interaction between RMPTC and Hg2+.

ACCEPTED MANUSCRIPT
Sensitivity is another very important criterion for a sensor. To investigate the
sensitivity of RMPTC with respect to Hg2+, fluorescence titration experiments were
then performed as shown in Fig. 4. The fluorescence intensity of RMPTC (50μM) at
584 nm was gradually increased with the addition of increasing amount of Hg2+ ions,
and consequently became saturated when the amount of Hg2+ reached 40μM.
However, a continuous decrease in the fluorescence intensity was observed for further
increments of Hg2+ concentration. The linear fluorescence enhancement of RMPTC
upon addition of Hg2+ was obtained in the range of 2.00×10-5-4.00×10-5mol/L (R2 =
0.9442, Fig. S6 in Supplemental Materials). So it seems that RMPTC is not an ideal
probe in quantitative determination of Hg2+.
3.6 UV-vis spectra studies on response of RMPTC to metal ions
The UV-vis spectra of RMPTC (50μM) in DMF-H2O (v/v=1:1, Tris–HCl，
pH=7.40) were recorded in the presence of various metal ions as shown in Fig. 6A. It
can been seen that there is the same trend as the fluorescence measurements. The free
RMPTC also exhibited no absorption near 565nm, which is the characteristic
absorbance of rhodamine B. However, upon addition of Hg2+ to the solution of
RMPTC, a new absorption band appeared at 565nm along with the increasing
intensity which was ascribed to the ring opened rhodamine moieties. Figure 6B shows
the spectral variation of RMPTC upon the gradual addition of Hg2+ by the UV-vis
titration experiments. A absorption band centered at 565 nm appeared and gradually
increased with the gradual increase of Hg2+ ion concentration. When the concentration
of Hg2+ was increased from 2.00×10-5 to 4.00×10-5 mol/L, the absorption peak
intensity at 565nm gradually increased. But with the sequential addition of Hg2+,
attainment of the plateau ( the inset in Fig. 6B) implied saturation in the binding
interaction between the two partners.
3.7 The competition experiments
To further identify a high selectivity of RMPTC as a fluorescent sensor, the
competition experiments were performed in the presence of Hg2+ (50μM) mixed with
other metal ion solutions with the same concentration (Fig. 7). It was noticed that the
enhancement in fluorescence intensity resulting from the addition of Hg2+ was not
affected too much by coexistence of other metal ions at this concentration. Meanwhile,
only Hg2+ rendered significant fluorescence turn-on responses among all the tested
metal cations, whereas all the coexistent metal ions had no noteworthy variance in the

ACCEPTED MANUSCRIPT
fluorescence intensity. The results indicated that the fluorescence response of RMPTC
towards Hg2+ was highly selectively than other competitive metal ions.
3.8 Effect of pH
The pH value is a crucial factor for the detection procedure. To evaluate the
potential application of the sensor RMPTC in different environments, the effect of pH
on the fluorescence intensity was investigated in a range of pH between 3.0-10.0. Fig.
8 is the effect of pH on the fluorescence intensity of RMPTC (plot a) and
RMPTC-Hg2+ complex (plot b) with a concentration of RMPTC or Hg2+ fixed at
50μM. As shown plot a in Fig. 8, RMPTC showed obvious fluorescence when the
value of pH is less than 4. But there was no any obvious and characteristic
fluorescence observed for RMPTC in the range of pH from 4.0 to 10.0, which
indicated the formation of stable spirocyclic RMPTC compound in the range of the
value of pH. However, it can be seen from plot b in Fig. 8 that the fluorescence
emission intensity of RMPTC was enhanced under the different value of pH from 4.0
to 8.0 in the presence of Hg2+, which suggested that RMPTC-Hg2+ complex remain in
a“turn-on”state within this pH range. And the maximum fluorescence response
toward Hg2+ was observed at the value of pH as 7.4. If the value of pH is higher than
8.0, there was no fluorescence enhancement for RMPTC-Hg2+ system. The reasonable
explanation is that the acidic medium(pH <4.0) led to the dissociation of the complex
and protonation of the ligand, and induced a decrease in emission intensity
accordingly. However, at higher pH values (pH > 8.0), the OH- ions and RMPTC
compete for Hg2+ ion. And Hg2+ ions might bind to the OH- and form the
corresponding hydroxide precipitation Hg(OH)2, which might decrease the actual
concentration of Hg2+ ions in the sample solution and therefore cause the drop of
fluorescence intensity[43]. Therefore, a best response toward Hg2+ ions could be
achieved at the pH 7.4. This result indicates that RMPTC can be used as a good
fluorescent probe for Hg2+ ions determination in nearly neutral conditions typical of
many biological system. Combined the results on the fluorescence and UV-vis
titration measurements, RMPTC can be applied to highly selectively and qualitatively
detect Hg2+ under physiological conditions, implying that we can further explore and
make full use of RMPTC for detection of Hg2+ in
samples.
3.9 Time-response studies
A short response time is necessary for the fluorescent sensor to detect metal ions

