The specific binding of a new 1,2,3-triazole to three blood proteins and it's appended rhodamine complex for selective detection of Hg2+

Article abstract of DOI:10.1016/j.saa.2019.117728

A synthesized compound, ethyl 2,5-diphenyl-2H-1,2,3-triazole-4- carboxylate (EDTC) was investigated on its physiochemical parameters and structural properties by using the quantum-chemical method. The results on the theoretical spectrum of EDTC were line with experimental fluorescence and absorption spectrum in large degree. Then EDTC was successfully synthesized to a novel rhodamine-based fluorescent probe (REDTC), which showed a distinct fluorescence enhancement upon the presence of Hg²⁺ in dimethyl formamide-water (DMF-H₂O) buffer solution (pH 7.4). Meanwhile, the triazole appended colorless compound turns to pink upon complex formation with Hg²⁺ ions as 1:2 molar ratios and enables naked-eye detection. The chromogenic mechanism was determined by using spectroscopic measurements and TD DFT calculation. The fluorescence imaging experiments of Hg²⁺ in HeLa cell revealed that the probe REDTC could be labeled and it could be used in biological systems. Also, the intermediate EDTC was developed as a sensitive fluorescent probe for specific studies on the interactions to three kinds of blood proteins including human serum albumin (HSA), human immunoglobulin (HIg) and bovine hemoglobin (BHb) by using a series of spectroscopic methods and molecular docking under the simulative physiological conditions. The interactions between EDTC and these proteins led to the distinct fluorescence enhancement. The thermodynamic measured results further suggested that hydrophobic forces play a dominating role in stabilizing the complexes, which are in correspondence with the results from molecular docking. The UV–visible, synchronous, and three-dimensional (3D) fluorescence measurements demonstrated that EDTC influences the conformational of proteins and the microenvironment surrounding HSA, HIg, or BHb in aqueous solution.

Full text of DOI:10.1016/j.saa.2019.117728

Journal Pre-proof
The specific binding of a new 1,2,3-triazole to three blood proteins and it's appended
2+
rhodamine complex for selective detection of Hg
Guohua Ding, Luyong Wu, Huajie Feng, Yanping Liu, Jianling Li, Hongzong Si,
Xiaojun Yao, Mengxiong He, Wenying He
PII:
S1386-1425(19)31118-7
https://doi.org/10.1016/j.saa.2019.117728
SAA 117728
DOI:
Reference:
To appear in: Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Received Date: 30 June 2019
Revised Date: 26 October 2019
Accepted Date: 28 October 2019
Please cite this article as: G. Ding, L. Wu, H. Feng, Y. Liu, J. Li, H. Si, X. Yao, M. He, W. He, The
specific binding of a new 1,2,3-triazole to three blood proteins and it's appended rhodamine complex
2+
for selective detection of Hg , Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
2019), doi: https://doi.org/10.1016/j.saa.2019.117728.
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The specific binding of a new 1,2,3-triazole to three blood proteins and it’s
2
+
appended rhodamine complex for selective detection of Hg
a
a
a
a
a
b
Guohua Ding , Luyong Wu , Huajie Feng , Yanping Liu , Jianling Li , Hongzong Si ,
c
a
a
Xiaojun Yao , Mengxiong He , Wenying He *
Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education,
Hainan Normal University, 571158 Haikou, China
a
b
c
Institute for Computational Science and Engineering, Qingdao University, 266071
Qingdao, China
College of Chemical and Chemical Engineering, Lanzhou University, 730000
Lanzhou, China
*Corresponding author at: Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal
University, Hainan Normal University, Haikou 571158, China.
E-mail address: hewenying@hainnu.edu.cn(WY He)

The specific binding of a new 1,2,3-triazole to three blood proteins and it’s
2
+
appended rhodamine complex for selective detection of Hg
a
a
a
a
a
b
Guohua Ding , Luyong Wu , Huajie Feng , Yanping Liu , Jianling Li , Hongzong Si ,
c
a
a
Xiaojun Yao , Mengxiong He , Wenying He *
a
Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education,
Hainan Normal University, 571158 Haikou, China
b
Institute for Computational Science and Engineering, Qingdao University, 266071
Qingdao, China
c
College of Chemical and Chemical Engineering, Lanzhou University, 730000
Lanzhou, China
ABSTRACT
A synthesized compound, ethyl 2,5-diphenyl-2H-1,2,3-triazole-4- carboxylate
(EDTC) was investigated on its physiochemical parameters and structural properties
by using the quantum-chemical method. The results on the theoretical spectrum of
EDTC were line with experimental fluorescence and absorption spectrum in large
degree. Then EDTC was successfully synthesized to a novel rhodamine-based
fluorescent probe (REDTC), which showed a distinct fluorescence enhancement upon
2
+
the presence of Hg in dimethyl formamide-water ( DMF-H O) buffer solution (pH
2
7
.4). Meanwhile, the triazole appended colorless compound turns to pink upon
2
+
complex formation with Hg ions as 1:2 molar ratios and enables naked-eye
detection. The chromogenic mechanism was determined by using
spectroscopic measurements and TD DFT calculation. The fluorescence imaging
*Corresponding author at: Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal University,
Hainan Normal University, Haikou 571158, China.
E-mail address: hewenying@hainnu.edu.cn(WY He)
1

