Benzo[e]indolium derivatives in aqueous solutions: Reaction with bisulfite and successive interaction with Cu2+ and Hg2+

Article abstract of DOI:10.1016/j.saa.2018.05.052

A new benzo[e]indolium derivative 1 including pyridyl and thienyl groups was synthesized and characterized, which failed to response to Hg²⁺ or Cu²⁺ in aqueous system. However, it is interesting that when it reacted with bisulfite in

Full text of DOI:10.1016/j.saa.2018.05.052

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 202 (2018) 324–332
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Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Benzo[e]indolium derivatives in aqueous solutions: Reaction with
bisulfite and successive interaction with Cu2+ and Hg2+
a
a
b,
Rongqing Zeng a, Qing Li a, Zhe Li a, Xianghong Li a, , Chaoyi Xie , Xianlong Su , Dingguo Tang
⁎
⁎
a
College of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan 430074, China
b
Key Laboratory of Catalysis, Materials Science of the State Ethnic Affairs Commission, Ministry of Education, South-Central University for Nationalities, Hubei, Wuhan 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new benzo[e]indolium derivative 1 including pyridyl and thienyl groups was synthesized and characterized,
which failed to response to Hg2+ or Cu2+ in aqueous system. However, it is interesting that when it reacted
with bisulfite in HEPES buffer, the in situ generated ensemble as 1-SO3H displayed dramatic absorption and fluo-
rescence changes after adding Cu2+ or Hg2+, which were contrary to the changes of 1 upon the addition of bisul-
fite ions. By contrast, the other two derivatives 2 and 3 showed the almost similar trends of spectral changes,
which were short of ligating atoms N or S. The further investigation of 1HNMR spectra changes of 1 showed
that C−SO3H bond may be interrupted because the good binding capacity of Hg2+ and Cu2+ with O of C
−SO3H weaken the binding force of C−SO3H, which resulted in the recovery of ethylene with benzo[e]indolium
block. The DFT calculations results further confirmed it.
Received 29 November 2017
Received in revised form 11 May 2018
Accepted 14 May 2018
Available online xxxx
Keywords:
Benzo[e]indolium
Pyridyl
Thienyl
Bisulfite
Absorption
Fluorescence
© 2018 Elsevier B.V. All rights reserved.
1. Introduction
enter the human body, bring damages to human organs including the
heart, brain, lungs, liver, and kidney [27,28]. An excessive intake of cop-
In recent years, optical molecular probes have been drawn much at-
tention because of their simplicity, sensitivity and virtues in real-time
observation and visualization. There have been many molecular probes
reported for anions [1–4], metal ions [4–6] and some small molecules
[4,7–9]. Benzoindolium derivatives, as one of the most important or-
ganic functional dyes, have been widely used in optical discs [10,11], bi-
ological analysis [12], organic solar cells [13] and nonlinear optical
materials [14] due to their good optical properties including wide spec-
tral spectrum and high extinction coefficient. Moreover, benzoindolium
derivatives also display potential applications in recognizing molecules
because of their water-solubility. Currently, they have been intensively
concerned in detecting HSO−3 /SO32− [2,15–21], HS− [22–24] and CN−
[15,24–26] for an increasing attention to the environment protection
and human healthy. The investigations of these benzoindolium were fo-
cused on the detection process including optical changes, detection
limits, response time and their nucleophilic addition reaction mecha-
nisms. It should be noted that most of these reported compounds
presented selective, sensitive, rapid, and a naked-eye response to
HSO−3 /SO23− over other physiologically anions and biothiols.
per leads to neurodegenerative diseases and oxidative stress probably
by its involvement in the procedure of reactive oxygen species [29].