ACCEPTED MANUSCRIPT
in practical application. Therefore, the time dependence on the fluorescence intensity
of RMPTC to Hg2+ was investigated by recording the change of the fluorescence
intensity at 584nm after the addition of Hg2+ to RMPTC (50 μM) in DMF-H2O
(v/v=1/1, pH 7.4) buffer solution (Fig.9). As shown Fig. 9, the fluorescence intensity
of RMPTC-Hg2+ system at 584nm gradually increased and reached the maximum
over a period of 3 min and remained unchanged within 12h, which indicated that the
reaction between RMPTC and Hg2+ was completed within 3 min. So the right
response time is 3 min which can be used to do the relevant spectroscopic
experiments. It demonstrated that sensor RMPTC could be used for the real-time
analysis of Hg2+ in aqueous media.
3.10 Determination the binding stoichiometry of RMPTC and Hg2+ complex
It is reported that rhodamine B and triazoles based derivatives can form the
complexs with different metal ions by displaying special color and various complex
ratio [37, 39]. To investigate the binding stoichiometry between RMPTC and Hg2+ ion,
the method of equimolar continuous variations (Job’s plot) [44] were therefore carried
out by maintaining the total concentration of RMPTC and Hg2+ constant (50μM) and
changing the mole fraction of RMPTC from 0 to 1. The result indicated that the
fluorescence intensity at 584nm of the RMPTC-Hg2+ complex was achieved at a mole
fraction of approximately 66.7% of Hg2+ ions, indicating that a 1:2 stoichiometry was
the likely binding mode of complex RMPTC with Hg2+ ions (Fig. 5).
In order to confirm whether our experiment is correct or not, we decided to
calculate molecular orbitals of RMPTC-Hg2+ complex. Based on Gaussian 09
program package, a plausible structure of RMPTC-Hg2+ complex was generated from
the available calculated data to demonstrate the reasonableness of 1:2 stoichiometry
for RMPTC binding to Hg2+ ions. And the molecular structure of RMPTC-Hg2+
complex was fully optimized and its structural energy was calculated by using B3LYP
method in terms of LANL2TZ basis set for Hg and 6-311G(d) basis set for H, C, N, O
and F respectively. The corresponding results were shown in Fig. 5B and Table S3. In
Fig. 5B, RMPTC behaved as two pairs of tongs, one is the ligand using one oxygen
atoms from carbonyl group and one nitrogen atom from triazoles group to bind one
Hg2+ ion, another one is the ligand using one oxygen atoms and one nitrogen atom
from hydrazide group to bind another Hg2+ ion. The molecular docking diagram
showed that RMPTC can provide suitable space to accommodate two Hg2+ ions with
two penta cyclic groups formed. The calculated data in Table 1 indicated that the