2
+
experiments of Hg in HeLa cell revealed that the probe REDTC could be labeled
and it could be used in biological systems. Also, the intermediate EDTC was
developed as a sensitive fluorescent probe for specific studies on the interactions to
three kinds of blood proteins including human serum albumin (HSA), human
immunoglobulin (HIg) and bovine hemoglobin (BHb) by using a series of
spectroscopic methods and molecular docking under the simulative physiological
conditions. The interactions between EDTC and these proteins led to the distinct
fluorescence enhancement. The thermodynamic measured results further suggested
that hydrophobic forces play a dominating role in stabilizing the complexes, which
are in correspondence with the results from molecular docking. The UV-visible,
synchronous, and three-dimensional (3D) fluorescence measurements demonstrated
that EDTC influences the conformational of proteins and the microenvironment
surrounding HSA, HIg, or BHb in aqueous solution.
Keywords: ethyl 2,5-diphenyl-2H-1, 2, 3-triazole-4-carboxylate; structural properties;
2
+
Hg ions; binding; blood protein.
1
. Introduction
Since the representative research on the Cu(I)-catalyzed azide-alkyne
cycloaddition reaction for the synthesis of 1,2,3-triazole in 2002[1], this organic
reaction as the so-called click chemistry has been widely applied in various fields of
biological, chemical, macromlecualr materials and pharmaceutical sciences due to its
manifold chemical versatility and orthogonality[2]. 1,2,3-triazoles, a class of
five-membered nitrogen heterocycles manifest a privileged structural component in a
great variety of bioactive molecules, which have recently been well investigated on
account of their low toxic effects and several pharmacological properties[3]. They can
2

also act as ligands capable of binding to various metal ions. Therefore, it is very
important to prepare novel 1,2,3-triazole derivatives with various functional
substituents in order to discovery compounds possessing coordination ability to
different transition metals as well as new types of biological activity[4,5,6]. Among
the various probes of detecting metal ions, rhodamine-based fluorescent probes have a
time-honored role due to their excellent spectroscopic properties, such as long
absorption, large extinction coefficient and emission wavelength, high fluorescence
quantum yield, and high light stability[7]. Up to now, several fluorescence probes
based on triazole-rhodamine complexes have also been reported and can be applied to
detect specific metal ions [8-10].
It is well known that heavy metal pollution is widespread and be derived from
extensive natural and anthropogenic sources, which can threat to the humanity's life
even at low concentrations because of the accumulative and toxic properties. As one
2
+
of the most prevalent toxic heavy metals, mercury, or mercury ion (Hg ) is
considerably harmful to the environment and the human body. Excessive intake of
mercury accumulation will cause multitudinous chronic diseases such as headache,
deafness, visual impairment, central nervous system, and even irreversible damage of
the brain. Therefore, developing a highly selective and rapid method for detecting
mercury ions is still a vital need in order to solve the problem of increasingly urgent
mercury pollution in water and the environment. Above all, the colorimetric sensors
have drawn greater attention due to the advantages of fast response, signal visibility,
and application for on-site and high throughput measurement[11-12]. In present work,
ethyl 2,5-diphenyl-2H-1,2,3-triazole-4-carboxylate (EDTC, Scheme 1) was a new
compound[13] and introduced to rhodamine B to synthesize a novel derivative named
2
+
REDTC, which exhibited excellent selectivity toward Hg with chromogenic reaction
3

over other metal ions.
On the other hand, given the biological significance of the l, 2, 3-triazoles, recent
studies have revealed that 1, 2, 3-triazole moieties and related heterocyclic
compounds have been identified as bioactive molecules, and occupied a prominent
field in medicinal chemistry because of their therapeutic properties and favorable
metabolic stability. Thus, their drug-like properties have been developed and made
full use in medicinal chemistry[2]. It is a well-known face that proteins are frequently
the ‘targets’ for therapeutically active pharmaceuticals of both synthetic and natural
origin[14]. The blood proteins serve on the shipping object for small molecule drugs,
and the bonding interaction between drugs and protein has a great impact on the
distribution of these drugs in the body and the corresponding model of metabolism
and excretion. So it is an important research area on the binding properties of small
molecule drugs to plasma proteins.
Among of various target proteins, three kinds of blood proteins including human
serum albumin (HSA), human immunoglobulin (HIg) and bovine hemoglobin (BHb)
were usually applied to as plasma model proteins[15]. As one of the model blood
protein, HSA is the most abundant multifunctional protein and takes up large
proportion of the total protein content of plasma. It plays a vital role in transporting
and depositing several exogenous and endogenous substances including dyes,
hormones, fatty acids, and numerous drugs in the blood stream to the related target
organ [16]. The second model protein is HIg, which involved in different cell actions
and human immune response. It is usually used as the biomarker of numerous
diseases such as cancers, liver disease, Alzheimer's disease, inflammatory bowel
disease, infectious disease and autoimmune disease, etc [17]. As the third model
protein, hemoglobin (Hb) is also one of the major functional proteins in the vascular
4

system of animals. It belongs to an iron-containing metalloprotein and responsible for
the transportation of oxygen in blood red cells. The changes in the abnormally high or
low amount of Hb in human serum can cause some possible healthy problems, such as
anemia and polycythemia[18]. The above three kinds of model proteins are
widespread used in biochemical and biophysical researches on the bonding
interactions between proteins and small molecules [15, 17,18]. The various
spectroscopic techniques have become more valuable methods to study these
interactions at the molecular level in consideration of the advantages of rapidness,
sensitivity, convenience, and high simplicity[19]. However, many types of research
are based on the phenomenon of fluorescence quenching of proteins by small
molecular drugs and thus led to the second structure changes of these
biomacromolecules [20-21]. In the present paper, it is interesting that we reported a
new array on the interaction of EDTC with three blood proteins by the fluorescence
enhancement.
In short, we designed and synthesized a novel rhodamine B derivative REDTC
by using EDTC and rhodamine B, which shown a distinct chromogenic reaction with
2
+
Hg over than other metal ions originated the opening of the spironolactone ring.
Then the structural properties and physiochemical parameters of EDTC were studied
by quantum-chemical methods. Finally, based on the fluorescence enhancement
mechanism, as a special probe, EDTC was used to study the interaction to HSA, HIg
and BHb by fluorescence spectroscopy, UV/vis absorption spectroscopy and
molecular modeling methods. These results are the first spectroscopic application and
bioactivity results on EDTC or REDTC at the molecular level, which can illustrate the
nature of EDTC or REDTC information for further exploitation.
2
. Materials and Methods
5