Hence, detection and recognition of these metal ions have gathered
much attention owing to its importance [4–6,30–34]. However, most
of them only can be operated in organic solvents or organic-aqueous
system that organic solvent is predominant. Therefore, the design and
development of optical probes that do function well in water is signifi-
cant and necessary. Some efforts have been devoted to exploring
benzoindolium derivatives as metal ions probes [35,36]. The results
showed that benzoindolium derivatives have advantages over other
common dyes when they were used as probe in water. However, few
of them were reported to detecting mercury or copper ions due to
their strong affinity with oxygen atom [37,38].
Herein, benzo[e]indolium derivative 1 involving pyridyl and thienyl
group was synthesized to detect metal ions in aqueous system (see
Scheme 1). By contrast, the other two benzo[e]indolium derivatives 2
and 3 were also synthesized according to the routes depicted as
Scheme 1. As expected, all these three compounds failed to detect
metal ions in aqueous system such as CH3CN/H2O (1: 8, v/v) or
CH3CN/HEPES solution (pH 7.0, 1: 8, v/v), despite that 1 can provide S
coordination atom and a nitrogen coordination atom. Interestingly,
their in-situ generated systems with bisulfites (abbreviated as 1-SO3H,
2-SO3H and 3-SO3H) are all susceptible to Cu2+ and Hg2+ in CH3CN/
HEPES solutions and showed obvious spectral changes. It is notable
On the other hand, heavy metals such as mercury and copper have
caused serious environmental problems. Mercury ions, when they
⁎
Corresponding authors.
that the presence of some common metal ions (Li+, K+, Na+, Ca2+
,
E-mail addresses: lixhchem@mail.scuec.edu.cn, (X. Li), tdgpku@scuec.edu.cn (D. Tang).
https://doi.org/10.1016/j.saa.2018.05.052
1386-1425/© 2018 Elsevier B.V. All rights reserved.

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325
Scheme 1. Synthetic routes of compounds 1–3.
Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Ag+, Pb2+, Co2+, Ni2+, Al3+) did not in-
duce any significant spectra changes of these ensembles in comparison
with Cu2+ and Hg2+. As results, all these in-situ generated system of
compounds 1−3 with bisulfite can be used as probes to detect Cu2+
and Hg2+, and their sensitivity was not significantly affected by these
commonly coexistent metal ions. Based on the structures of compounds
1, 2 and 3, it can be found that the sensing unit mainly depended on the
-SO3H group. The 1HNMR spectral changes of 1 and DFT calculations of
three compounds verified that the presence of the -SO3H group that
probably donated electrons to metal ions Cu2+ or Hg2+ resulted in frac-
ture of C-SO3H and recovery of the benzo[e]indolium block with
ethylene.
dichloromethane (v/v, 1:1). After the mixture was stirred at room temper-
ature for 12 h, the silver(I) salt was removed by centrifugation. Then the
clear solution was collected and concentrated to afford an orange solid.
Yield: 0.43 g (68%).
2.2.2. Synthesis of 5-(2-Pyridyl)thiophene-2-aldehyde
In the solution of 2-bromopyridine (0.50 g, 3.16 mmol) in 40 mL of
1,4-dioxane, K2CO3 aqueous solution (15 mL, 3 M) and 10 mL of ethanol
were added. After the addition of 5-formylthiophene-2 boronic acid
(0.59 g, 3.79 mmol), PPh3 (0.067 g, 0.19 mmol) and Pd(PPh3)4 (0.22 g,
0.19 mmol), the resulting suspension was refluxed under N2 for 24 h.
Then, the mixture was cooled and extracted with dichloromethane.
The crude compound was purified by column chromatography on silica
gel by using petroleum ether/dichloromethane as eluent. Finally, a pale
yellow solid was obtained. Yield: 0.26 g (43%). 1H NMR (CDCl3,
400 MHz): 9.94 (s, 1H), 8.65 (d, J = 2.27 Hz, 1H), 7.79–7.75 (m, 3H),
7.70–7.69 (m, 1H), 7.29–7.28 (m, 1H).