ACCEPTED MANUSCRIPT
formation of RMPTC-Hg2+complex as 1:2 stoichiometry caused the energy difference
of ΔE(a.u.) and ΔE(ev), which were more small than that of as 1:1 stoichiometry. The
results illustrated that RMPTC binding with two Hg2+ ions was more stable than that
of one Hg2+ ion and confirmed theoretically the experimental result.
3.11 Proposed response mechanism of RMPTC to Hg2+
When designing florescence sensor systems (the host) for the recognition of
small molecules such as cations, anions, or amino acids (the guest), it is significant to
utilize specific chemical interactions or host-guest reactions that cause a change in the
luminescent emission properties of the system. If the specific interaction between the
host and the guest is noncovalent and reversible, and the interaction can be disrupted
under certain conditions, the indicator is referred to as a chemosensor[45-47]. A large
number of studies related to fluorescent chemosensors based on spiroring-opening
processes of rhodamine have been published[24,37,38,42]. If the binding interaction
between the host and the guest is based on an irreversible chemical reaction, the
indicator is described as a chemodosimeter. For chemodosimeters, it is necessary that
there are at least two functional units. One of two units is the reaction site, in which
the host binds to the detection object. And the second unit is responsible for a
spectroscopic signal that relies on the binding to the analyte. In principle, both the
interaction between the analyte and the chemodosimeter and the variation in
detectable signal are irreversible[45]. Generally, rhodamine derivative shows a red
color change and strong fluorescence in acidic solutions by activation of a carbonyl
group in a spirolactone or spirolactam moiety. In a similar way, an appropriate ligand
on a spirolactam ring can induce a color change as well as a fluorescence change upon
addition of metal ions, even though this process is somewhat dependent on the solvent
system [42].
In the present study, based on the above spectral analysis and molecular docking,
a plausible binding mode of RMPTC•Hg2+complex is depicted in Scheme 2. The
rhodamine derivative RMPTC is nonfluorescent and colorless, which exhibited the
typical closed ring effect for rhodamine B units due to the combination of MPTC. The
interaction of RMPTC with Hg2+ made rhodamine B ring opened with two
penta cyclic groups formed. The reaction site of chemodosimeter RMPTC, as the first
unit, was located in oxygen atom of carbonyl group and nitrogen atom of triazole
group of MPTC unit, which bound to one Hg2+ ion. The second unit providing the
spectroscopic signal was situated in nitrogen atoms and oxygen atom of rhodamine B

ACCEPTED MANUSCRIPT
hydrazide unit, which was responsible for interacting with another Hg2+ ion. This
binding mode has been confirmed by the fluorescence measurement observed and
structural computation as the induced conformational alterations of RMPTC upon 1:2
complexation with Hg2+. The complex of RMPTC with Hg2+ was verified by ESI–MS
testing (Fig.10). It can be seen that there is a strongest peak at m/z 996.6 , which
probably is assigned to [Hg+Na+2Cl+2CH3OH-4H] with less feasibility. However, a
peak at m/z 1081.6 has stronger intensity, which seems more reasonable and can be
assigned to [RMPTC+2Hg+ K-H]. The observed and calculated isotopic patterns
agree well with each other(Fig.10, inset). Combined the results on the spectral
experiment and computed calculation, the present ESI-MS detection confirmed the
formation of RMPTC-Hg2+ complex in 1:2 ratio again. Thus, the interaction of
RMPTC with Hg2+ could trigger the formation of the ring-opened form of RMPTC
from the spirolactam form. Moreover, RMPTC can serve as a “naked-eye”
chemodosimeter targeted toward Hg2+, similar to some reports on rhodamine-based
fluorescence probes for specific recognition of metal ions [48-50].
4. Conclusion
In summary, one new of the 1,2,3-triazoles, 5-methyl-2-phenyl-2H-1,2,3
-triazole-4-carboxylic acid (MPTC), was investigated in terms of structural,
fluorescence spectroscopic, UV-visible spectroscopic and theoretical analysis by
using HF/6-31G(d), CIS/6-31G(d) and B3LYP/6-31G(d) methods. The experimental
results are in line with the theoretical values derived from structural parameters,
providing guidance for utilizing MPTC in photophysical chemistry due to its special
optical property. Furthermore, a novel rhodamine-based colorimetric and turn-on
fluorescent chemodosimeter RMPTC was successfully developed and its sensing
ability for a wide range of metal ions was investigated. It selectively responds to Hg2+
by enhancement of the fluorescence and UV-vis absorbance intensity with 1:2
stoichiometry and also facilitates “naked-eye” detection of Hg2+. The results
demonstrate that RMPTC has a promising future for reliable and qualitative detection
of Hg2+ ions in aqueous solutions under physiological conditions. It is also anticipated
that the chemodosimeter RMPTC will significantly promote the studies on the effects
of Hg2+ ions in biological systems.
Acknowledgments
This work is financially supported by National Natural Science Foundation of
China (No. 21562019); Natural Science Foundation of Hainan Province (No.