2
.1. Materials and apparatus
EDTC was provided by Organic Chemistry Laboratory of Hainan Normal
−
3
University, China, and the stock solution (1.0×10 mol /L) was prepared in methanol.
Both of blood proteins, including HSA(10 g/50 mL) and HIg(2.5g/50 mL) were
purchased from Shanghai Bio Science & Technology Co. BHb(solid) was purchased
from Worthington Biochemical Corporation. All protein stock solutions were
-5
dissolved in 20 mM, Tris-HCl buffer pH 7.4, and the concentration is 3.0×10 mol /L.
3
+
2+
2+
2+
2+
Sodium hydroxide (NaOH) and chloride salts including Al , Pb , Mg , Mn , Hg ,
3
+
2+
2+
2+
2+
+
2+
2+
2+
+
+
Fe , Zn , Mo , Cd , Cu , Na , Ni , Co , Ca , K and Ag , and were acquired
from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All other reagents
were of analytical reagent grade and obtained commercially without further
purification. Doubly distilled water was used throughout the experiment.
NMR spectra were measured on a Bruker Avance 400MHz spectrometer at room
temperature. Fluorescent spectra were obtained by using an RF-5301PC spectrofluoro
photometer (Shimadzu, Japan) equipped with a xenon lamp source and a water bath.
UV-vis absorption spectra were recorded on
a
Hitachi U3900/3900H
spectrophotometer (Hitachi, Japan) equipped with 1.0 cm quartz cells. The 3D
fluorescence spectra were recorded on an F-7000 spectrofluorophotometer (Hitachi,
Japan). The pH measurement was performed on a PHS-3C pH meter (Shanghai,
China).
2
.2 Synthesis
As shown in Scheme S1, the first step is to synthesize rhodamine B hydrazide 1
according to the literature [22]. Rhodamine B (1.0 g, 2.1 mmol) was dissolved in 20
mL ethanol in a 50mL flask, 80% hydrazine hydrate (2 mL) was then added with
stirring at room temperature and the stirred mixture was heated to reflux for 3 h . Then
6

water (50 mL) added to the mixture was extracted with ethyl acetate (50 mL) 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, CDCl ):δ (ppm) = 1.16 (t, J = 6.8 Hz, 12H),3.34 (q, J = 7.2 Hz, 8H), 3.61 (s,
3
2
H), 6.30 (m, 2H), 6.45(m, 4H), 7.1 (m, J = 2.64 Hz, 1H), 7.44 (m, 2H), 7.94(m, 1H);
1
3
CNMR (400 MHz, CDCl ) δ (ppm) = 166.03, 153.75, 151.45, 148,78,132.39,
3
1
6
29.91, 128.00, 127.95, 123.71, 122.83, 107.94, 104.47, 97.89, 77.32, 77.00, 76.68,
5.82, 44.25, 12.50.
The second step is a synthesis of REDTC as follows: A mixture of EDTC (100
mg, 0.34 mmol), and potassium hydroxide (100 mg, 1.79 mmol) in 10 mL of a 1:1
mixture of ethanol: water was stirred for four hours, then heated to reflux for two
hours. After cooling to room temperature, hydrochloric acid was added dropwise with
stirring until the pH of the solution was below 1. The mixture was poured into water
and extracted with ethyl acetate. The organic phase was washed three times with
water and dried over Na SO . The solvent was removed by evaporation, affording a
2
4
solid of 2.3mL SOCl was added to the flask of compound 2, and the reaction mixture
2
was refluxed for three hours at 90℃ . Then the residuary SOCl was distilled out and
2
obtained the residue 3. The residue 3 was dissolved in 10 mL dichloromethane and
was mixed with intermediate 1 (150mg, 0.33mmol) and 1 mL triethylamine. The
mixture was stirred at room temperature for 12h then extracted with ethyl acetate.
After drying, the crude product was purified on a silica column using petroleum ether:
1
ethyl acetate (3:1, v/v) as the eluent to afford product REDTC (148.7 mg,61.98%). H
NMR (400 MHz, CDCl ):δ (ppm) = 1.36 (t, J = 7.2 Hz, 12H),3.34 (q, J = 7.2 Hz, 8H),
3
6
7
.37 (m, 3H), 6.77 (m, 2H), 7.32(m, 1H), 7.35 (m, 4H), 7.45 (m,2H), 7.53(m,2H),
1
3
.52 (m,2H), 7.84(m,3H), 7.87 (m, 1H); CNMR (100 MHz, CDCl3) δ(ppm) =
7

1
1
1
2
2
71.07, 164.97, 158.32, 153.82, 151.18, 149.44, 148.93, 138.96, 137.32, 133.15,
29.46, 129.34, 129.20, 129.04, 129.01, 128.78, 128.33,128.25, 128.02, 124.13,
23.51, 119.18, 107.99, 104.44, 97.59, 77.32, 77.00, 76.68, 66.32, 60.31, 44.31, 29.62,
+
0.96, 14.12, 12.53. HR-MS (ESI) calcd.for [M+H] 704.3349 , found 704.3348.
.3 Quantum chemical calculation of EDTC
The molecular structure of EDTC in the ground state was fully optimized by using
GaussView5.0 program package at Hartree-Fock Restricted algorithm level. The
Configuration Interaction with Single excitations (CIS) method was carried out to
fully optimize the configurations of the lowest excited state. The molecular orbital of
EDTC was analyzed by HF/6-31G (d) method. The theoretical absorption spectra and
fluorescence emission spectra of EDTC was calculated by using
the time-dependent density functional theory (TD DFT). Then the values of electron
affinity and ionization potential of EDTC were obtained by Gaussian 09 software at
the DFT//B3LYP/6-31G* algorithm level [23]. Also, based on Gaussian 09 program
package, B3LYP method was applied to fully optimize the molecular structure of
REDTC 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 [24].
2
.4 General fluorescence spectra and UV-vis measurements on REDTC
2
+
The solutions of REDTC and Hg of different concentrations were prepared by
stepwise dilution of a concentrated stock solution (0.5 mM for REDTC and 5 mM for
2
+
Hg ) in acetonitrile and water, respectively. The spectral analyses were carried out in
DMF-H O buffered (Tris-HCl, v/v, 2/8, pH 7.40) solution at room temperature. The
2
-5
concentration of REDTC for fluorescence and UV-vis measurement was 1×10 mol
L. The solutions of different metal ions were prepared with chloride salts in water
/
solution. Fluorescence and UV-vis spectrophotometric titrations were done by
8