2. Experimental Section
2.1. Materials and Physical Measurements
All solvents were commercially available and used without further
purification unless specified. 2,3,3-Trimethyl-1-ethylbenzo[e]indolium
iodide [15] and 4-(2-pyridyl)benzaldehyde [39] were prepared by the
methods described in the literatures and confirmed by 1H NMR spectra.
Herein, NMR spectra were recorded on Bruker Avance–400 and TCI
IIITM 600 MHz spectrometers at room temperature. Dimethyl sulfoxide
(DMSO d6) or CDCl3 was used as solvent with tetramethylsilane (TMS)
as an internal standard. Mass spectra were obtained on a G6520 Q-
TOF LC/MS spectrometer. UV–Vis absorption spectra were measured
using a Shimadzu UV-2550 spectrophotometer. All fluorescence spectra
were measured on a Hitachi F-7000 fluorescence spectrometer at room
temperature. The PMT voltage was 400 V, and the excitation and emis-
sion slit width were 10 and 10 nm, respectively. The excitation wave-
lengths used for compound 1, 2 and 3 were 380, 371 and 320 nm,
respectively. Quantum efficiency measurements in HEPES buffers
were carried out at room temperature with λex = 350 nm. The solution
of quinine sulfate in 0.1 mol dm−3 sulfuric acid (Φ = 0.58) was used as a
reference [40].
2.2.3. Synthesis of 1, 2, and 3
2.2.3.1. Synthesis of 1. Under nitrogen atmosphere, 10 mL of dry ethanol
solution containing 5-(2-pyridyl)thiophene-2-aldehyde (0.10 g,
0.94 mmol) and 2,3,3-trimethyl-1-ethylbenzo[e]indolium tetrafluoro-
borate (0.29 g, 0.90 mmol) was refluxed with stirring for 12 h. After
cooling, the precipitate was filtrated, collected and washed with petro-
leum ether/ethyl acetate (v/v, 3:1) to afford a red solid. Yield: 70 mg
(27%). 1H NMR (CD3CN, 400 MHz): 8.63 (d, J = 4.83 Hz, 1H), 8.59
(d, J = 15.98 Hz, 1H), 8.42 (d, J = 8.60 Hz, 1H), 8.24 (d, J =
8.86 Hz, 1H), 8.19 (d, J = 8.22 Hz, 1H), 8.00–7.97 (m, 2H), 7.93–7.91
(m, 3H), 7.83 (t, J = 7.05 Hz,1H), 7.76 (t, J = 7.16 Hz, 1H), 7.41–7.39
(m, 1H), 7.24 (d, J = 15.89 Hz, 1H), 4.68–4.62 (m, 2H), 2.22 (s, 6H),
1.62 (t, J = 7.37 Hz, 3H); 13C NMR (CD3CN, 100 MHz): 181.48, 154.30,
150.72, 149.92, 145.23, 141.34, 138.93, 138.86, 138.24, 137.39, 133.69,
131.42, 130.12, 128.45, 127.46, 127.23, 126.81, 124.11, 123.21,119.91,
112.49, 109.85, 99.86, 54.04, 42.66, 25.34, 13.19. MS (Maldi-TOF,
CHCA): m/z calculated for 409.17 (M-BF−4 ), Found: 409.18.