ACCEPTED MANUSCRIPT
20162027); The Program of Hainan Association for Science and Technology Plans to
Youth R & D Innovation (HAST201621); Hainan province natural science foundation
of innovative research team project（2016CXTD007); The postgraduate Innovation
Research Project of Hainan Normal University(Hsyx2017-14).
Reference
[1] C. W. Tornøe, C. Christensen, M. Meldal, Peptidotriazoles on solid phase:ꢀ
[1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions
of terminal alkynes to Azi, J. Org. Chem. 67 (2002) 3057-3064.
[2] N. N. Fu, S. L. Wang, Y. Q. Zhang, C. X. Zhang, D. L. Yang, L. X. Weng, B. M.
Zhao, L. H. Wang, Efficient click chemistry towards fatty acids containing
1,2,3-triazole: Design and synthesis as potential antifungal drugs for Candida
albicans，Eur. J. Med. Chem. 136 ( 2017) 596-602.
[3] J. Totobenazara, A. J. Burke, New click-chemistry methods for 1, 2, 3-triazoles
synthesis: recent advances and applications, Tetrahedron Lett. 56 (2015)
2853-2859.
[4] A. C. García-Álvarez, E. Bernabé-Pablo, J. Barroso-Flores, V. Jancik, D.
Martínez-Otero, T. Morales-Juárez, M. Moya-Cabrera, Multinuclear rare-earth
metal complexes supported by chalcogen-based 1,2,3-triazole，Polyhedron 135
(2017) 10-16.
[5] G. Aromi, L.A. Barrios, O. Roubeau, P. Gamez, Triazoles and tetrazoles: prime
ligands to generate remarkable coordination materials, Coord. Chem. Rev. 255
(2011) 485-546.
[6] A. Maisonial, P. Serafin, M. Traıkia, E. Debiton, V. Thery, D. J. Aitken, P.
Lemoine, B. Viossat, A. Gautier, Click chelators for platinum-based anticancer
drugs, Eur. J. Inorg. Chem. 2 (2008) 298-305.
[7] N. T. N. Anh, A. D. Chowdhury, R. A. Doong, Highly sensitive and selective
detection of mercury ions using N, S-codoped graphene quantum dots and its
paper strip based sensing application in wastewater, Sensor. Actuat. B 252 (2017)
1169-1178
[8] S. Squadrone, A. Benedetto, P. Brizio, M. Prearo, M. Abete, Mercury and
selenium in European catfish (Silurus glanis) from Northern Italian Rivers: can
molar ratio be a predictive factor for mercury toxicity in a top predator?
Chemosphere 119 (2015) 24–30