successive addition of corresponding chemical reagent using a microliter syringe. To
2
+
evaluate the selectivity of REDTC for detection of Hg , a series of competitive metal
2
+
2+
+
+
+
2+
2+
2+
2+
3+
2+
2+
ions, including Pb , Mn , K , Na , Ag , Ca , Cd , Co , Cu , Fe , Zn , Ni ,
2
+
+
2+
Hg , Li and Mg , at a final concentration of 500 nM, were tested following the
identical experimental procedure.
2
.5 Cell culture and fluorescence imaging
HeLa cells were cultured on a 6-well plate with a density of 2 × 10 cells per well
3
in cell incubator at 37℃ with a humidified atmosphere for 12 h before staining. After
removing culture media, cells were additionally washed with PBS buffer three times.
Then HeLa cells were incubated with chemosensor REDTC(10µM) in the culture
2
+
media containing ethanol for 30 min at 37℃ . Experiments to assess Hg uptake were
2
+
performed in the same culture media supplemented with 25 µM Hg for 30 min.
Moreover, the fluorescence images were acquired through fluorescence microscopy.
2
.6 Analytical procedure of the interaction of EDTC with three proteins
Fluorescence measurements: The stock solutions of HSA, HIg, and BHb were
-7
diluted to 5 µM, 5µM, and 6×10 mol/L respectively. The intrinsic fluorescence
studies were done in a 1 cm path length cuvette. The HSA/HIg /BHb alone and in the
presence of EDTC samples were excited at 278 nm, and emissions were taken in the
range of 280 to 500 nm. The test solutions containing 0.05 mol /L Tris-HCl buffer
(pH 7.40) and HSA/HIg /BHb solution were titrated by successive additions of
-4
1
.0×10 mol /L EDTC, respectively. Moreover, the excitation and emission slit width
was set 5 nm for HSA and BHb while 10 nm for HIg. All fluorescence spectra were
measured at three temperatures (300 K, 310 K, and 320 K), which were kept by
recycling water throughout the experiment. The synchronous fluorescence spectra
were tested by a constant scanning interval ∆λ of 15nm and 60nm under the same
9

concentration condition at room temperature.
D fluorescence measurements: The stock concentration of EDTC, BHb, HSA
3
-4
-7
-7
and HIg were 1.0×10 mol /L, 5 µM, 2×10 mol /L, and 1×10 mol /L respectively.
The 3D emission spectra of three kinds of blood proteins in the absence and presence
of EDTC were recorded at the exiting wavelength ranging from 200 to 350 nm and
the emission wavelength ranging from 200 to 700 nm. The excitation and emission
slit widths were set 5 nm.
UV-vis absorption measurements: The UV-vis absorption spectra of free
-7
-7
-7
HSA(4×10 mol /L), HIg(1×10 mol /L) and BHb(2×10 mol /L) in the absence and
presence of increasing amount of EDTC were performed at the wavelength range of
2
00-600 nm in a pH 7.4 tris-HCl buffer solution at room temperature. The absorbance
titrations were performed by varying the concentrations of EDTC (0-11.67 µM for
HSA, 0-20 µM for HIg and 0-10 µM for BHb，respectively).
Molecular docking: The crystal structure of HSA (PDB ID: 5ID7), HIg (PDB ID:
5
DZN), and BHb (PDB ID: 3WHM) were obtained from the Protein Data Bank. The
three-dimensional structure of the EDTC molecule was constructed in SYBYL
software. The credible binding modes were computed by using SurflexDock program
in SYBYL 7.3 software as follows: The polar hydrogen atoms were added, and the
structure of water molecules were removed from the crystal structure. Then the
docking simulations were initiated by SYBYL software. Then a binding pocket was
established by introducing the Kollman-all atom charges to protein atoms, and EDTC
molecule was docked into the prepared protein. Finally, Surflex-Dock produced the
top 20 options of binding conformation for each ligand ranked by total scores [23].
3
. Results and discussion
1
0

3
.1 The structural and photochemical properties of EDTC
Generally, Frontier Molecular Orbitals (FMO's) including the highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are
both of the most important tools used in quantum chemistry. Based on FMO's theory,
the interaction between HOMO and LUMO levels of the reacting species results in
the formation of a transition state. Moreover, the higher the LUMO energy shows the
harder the acceptance of electrons from electron-donating substitutes [25]. Fig. S1 (A)
showed the calculated ground-state geometry optimization of EDTC by
GaussView5.0 software at the Hartree-Fock Restricted algorithm level, which
displayed that the structure of EDTC is nonplanar. The compound EDTC consists of
3
7 atoms, and the geometrical parameters of EDTC including bond length, bond angle
and dihedrals are listed in Table S1 (as shown in Supplemental Materials) by using
HF/6-31G(d), CIS/6-31G(d), and B3LYP/6-31G(d) basis sets respectively. From
Table S1, it can be seen that the calculated values of bond lengths and bond angles are
semblable at these three levels of calculations in great degree. The range of bond
length is between 1.07 Å and 2.39 Å, and that of the bond angle varied from 90.70 to
1
57.85°. However, the dihedrals seem somewhat different given using various
computing methods. It is noticeable, the C-C bond lengths in two benzene rings (Tag
C6-C17) are between 1.36 and 1.48 Å at three levels, revealing that bonds are longer
than the C=C double bond (1.34 Å), but much shorter than the typical C-C single
bond (1.54 Å) and some bonds are shorter than that of normal C-N bond (1.47 Å)[26].
These information further confirmed that the molecular structure of EDTC is no
planar.
Fig. S1 (B-1) and Fig. 1 (B-2) showed LUMO and HOMO of EDTC,
respectively. The calculated value of LUMO (-0.0675eV) is higher than that of
1
1