2.2. Synthesis and Characterizations
2.2.3.2. Synthesis of 2. According to the similar synthetic procedure for 1,
compound 2 was prepared by 4-(2-pyridyl)benzaldehyde. Herein, 2
was obtained as an orange-red solid. Yield: 1.05 g (70%). 1H NMR
(CDCl3, 400 MHz): 8.71–8.70 (m, 1H), 8.38 (d, J = 8.5 Hz, 2H),
2.2.1. Synthesis of 2,3,3-Trimethyl-1-ethylbenzo[e]indolium Tetrafluoroborate
2,3,3-Trimethyl-1-ethylbenzo[e]indolium iodide (0.71 g, 1.95 mmol)
and AgBF4 (0.39 g, 2.01 mmol) were dissolved in 20 mL of methanol/

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8.30–8.22 (m, 4H), 7.95 (d, J = 16.2 Hz, 1H), 7.87–7.70 (m, 2H),
7.71–7.68 (m, 1H), 7.62–7.59 (m, 3H), 7.32–7.28 (m, 2H), 5.11 (q, J =
7.3 Hz, 2H), 1.89 (s, 6H), 1.66 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz,
CD3CN): 182.90, 155.66, 152.92, 150.50, 144.13, 139.92, 138.70,
137.82, 135.27, 134.37, 132.02, 131.22, 130.67, 129.07, 128.22, 128.16,
127.99, 127.69, 124.02, 123.98, 123.86, 121.59, 113.23, 112.54, 54.94,
43.66, 25.71, 18.32, 13.89.
2.2.3.3. Synthesis of 3. According to the similar synthetic procedure for 1,
compound 3 was prepared by benzaldehyde and obtained as an orange-
yellow solid. Yield: 0.29 g (71%). 1H NMR(CD3CN, 400 MHz): 8.43 (t,
2H), 8.27 (d, J = 9.00 Hz, 1H), 8.20 (d, J = 8.18 Hz,1H), 8.09 (d, J =
7.45 Hz, 2H), 7.94 (d, J = 8.97 Hz, 1H), 7.85 (t, J = 7.19 Hz, 1H),7.77
(t, J = 7.78 Hz, 1H), 7.69–7.61 (m, 3H), 7.54 (d, J = 16.51 Hz, 1H),
4.73 (q, J = 7.45 Hz, 2H), 2.07 (s, 6H), 1.64 (t, J = 7.34 Hz, 3H). 13C
NMR (150 MHz, CD3CN): 183.12, 153.68, 139.87, 138.68, 134.83,
134.37, 133.96, 132.00, 130.70, 130.66, 129.97, 129.06, 128.21, 127.69,
123.86, 113.25, 112.35, 54.96, 43.66, 25.67, 18.31, 13.85.
Fig. 1. The absorption spectra of compounds 1, 2 and 3 in CH3CN/H2O (v/v, 1:8) at room
temperature, respectively.
2.3. Preparation of Solutions
The stock solutions (2.0 mM) of 1, 2 and 3 were prepared in CH3CN,
and then were diluted with HEPES buffers (10 mM, pH 7.0) to afford so-
lutions (10 μM) with acetonitrile and buffer at volume ratio of 1:8, re-
spectively. The adducts of the reactions between these three benzo[e]
indolium and bisulfite were abbreviated as 1-SO3H, 2-SO3H and 3-
SO3H, which were in-situ obtained by adding 2.5, 1.5 and 1.5 equiva-
lents of HSO−3 in CH3CN/HEPES solutions of 1, 2 and 3, respectively.
Herein, the equivalents of HSO−3 were determined by the titration ex-
depicted in Fig. S1−S6 (Supporting information). It should be noted
that all these compounds displayed good absorptions between
350–500 nm. As shown in Fig. 1, the maximum absorption peaks of 3,
2 and 1 are apparently red-shift with the increasing molar extinction co-
efficients, which appeared at 420, 432 and 476 nm, respectively. The re-
sults should be attributed to the increasing length of the conjugated
structures.