ACCEPTED MANUSCRIPT
[9] J. Y. Chen, W. Su, E. J. Wang, Highly selective rationmetric fluorescent probe
for Hg2+ based on fluorescence Resonance Energy Transfer between
1,8-naphthalimide and rhodamine B, Chem. J. Chine. U. 37 (2016) 232-238.
[10] J. Ge, X. Geng, Y. H. Du, J. J. Chen, L. Zhang, D. M. Bai, D. Y. Ji, Y. L. Hu, Z.
H. Li, Highly sensitive fluorescence detection of mercury (II) ions based
onWS2nanosheets and T7 exonuclease assisted cyclic enzymaticamplification，
Sensor. Actuat. B 249 (2017) 189-194
[11] C. Y. Song, B. Y.Yang, Y. Zhu, Y. J.Yang, L. H. Wang ，Ultrasensitive sliver
nanorods array SERS sensor for mercury ions，Biosens. Bioelectron. 87(2017)
59-65.
[12] H. Yang, Z. G. Zhou, K. W. Huang, M. X. Yu, F. Y. Li, T. Yi, C. H. Huang,
Multisignaling optical-electrochemical sensor for Hg2+ based on a rhodamine
derivative with a ferrocene unit. Orga. Lett. 9 (2007) 4729-4732.
[13] Y. Chen, B. Chen, Y. F. Han, A novel rhodamine-based fluorescent probe for
the fluorogenic and chromogenic detection of Pd2+ ions and its application in
live-cell imaging, Sensor. Actuat. B 237 (2016) 1-7.
[14] Y. S. Jeong, J. Y. Yoon, Recent progress on fluorescent chemosensors for metal
ions, Inorg. Chim. Acta 381 (2012) 2-14.
[15] K. Wechakorna, K. Suksenc, P. Piyachaturawat, P. Kongsaeree, Rhodamine
-based fluorescent and colorimetric sensor for zinc and its application in
bioimaging, Sensor. Actuat. B 228 (2016) 270-277.
[16] S. Erdemir, S. Malkondu,A simple triazole-based “turn on” fluorescent sensor
for Al3+ ion in MeCN–H2O and F- ion in MeCN, J. Lumin. 158 (2015) 401-406.
[17] H. Wang, B. B. Chen, S. Q. Zhu, Chip-based magnetic solid-phase
microextraction online coupled with MicroHPLC–ICPMS for the
determination of mercury species in cells, Anal. Chem. 88 (2016) 796-802.
[18] N. Amiri, M. K. Rofouei, J. B. Ghasemi, Multivariate optimization,
preconcentration and determination of mercury ions with (1-(p-acetyl
phenyl)-3-(o-methyl benzoate)) triazene in aqueous samples using ICP-AES,
Anal. Methods-UK 8 (2016) 1111-1119.
[19] S. Lee , B. A. Rao, Y. A. Son, A highly selective fluorescent chemosensor for
Hg2+ based on a squaraine–bis(rhodamine-B) derivative: Part II, Sensor. Actuat.
B 210 (2015) 519-532.

ACCEPTED MANUSCRIPT
[20] S. Jadhav, E. Bakker, Selectivity behavior and multianalyte detection capability
of voltammetric ionophore-based plasticized polymeric membrane sensors,
Anal. Chem. 73 (2001) 80-90.
[21] Y. W. Wang, L. X. Wang, F. P. An, H. Xu, Z. J. Yin, S. R. Tang, H. H. Yang, H.
B. Song, Graphitic carbon nitride supported platinum nanocomposites for rapid
and sensitive colorimetric detection of mercury ions, Anal. Chim. Acta 980
(2017) 72-78.
[22] G.Liu, J.Shao, A C2-symmetric ratiometric fluorescence and colorimetric anion
sensor based on pyrrole derivative, J. Lumin. 131 (2011) 1397-1400.
[23] L. Y. Wu, S. Guo, X. H. Wang, Z. F. Guo, G. G. Yao, Q. Lin, M. S. Wu,
Tandem synthesis of 2-aryl-1,2,3-triazoles from α-arylhydrazonoketones with
NH4OAc via copper-catalyzed aerobic oxidation, Tetrahedron Lett. 56 (2015)
2145-2148.
[24] F. Ge, H. Ye, J. Z. Luo, S. Wang, Y. J. Sun , B. X. Zhao, J. Y. Miao, A new
fluorescent and colorimetric chemosensor for Cu(II) based on rhodamine
hydrazone and ferrocene unit, Sensor. Actuat. B 181 (2013) 215-220.
[25] X. X. Wang, X. L. Li, W. Shi, S. Wei, J. P. Giesy, H. X. Yu, Y. L. Wang.
Docking and CoMSIA studies on steroids and non-steroidal chemicals as
androgen receptor ligands, Ecotoxicol. Environ. Saf. 89 (2013) 143-149.
[26] Y. C. Chou, The low-lying singlet electronic excited states of ZrO2: A
symmetry adapted cluster-configuration interaction (SAC-CI) study, Comput.
Theor. Chem. 1069 (2015) 112-118.
[27] S. M. Xu, Y. Liu, H. Yang, K. Zhao, J. Li, A. Deng, Fluorescent nitrogen and
sulfurco-doped carbon dots from casein and their applications for
sensitivedetection of Hg2+ and biothiols and cellular imaging, Anal. Chim. Acta
964 (2017) 150-160.
[28] X. H. Li, H. L. Cui, R. Z. Zhang, X. Z. Zhang, Theoretical investigation on the
non-linear optical properties, vibrational spectroscopy and frontier molecular
orbital of (E)-2-cyano-3-(3-hydroxyphenyl)acrylamide molecule, Spectrochim.
Acta A 137 (2015) 321-327.
[29] N. Anusuya, P. Sounthari, J. Saranya, K. Paramswar, S. Chitra, Quantum
chemical study on the corrosion inhibition property of some heterocyclic azole
derivatives, Orient. J. Chem. 31 (2015) 1741-1750.