HOMO (-0.2402eV), which suggested that EDTC has weaker ability to accept
electrons from other electron donating group. Simultaneously, some physicochemical
parameters were also calculated by the diffuse functions B3LYP/6-311G+ (d) at the
DFT//B3LYP/6-31G* algorithm level such as adiabatic ionization potential
(
6.2688eV), vertical ionization potential (6.458 eV), vertical electron affifinity
2.0962eV) and adiabatic electron affinity (2.7231eV) respectively. These values
(
revealed that it is easy for EDTC to lose an electron or the electron accepting the
ability of EDTC is very weak [27].
Usually, the HOMO-LUMO gap is an important indicator related to the
luminescent properties [28]. The present computed parameters indicated that the
electron transition of EDTC focused primarily on HOMO→LUMO transition,
suggesting that EDTC had luminescent properties and confirmed by the
experimental spectra as followings. Fig. (1A) is the predicted electronic absorption
spectra of EDTC by time-dependent DFT (TD-DFT) calculations. The corresponding
UV-vis absorption spectra of EDTC in methanol were showed in the inset of Fig.
1
(A). We can see that both of the two graphs have the semblable profiles with double
peak, but there is some difference in the position of their maximum absorption peaks.
Nevertheless it is noted that the second biggest absorption peak is at 286 nm, which is
very close to that of the experimental measurement (283 nm). It is reported that the
absorption peak observed at about 280 nm was likely due to π-π* transition and the
formation of polyene π bonds by one triazole ring and two benzene rings of EDTC
[
29]. Fig. 1 (B) is the calculated fluorescence emission spectra of EDTC by TD DFT
and those experimented in methanol (illustration in Fig. 1(B)). The position of
the maximum fluorescence emission peak is located at 374 nm for the calculated
theoretical spectra and 350 nm for the corresponding measured spectra, respectively.
1
2

The reasonable excuse about the distinction is that the conjugated aromatic compound
EDTC caused π→π* transition from the ground state to the excited state. In general,
the polarity of the ground state is weaker than that of the excited state, and low polar
solvent can have more impact on stabilizing the ground state. So it led to the shift
the position of maximum fluorescence emission towards the longer wavelength side
[
30]. Herein, As a high polar molecule, methanol is used as a solvent of EDTC and
therefore resulted in the blue-shift effect with the maximum fluorescence emission
peak from 374 nm to 350 nm.
70000
60000
50000
40000
30000
20000
10000
0
0
.7
0.6
A
0.5
283 nm
0.4
0
.3
.2
.1
0
0
0.0
2
00
225
250
275
300
325
350
375
Wavelength(nm)
2
86 nm
150
200
250
300
350
Wavelength(nm)
1
3

40000
35000
30000
25000
20000
15000
10000
6
5
4
3
2
1
00
00
00
00
00
00
B
350 nm
374 nm
b
a
0
2
00
250
300
350
400
450
500
Wavelength(nm)
5000
0
200 250 300 350 400 450 500 550 600 650 700
Wavelength(nm)
Fig. 1 (A) The electronic absorption spectra of EDTC predicted by TD-DFT
calculations. Inset: the absorption spectra of EDTC measured in methanol.
(
B) The fluorescence emission spectra of EDTC predicted by TD-DFT
calculations. Inset: the fluorescence emission spectra of EDTC measured
in methanol.
Based on the above results on the UV-Vis absorption and fluorescence spectra, it
proclaimed that the computed results are consistent with the experiment results to a
great extent. Taking into account the characteristic structure of EDTC, it is inferred
that EDTC should have larger fluorescence quantum efficiency because of it is
fluorescent emission peak at 350 nm and is an expectant blue light emitting material
in the field of organic light-emitting diodes [31].
3
.2 Synthesis and spectroscopic characterization of REDTC
A number of highly selective chemosensors for the detection of various metal
2
+
2+
3+
2+
2+
ions (such as Cu , Cd , Fe , Zn and Hg etc) based on click produced triazole
have been applied and designed [32-35]. Theoretically, given the triazole unit
containing ester group, it is possible and feasible for EDTC to synthesize with
1
4

rhodamine B hydrazide[36]. So EDTC was explored and synthesized with the
rhodamine hydrazide to successfully produce a new rhodamine B derivative REDTC
by a serious of synthetic reaction (Scheme S1). REDTC were characterized by
1
13
HNMR(Fig. S2), C NMR(Fig. S3), and HRMS(Fig. S4) respectively.
The spectral signature of REDTC was investigated by UV-visible absorption and
fluorescence measurements in DMF/H O (1:1, v/v, Tris-HCl buffer，pH=7.4) buffer
2
solution (Fig. S5 and Fig. S6). As shown in Fig. S5, the maximum absorption
wavelength was found at 275nm, which attributed to the formation of π-π* transition
and polyene π bonds by one triazole ring and more benzene rings[29]. However, the
characteristic absorption band of rhodamine B at about 560 nm was not observed,
demonstrating its existence in spirolactam form [37]. Fig. S6 is the fluorescence
spectra of REDTC measured in the same solution, which showed that the positions of
the maximum excitation and emission wavelengths were at 262 nm and 354 nm
respectively. Compared to the fluorescence spectroscopy of EDTC(inset in Fig. 1B), it
seems that REDTC is a higher polar molecule with the shift of the maximum
fluorescence emission peak towards to shorter wavelength from 281 nm to 262 nm
[
30].
3
.3 Spectral characteristics of REDTC for metal cations
It is reported that rhodamine spironolactone or spirolactam compounds are
colorless and nonfluorescent, while the corresponding spironolactone/lactam with
ring-opening can generate distinct fluorescence emission and show a pink color. In
general, a suitable ligand on a spirolactam ring can bring about a color variation along
with a fluorescence change in the presence of some metal ions[38]. To get an insight
into the binding properties of REDTC toward diverse metal cations, the fluorescence
spectra of REDTC (10µM) in DMF-H O buffered (20 mM, pH 7.40, 1/1, v/v) solution
2
1
5