ACCEPTED MANUSCRIPT
[30] Ozlem Mıhçıokur, Talat Ozpozan,Molecular structure, vibrational spectroscopic
analysis (IR & Raman), HOMO-LUMO and NBO analysis of anti-cancer drug
sunitinib using DFT method, J. Mol. Struct. 1149 (2017) 27e41
[31]T. K. Kuruvilla , J. C. Prasana, S. Muthu, J. George, S. A. Mathew,Quantum
mechanical and spectroscopic (FT-IR, FT-Raman) study, NBO analysis,
HOMO-LUMO, first order hyperpolarizability and molecular docking study of
methyl[(3R)-3-(2-methylphenoxy)-3-phenylpropyl]amine by density functional
method, Spectrochim. Acta Part A 188 (2018) 382-393
[32] A.J. Rasool Chaudhry, R. Ahmed, A. Irfan, S. Muhammad, A. Shaari, A. G.
Al-Sehemi,Effect of heteroatoms substitution on electronic, photophysical and
charge transfer properties of naphtha [2,1-b:6,5-b0 ] difuran analogues by
density functional theory, Comput. Theor. Chem. 1045 (2014) 123-134.
[33] H. A. Kurtz, J. J. P. Stewart, K. M. Dieter, Calculation of the nonlinear optical
properties of molecules, J. Computat. Chem. 11 (1990) 82-87.
[34] M. H. Zhu, P. Hu, Instrumental analysis, 4th ed, Higher Education Press,
Beijing, 2008.
[35] J. G. Xu, Z. B.Wang, Fluorescence Analysis, 3rd ed, Science Press，Beijing,
2006.
[36] W. M. Yan,Q .Y. Wang, Q. Lin, M. Y. Li, J. L. Petersen, X. D. Shi,
N-2-Aryl-1,2,3-triazoles: a novel class of UV/Blue-light-emitting fluorophores
with tunable optical properties, Chem. Eur. J. 17(2011)5011-5018.
[37] V. Tekuri, D. R. Trivedi, A new colorimetric chemosensors for Cu2+ and Cd2+
ions detection: Application in environmental water samples and analytical
method validation, Anal. Chim. Acta 972 (2017) 81-93
[38] J. Zhang , C.W. Yu , S.Y. Qian , G. Lu, J. L. Chen, A selective fluorescent
chemosensor with 1, 2, 4-triazole as subunit for Cu (II)and its application in
imaging Cu (II) in living cells, Dyes Pigments 92 (2012) 1370-1375.
[39] K. Shaha, N. U. Aina, F. Ahmeda, I. Anis, M. R. Shah, A new highly selective
chemosensor for the detection of iron ion in aqueous medium based on click
generated triazole, Sensor. Actuat. B 249 (2017) 515-522
[40] C. Wu, Y. Ikejiri , J. L. Zhao, X. K. Jiang, X. L. Ni , X. Zeng, C. Redshaw, T.
Yamato, A pyrene-functionalized triazole-linked hexahomotrioxacalix [3] arene

ACCEPTED MANUSCRIPT
as a fluorescent chemosensor for Zn2+ ions, Sensor. Actuat. B 228 (2016)
480-485.
[41] W. Q. Chen, G. Y. Jin, Synthesis and biological activity of substituted
carbamates and hydrzines containing bicyclophosphate, Chin. J. Appl. Chem.
17 (2000) 479-482.
[42] X. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Fluorescent chemosensors
based on spiroring-opening of xanthenes and related derivatives, Chem. Rev.
112 (2012) 1910-1956.
[43] S. Erdemir, S. Malkondu, O. Alici, A highly selective and sensitive
benzothiazole-based‘turn-on’fluorescent sensor for Hg 2+ ion, Color. Technol.,
131, 32-37.
[44] E. J. Olson, P. Buhlmann, Getting more out of a job plot: determination of
reactant to product stoichiometry in cases of displacement reactions and n:n
complex formation, J. Org. Chem. 76 (2011) 8406-8412.
[45] Y. M. Yang, Q. Zhao, W. Feng, F. Y. Li, Luminescent chemodosimeters for
bioimaging, Chem. Rev. 113 (2013) 192-270.
[46] D. G. Cho, J. L. Sessler, Modern reaction-based indicator systems, Chem. Soc.
Rev. 38 (2009) 1647-1662.
[47] D. T. Quang, J. S. Kim, Fluoro- and chromogenic chemodosimeters for heavy
metal ion detection in solution and biospecimens, Chem. Rev. 110 (2010)
6280-6301.
[48] C. C. Jiang, M. Wang, Y. X. Wang, X. N. Tang, Y. M. Zhang, H. Zhang, L. Ma,
J. Y. Wang ， Synthesis and evaluation of two novel rhodamine-based
fluorescence probes for specific recognition of Fec+ ion, Tetrahedron Let. 58
(2017) 2560-2565.
[49] L. Lan, Q. Niu, Z. Guo, H. Liu, T. Li, Highly sensitive and fast responsive
turn-on fluorescent sensor for selectively sensing Fe3+ and Hg2+ in aqueous
mediabased on an oligothiophene derivative and its application in real water
samples, Sensor. Actuat. B 244 (2017) 500-508.
[50] S. Erdemir, S. Malkondu, O. Alici, A highly selective and sensitive
benzothiazole-based ‘turn-on’ fluorescent sensor for Hg2+ ion, Color. Technol.
131 (2015) 32-37.

ACCEPTED MANUSCRIPT
Figure captions:
Scheme 1 Synthesis route of RMPTC
Scheme 2 Proposed recognition mechanism of RMPTC towards Hg2+.
Fig.1 (A) The optimized structure of MPTC; (B1) Frontier Molecular Orbital of
ground state on LUMO；(B2) Frontier Molecular Orbital of ground state
on HOMO;
Fig. 2 (A) The electronic absorption spectra of MPTC predicted by TD-DFT
calculations. Inset: the absorption spectra of MPTC measured in methanol.
(B) The fluorescence emission spectra of MPTC predicted by TD-DFT
calculations. Inset: the fluorescence emission spectra of MPTC measured in
methanol.
Fig. 3. Fluorescence spectra (A) and fluorescence intensities (B) at 584nm of RMPTC
(10μM) in DMF-water (v/v=1/1, Tris-HCl, pH 7.4) in the presence of various
cations (40μM) (λex=563 nm). Inset: Photographs of the RMPTC in the
presence of Hg2+ and other metal ions.
Fig. 4 Fluorescence spectra (λex=563 nm) of RMPTC (10μM) upon the addition of
Hg2+ in DMF-water (v/v=1/1, Tris-HCl, pH 7.4). The illustration: graph of the
fluorescence intensity at 584 nm as a function of Hg2+ concentration.
Fig. 5 (A)The Job plot of RMPTC with Hg2+ ion using fluorescence.
（B）The optimized structure of RMPTC binding to two of Hg2+ ions.
Fig. 6A Absorption spectra of RMPTC (50μM) in the presence of different metal ions
(40μM) in DMF-water (v/v=1/1, Tris-HCl, pH 7.4)
Fig. 6B Absorption spectra of RMPTC (50μM) upon the addition of Hg2+ (0-50μM)
in DMF-water (v/v=1/1, Tris-HCl, pH 7.4).
Fig. 7 Fluorescent selectivity of RMPTC (50μM) toward Hg2+ over various
competitive metal ions (5.0μM) in DMF-water (v/v = 1/1, Tris-HCl, pH7.40).
The black bars represent the intensity of the solution upon the addition of
Hg2+(50μM).The red bars represent the intensity of RMPTC in the presence of
other metal ions(50μM) and Hg2+(50μM).
Fig. 8 Effect of pH on the fluorescence intensity of RMPTC (a) and RMPTC-Hg2+
complex (b).
Fig. 9 Time response of RMPTC to Hg2+ in DMF-water (v/v = 1/1, Tris-HCl, pH7.40 )
from 0 to 60min.
Fig. 10 (A) ESI-MS of RRMPTC in the presence of Hg2+ and trace amounts of Cl-.