3
+
2+
2+
2+
2+
upon addition of a wide range of metal cations including Al , Pb , Mg , Mn , Hg ,
3
+
2+
2+
2+
2+
+
2+
2+
2+
+
+
Fe , Zn , Mo , Cd , Cu , Na , Ni , Co , Ca , K and Ag at the excitation 562
nm were recorded(Fig. 2A). In Fig. 2A, it showed that there was no obvious
characteristic emission observed for free REDTC. By comparison to various metal
2
+
ions, only Hg can induce a significant fluorescence enhancement observed at 577
+
nm accompanied with a color change from colorless to pink, while Ag could
simultaneously cause a very weak fluorescence enhancement.
6
5
4
3
2
1
00
00
00
00
00
00
0
A
2
+
Hg
3
+
2+
2+
2+
REDTC, Al , Ca , Cd , Co ,
2
+
3+ 2+ 2+
+
Cu , Fe , K , Mg , Mn ,
2+ 2+ 2+
2
+
+
Mo , Na , Ni , Pb , Zn
+
Ag
5
70 585 600 615 630 645 660 675 690
Wavelength (nm)
1
6

Fig. 2. (A) Fluorescence spectra (a) and fluorescence intensities (b) at 577nm of
-5
-1
REDTC (1×10 mol L ) in DMF-water (v/v=1/1, Tris-HCl, pH 7.4) in
-5
-1
the presence of various cations (5×10 mol L ) (
B) Relative fluorescence intensity of the REDTC in the presence of Hg
and other metal ions. Inset: Photographs of the REDTC in the presence
λex=562 nm).
2
+
(
2
+
of Hg and other metal ions.
2
+
The addition of Hg to REDTC resulted in a 180-fold increase in fluorescence
intensity at 577 nm (Fig. 2B). The inset in Fig. 2B showed photographs of the
2
+
3+
REDTC in the presence of Hg and other metal ions. Other metal ions, such as Al ,
2
+
2+
2+
2+
3+
2+
2+
2+
2+
+
2+
2+
+
Pb , Mg , Mn , Hg , Fe , Zn , Mo , Cd , Cu , Na , Ni , Co , K and
2
+
Ca ions, did not show any significant color and spectral variation under same
+
+
conditions except for Ag , suggesting that Ag has a slight capacity to induce the
spirolactam ring-opening of REDTC to some extent. However, it seems that REDTC
2
+
possibly generated a new compound with Hg and led to a ring opening from the
spirolactam (nonfluorescent) to ring-opened amide (fluorescent). The result indicated
2
+
that REDTC is a more sensitive response to Hg ion than other metal ions.
2
+
To further detect sensibility of REDTC on Hg ions, the fluorescence titration
1
7

2
+
experiments were carried out upon the gradual addition of Hg ions (Fig. 3A). As the
2
+
increasing concentration of Hg ions, it was observed a significant enhancement in
the fluorescence intensity of REDTC at 584 nm. However, with the addition of the
2
+
increasing amount of Hg ions, the fluorescence intensity of REDTC gradually
2
+
became saturated when the concentration of Hg reached 46.7
µ
M. The linear
2
+
fluorescence intensity vs the concentration of Hg was obtained in the range of
-5
-5
2
2
.80×10 -4.67×10 mol /L(R =0.9840, Fig. S7). Moreover, the limit of detection of
-5
-9
REDTC (1×10 mol/L) was determined to be 1.61 ×10 mol/L (3
σ/K). The detection
limit is equal to 3 /K, where is the standard deviation of the blank solution, K is the
σ
σ
slope of the curve of the fluorescence intensity versus the sample concentration [39].
However, a continuous decrease in the fluorescence intensity was observed for further
2
+
increments of Hg concentration. As a result, it seems that RMPTC is special probe in
2
+
qualitative detection on Hg under physiological conditions rather than an ideal
2
+
probe in the quantitative determination of Hg .
6
5
4
3
2
1
00
00
00
00
00
00
0
6
5
4
3
2
1
00
00
00
00
00
00
0
A
2
.5
3.0
3.5
4.0
4.5
5.0
5.5
2+
-5
-1
[Hg
]/10 mol L
575
600
625
650
675
700
Wavelength(nm)
1
8

8
7
6
5
4
3
2
1
00
00
00
00
00
00
00
00
0
B
0
.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
2
+
[REDTC]/[REDTC+Hg ]
Fig. 3 (A) Fluorescence spectra (λex=562 nm) of REDTC (1×10-5 mol /L) upon the
2
+
addition of Hg in DMF-water (v/v=1/1, Tris-HCl, pH 7.40). The
illustrationis: graph of the fluorescence intensity at 577 nm as a function of
2
+
Hg concentration.
(
B) The Job’s plot according to the method for continuous variations,
2
+
indicating the stoichiometry for REDTC-Hg (the total concentration of
2
+
REDTC and Hg is 50µM).
2
+
In order to obtain the coordination number of REDTC to Hg ions, the job’s
plot was performed by using the equimolar continuous variations method[40], and the
corresponding results were shown in Fig. 3B. It can be seen that the fluorescence
2
+
intensity at 577nm of the REDTC-Hg complex was achieved at a approximate mole
2
+
ratio of 66.7% of Hg ions, which means that a ratio of 1: 2 stoichiometry was the
2
+
probable binding mode of complex REDTC with Hg ions (Fig. 3B) and it confirmed
by TD DFT calculations later in this article on proposed mechanism.
The UV-vis spectra of REDTC (50
µM) in DMF-H O (v/v, 1/1, pH 7.40) buffer
2
solution was measured with the addition of different metal ions (Fig. S8). In Fig. S8,
1
9