ACCEPTED MANUSCRIPT
Inset: enlarged patterns for the [RMPTC+2Hg+ K-H ].
(B) The calculated ESI-MS of RMPTC in the presence of Hg2+ and Cl-.
Inset: calculated isotopic patterns for the [RMPTC+2Hg+ K-H ].

ACCEPTED MANUSCRIPT
Table 1 Comparison on energy changes of RMPTC binding to Hg2+ using the
B3LYP functional in Gaussian 09 .
Energy change
ERMPTC:Hg2+(a.u.
RMPTC:Hg2+
EREMPC(a.u.)
EHg2+(a.u.)
ΔE(a.u.)
ΔE(ev)
)
1:1
1:2
-2080.91
-2080.91
-41.79
-83.59
-2123.15
-2165.02
-0.44
-0.53
-12.11
-14.37

ACCEPTED MANUSCRIPT
Scheme 1 Synthesis route of RMPTC

ACCEPTED MANUSCRIPT
Scheme 2 Proposed recognition mechanism of RMPTC towards Hg2+.

ACCEPTED MANUSCRIPT
Fig.1 (A) The optimized structure of MPTC; (B1) Frontier Molecular Orbital of
ground state on LUMO; (B2) Frontier Molecular Orbital of ground state on
HOMO.

ACCEPTED MANUSCRIPT
30000
25000
20000
15000
10000
5000
0
0.8
A
277 nm
0.6
286 nm
0.4
0.2
0.0
200
250
300
350
400
Wavelength (nm)
150
200
250
300
350
400
450
Wavelength (nm)
40000
30000
20000
10000
0
500
400
300
B
346 nm
a
b
365 nm
200
100
0
200 250 300 350 400 450 500 550
Wavelength (nm)
200
300
400
500
600
700
Wavelength (nm)
Fig. 2 (A) The electronic absorption spectra of MPTC predicted by TD-DFT
calculations. Inset: the absorption spectra of EDTC measured in methanol.
(B) The fluorescence emission spectra of MPTC predicted by TD-DFT
calculations. Inset: the fluorescence emission spectra of EDTC measured in
methanol.

ACCEPTED MANUSCRIPT
4000
3500
3000
2500
2000
1500
1000
500
A
2+
Hg
3+
2+
2+
2+
None, Al , Ca , Cd , Co
,
2+ 3+ 2+ 2+
+
Cu , Fe , K , Mg , Mn
2+ 2+ 2+
,
+
2+
Mo , Na , Ni , Pb , Zn
0
575
600
625
650
675
700
725
750
Wavelength (nm)
Fig. 3. Fluorescence spectra (A) and fluorescence intensities (B) at 584nm of RMPTC
(10μM) in DMF-water (v/v=1/1, Tris-HCl, pH 7.4) in the presence of various cations
(40μM) (λex=563 nm). Inset: Photographs of the RMPTC in the presence of Hg2+ and
other metal ions.

ACCEPTED MANUSCRIPT
3000
2500
2000
1500
1000
500
3000
2500
2000
1500
1000
500
0
20 25 30 35 40 45 50 55 60
[Hg2+] (10-6 mol/L)
0
575 600 625 650 675 700 725 750
Wavelength (nm)
Fig. 4 Fluorescence spectra (λex=563 nm) of RMPTC (10μM) upon the addition of
Hg2+ in DMF-water (v/v=1/1, Tris-HCl, pH 7.4). The illustration: graph of the
fluorescence intensity at 584 nm as a function of Hg2+ concentration.

ACCEPTED MANUSCRIPT
6000
A
5000
4000
3000
2000
1000
0
0.0
0.2
0.4
0.6
2+
0.8
1.0
[RMPTC]/[RMPTC+Hg
]
Fig. 5 (A) The Job plot of RMPTC with Hg2+ ion using fluorescence.
（B）The optimized structure of RMPTC binding to two of Hg2+ ions.