comparing to the fluorescence spectra, it showed similar results, and the free REDTC
also exhibited no absorption near 563 nm. However, it is noted that there is an
2
+
obvious absorption band appeared at 563 nm in the presence of Hg , which was
ascribed to the ring-opened rhodamine moieties. Fig. S9 exhibited the spectral change
2
+
of REDTC upon the increasing Hg ions by the UV-vis titration measurements. It can
2
+
be seen that the absorption strength of REDTC-Hg ions system is gradually
2
+
increased at the position of 563 nm with the increasing Hg ions concentration from
-5
-5
2+
2
.00×10 to 4.33×10 mol/ L. However, with the continuous addition of Hg
ions, the emergence of platform (inset in Fig. S9) suggested saturation in the reaction
2
+
between REDTC and Hg ions.
3
.4 The competition experiments, the effect of pH and reaction time
2
+
To evaluate the selectivity of REDTC for Hg , the competition experiments
under the same conditions were examined in the presence of other metal cations
3
+
2+
2+
2+
2+
3+
2+
2+
2+
2+
+
2+
2+
including Al , Pb , Mg , Mn , Hg , Fe , Zn , Mo , Cd , Cu , Na , Ni , Co ,
+
2+
K and Ca ions (Fig. S10). It can be seen that the increasing fluorescence intensity
2
+
resulting from the addition of Hg ions did not change much in the coexistence of
other metal ions at the same concentration. The results showed that the presence of
2
+
cations does not remarkably interfere with the detection of Hg ; thus, REDTC could
2
+
be used as a highly selective Hg fluorescent probe.
In order to further study the influence of the different acid-base concentration
on the spectra of REDTC and found an appropriate pH span in which REDTC could
2
+
selectively detect Hg efficiently, the acid titration experiments were performed.
The effect of pH on the emission intensity was examined at a range from 3.0 to 10.0
-5
2+
-5
with a concentration of REDTC fixed at 1×10 mol /L and Hg at 5×10 mol/ L,
respectively (Fig. S11). It can be seen that REDTC showed evident fluorescence when
2
0

the value of pH is less than 4 whereas there was no distinct fluorescence observed for
REDTC within the limits of the pH range from 4.0 to 10.0. The result implied that the
REDTC compound is at the stable spirocyclic state in the pH range of the value of pH.
2
+
On the contrary the addition of Hg led to the fluorescence enhancement over a
comparatively wide pH range (4.0-8.0), which was attributed to the opening of the
2
+
rhodamine ring. Moreover, the optimal fluorescence response toward Hg was
obtained at the value of pH as 7.4. Consequently, probe REDTC might be used to
2
+
detect Hg in approximate physiological conditions and the spectral studies were
carried out in a DMF/H2O solution (1:1, v/v, tris-HCl, pH 7.4).
Besides high sensitivity and selectivity, quick response time is another necessary
factor for the detection in real-time. In order to study the response time of REDTC to
2
+
Hg , the changes of the fluorescence intensity at 577 nm on the binding process of
2
+
Hg ion to REDTC was recorded (shown in Fig. S12). It can be seen that the
fluorescence intensity of REDTC reached a stable value in less than 3 min and keeps
almost unchanged within 12h. So the appropriate response time is 3 min, which can
be applied to do the corresponding spectroscopic measurements.
2
+
3
.5 Proposed mechanism of REDTC response to Hg
2
+
Combining the above results, a plausible binding mode of REDTC•Hg complex
is depicted in Scheme 1. This binding mode is consistent with the fluorescence
changes observed as the induced conformational alterations of the triazole groups
upon 1:2 complexes, which was supported by the ESI-MS spectra and TD DFT
calculations. The peak at m/z 1103.2 in the mass spectrum was assignable to the mass
of[[REDTC+2Hg-2H ] (Fig. 4A(a)), and it was well matched with theoretical mass
spectral simulation (calc. 1103.3) (Fig. 4A(b)).
2
1

2
+
Scheme 1 Proposed recognition mechanism of REDTC towards Hg .
To further certify whether the above results are correct or not, the molecular
2
+
orbitals of REDTC-Hg complex was calculated by computational chemistry. The
2
+
rational structure of REDTC-Hg complex was obtained by using the Gaussian 09
program package, and the related results were listed in Table 1 and Fig. 4B. From Fig.
2
+
4
B, it can be seen that REDTC have two pairs of tongs to bind two Hg ions, one of
them is the bond by one oxygen atoms and one nitrogen atom from hydrazide group,
another one is the bond by another nitrogen atom from triazoles group and another
oxygen atoms from carbonyl group. The computer molecular diagram exhibited that
2
+
REDTC can provide appropriate place to locate two Hg ions with two penta cyclic
groups generated. The corresponding parameters in Table 1 showed that the values of
2
+
energy difference of
∆E(a.u.) and ∆E(ev) for REDTC-Hg complex as 1:2
stoichiometry were smaller than that of as 1:1 stoichiometry, which indicated that the
2
+
structure of 1:2 stoichiometry for REDTC binding to Hg ions is more stable than
that of 1:1 stoichiometries.
2
2

2
+
-
Fig. 4 (A) a: ESI-MS of REDTC in the presence of Hg and trace amounts of Cl .
Inset: enlarged patterns for the [REDTC+2Hg-2H ].
2
+
-
b: The calculated ESI-MS of REDTC in the presence of Hg and Cl .
Inset: calculated isotopic patterns for the [REDTC+2Hg-2H].
2
+
(
B) The optimized structure of REDTC-Hg
2
3

2
+
Table 1 Comparison on energy changes of REDTC binding to Hg using the
B3LYP functional in Gaussian 09 .
2
+
EEDTC-Hg (a.u.
2
+
EL1(a.u.)
EHg (a.u.)
)
E(a.u.)
E(eV)
EDTC-Hg -2272.68
-41.794
-2314.92
-2356.79
-0.44
-0.52
-12.12
-14.22
EDTC-2Hg -2272.68
-83.59
In principle, in order to design an ideal fluorescence sensor systems (the host)
for the detection of various analytes (the guest), it is important to develop specific
chemical reactions or host-guest interactions that bring about a variation in the
spectral properties of the reaction system. If the characteristic reaction between the
host and the guest is based on a reversible and noncovalent chemical reaction, and the
interaction can be affected under possible conditions, the indicator has named a
chemosensor. On the contrary, the binding mode between the guest and the host is an
irreversible chemical reaction; the indicator is acted as a chemodosimeter. For
chemodosimeters, it is crucial that there are two or more functional groups. One group
is the reaction site where the host interacts to the detection object. Moreover, another
one is used to offer a luminescent signal that is based on the interaction to the analytes.
Both the interaction between the chemodosimeter and analyte and the change in a
detectable signal are irreversible [41-42]. Herein, EDTC unit as the reaction site and
rhodamine B unit providing spectroscopic signal form the chemodosimeter for
2
+
2+
detecting Hg . So the binding of REPPC to Hg ions could cause the formation of
the ring-opened state of REPPC from the spirolactam state. Therefore, REDTC would
2
+
act as a “naked-eye” chemodosimeter probed toward Hg , same as to some studies on
rhodamine-based fluorescence sensors for characteristic detection of metal ions
[
41-43].
2
4

2
+
3
.6 Determination of Hg in HeLa cell.
In order to examine the practical properties of REDTC in biological samples,
2
+
the probe was applied for detecting Hg in HeLa cell by fluorescence microscopy. As
2
+
depicted in Fig. 5, a bright field transmission image of cells with Hg and REDTC
confirmed that the cells were viable throughout the imaging experiments, implying
2
+
the probe REDTC can penetrate the cell membrane and image Hg ions in HeLa cells,
with the switching-on fluorescent signal.
(
a)
(c)
(d)
(e)
(f)
(b)
2
+
Fig. 5 Fluorescence imaging of Hg in HeLa cell with REMPC.
(a, c, and e) Brightfield transmission images;
(b) images of cells incubated with the FBS culture solution as the sample
blank;
(
(
d) images of cells incubated with REMPC(10
f) images of cells incubated with REMPC(10
µ
M);
2
+
µ
M) and Hg (20µM).
2
+
3
.7 Compare to other triazoles on detection of Hg ions
Based on the above results, there are several similar aspects
2
+
+
compared to references on the triazole-based fluorescence probes for detecting Hg
ions[19, 44-45]. Most of these probes showed excellent selectivity detecting Hg
2
2
5

with colour variation from colourless transparent to pink. Moreover, they could
enhance the fluorescence signal in methanol or weakly neutral aqueous solution.
2
+
The stoichiometry of the complex formed between triazoles and Hg is 1:1 or 1: 2
ratio in most cases. But there are some differences as well as the following examples.
Some triazole-based probes caused diverse change in colour of the solution from
colourless transparent to blue-green[46-48], yellow[49-50], or purple[51]. But other
probes showed the fluorescence quenching or enhancement without any change in
colour[52-53]. Also, there are some differences in the sensitivity and detection limit.
2
+
The present work indicated that the probe REDTC showed a high selectivity for Hg
ions but a narrower linear range and a lower sensitivity. However,
the advantage of the probe REDTC is that it possessed a high selectivity for sensing
2
+
Hg ions in neutral aqueous solution (pH 7.4) with the triazole appended colorless
chemosensor turned to pink and enabled naked-eye detection, which further guides
2
+
us to explore simple and high-selectivity Hg probes in aqueous solutions under
physiological conditions.
3
.8 The interactions of EDTC binding three blood proteins
The present results illustrated that EDTC could synthesize with rhodamine B to
2
+
form a novel compound which can be used to detect the chromogenic reaction of Hg
ion. On the other hand, taking account of the reason that the 1,2,3-triazoles may be
further exploited and developed as new drugs, it is necessary to study the interactions
with some biological macromolecules. Since the functions of the drugs in the body are
the results of their interactions with crucial macromolecules in the organism. Protein
is also an essential research object in life science and one of the most vital transport
carriers of drugs. Thus, the investigates on small molecular drugs binding targeted
2
6

proteins have become more and more important subjects in the field of chemistry, life
science and clinical medicine, etc[54].
The selectivity of EDTC (1.67 µM) in tris buffer (pH = 7.4) was evaluated with
various amino acids (including Tyr, Lys, Glu, Val, Arg, Met, Cys, Leu, Asn, Pro, Thr,
Ala, and Gly, at 1.67 µM) and with three model blood proteins (including HSA, HIg
and BHb) (as shown in Fig. S13). From Fig. S13, it can be seen that the distinct
decreases were observed after the addition of BHb, while a significant enhancement
in fluorescence intensity at 349 nm and 330 nm was created upon the addition of HSA
or HIg under identical conditions. However, there was not an obvious variation of
fluorescence intensity after the addition of other amino acids. The results make clear
that EDTC exhibited excellent selectivity for HSA/HIg/ BHb in tris buffer (pH = 7.4).
Herein, the binding interactions of EDTC with HSA, HIg, and BHb were
evaluated by comparing the intrinsic fluorescence intensity of protein before and after
the addition of EDTC (Fig. 6A-6C)[55]. These figures showed that the fluorescence
intensity of EDTC-protein systems regularly enhanced with increasing concentration
of EDTC when excitation wavelength was set at 278 nm. Moreover, the various red
shifts of the position of maximum emission wavelength were obtained for all the three
proteins, which was 348nm to 356nm for HSA, 331nm to 356nm for HIg and 325nm
to 355nm for BHb
，respectively. Fig. 6D is the fluorescence emission spectra of
EDTC under the same experimental condition as three EDTC-protein systems. It also
showed that the fluorescence intensity of EDTC enhanced regularly with increasing
concentration of EDTC. It is worth noting that the inset in Fig. 6D can reveal some
significant information on the interactions of EDTC and these proteins. Take
EDTC-HSA system for example, when the excitation wavelength is set at 278 nm, the
value of fluorescence intensity of HSA is 101.76 at 347 nm, the corresponding values
2
7

of EDTC is at 365 nm ( 175.00), and EDTC-HSA is at 355nm (156.15), respectively.
It is obvious that there is an interaction between EDTC and HSA, which is similar to
EDTC-HIg or EDTC-BHb systems. Therefore, the results suggested that there were
strong interactions between the three kinds of proteins and EDTC which were
responsible for enhancing the fluorescence of the proteins.
0
0
0
0
.08
.07
.06
.05
1
1
1
1
1
80
60
40
20
00
A
0.04
i
0
0
0
.03
.02
.01
3
00K
310K
20K
3
0
.0
0.1
0.2
0.3
0.4
0.5
0.6
a
-
1
1
/[Q] µM
8
6
4
2
0
0
0
0
0
3
00
350
400
450
500
550
Wavelength (nm)
2
8