As anticipated, only the addition of Hg2+ into the CH3CN solution of
1 caused a minor blue-shift in absorption (see Fig. 2), and the compound
2 and 3 exhibited no response to Hg2+ in CH3CN. The results are in good
agreements with the chalcogenophilicity of mercury [44]. However,
among some common metal ions including Hg2+, all these three com-
pounds including compound 1 displayed no obvious absorption
changes between 400–500 nm in CH3CN/H2O (1:8, v/v) or CH3CN/
HEPES solution (pH 7.0, v/v, 1:8) for the solvation of water molecules
with metal ions (Fig. S7). It should be noted that compounds 2 and 3 ex-
hibited very weak emissions, respectively. And the emission of 1
can hardly be observed. As shown in Fig. S8, the additions of Hg2+ and
Cu2+ to the aqueous solutions of 2 and 3 resulted in negligible emission
changes (Fig. S8).
periments. The aqueous solutions of cation (Li+, K+, Na+, Ca2+, Mg2+
,
)
Ba2+, Zn2+, Cd2+, Mn2+, Cu2+, Hg2+, Ag+, Pb2+, Co2+, Ni2+, Al3+
were prepared with their corresponding chlorides and nitrates, respec-
tively. And HEPES buffers at the range of pH 2.49–10.02 were obtained
by adjusting the concentration ratio of HEPES and NaOH or HCl, which
were used to perform the pH dependent experiments.
2.4. Detection Limits
The detection limit [41] was calculated based on the absorption or
emission titration. To determine the S/N ratio, the absorption or emis-
sion intensity of the probe solution was measured by 10 times. Then
the standard deviation of the blank measurements was determined.
The detection limit was calculated with the equation: detection limit
= 3S0/S, where 3 is the factor at the 99% confidence level, S0 is the stan-
dard deviation of the blank measurements, and S is the slope of the cal-
ibration curve for absorption or emission changes versus metal ions or
bisulfite ions concentration.
2.5. Theoretical Calculations
All the calculations were carried out using hybrid density functional
theory (DFT-B3LYP) method coupled with 6-31G(d) basis set as imple-
mented in Gaussian 09 program of package [42]. Time-dependent den-
sity functional theory (TDDFT) calculations were performed to evaluate
the electronic excitation energies of the three benzo[e]indolium com-
pounds and their complexes with HEPES, HSO−3 and Cu2+ considering
the PCM solvent effect of water. The UV–Vis absorption spectra were
convolved by using GaussSum program [43].
3. Results and Discussion
3.1. The Interactions of Compounds 1, 2 and 3 With Metal Ions
Considering that metal ions could combine with I−, herein, the com-
mon counter anions I− in the benzo[e]indolium was replaced by BF4−
(see Scheme 1). The 1HNMR and 13CNMR spectra of 1, 2 and 3 were
Fig. 2. Absorption changes of 1 (10 μM) in the presence of Hg2+ in CH3CN solution at room
temperature.

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3.2. The Reactions Between 1–3 and HSO3−
absorption intensities decreased, which were in consistent with the ti-
tration experiments results (Fig. 3, S9 and S10). The subsequent addi-
tions of Hg2+ or Cu2+ into the in situ generated ensembles of
compounds 1, 2, and 3 with bisulfite made their absorption intensity
change at different pH range. As results, all these ensembles displayed
best response in the physiological pH region. Therefore, HEPES buffer
(pH 7.00) was suitable and chosen as the sensing system.
Fig. 3 describes the UV–Vis absorption spectral changes of 1 (10 μM)
with different concentrations of HSO−3 in the CH3CN/HEPES solution.
With the increasing amounts of HSO−3 , the absorption intensities of 1
are decreasing gradually between 360–600 nm. Meanwhile, the intensi-
ties of absorption bands centered at 318 and 262 nm are rising. In com-
parison with 1, compounds 2 and 3 displayed gradual decreasing
absorption intensities between 350 and 550 nm (see Fig. S9 and S10),
which are in agreements with the previous reports [15,17]. As results,
all these spectra changes were along with the solutions colors fading
(see Fig. S11), which could be attributed to that the π-conjugation was
interrupted by the 1,4-addition reactions between these compounds
and HSO−3 [15–21]. Apparently, these color changes can be sensitively
observed by the naked eyes. On the other hand, through the absorption
titration curves of these three compounds with bisulfite, it can be seen
that the equilibrium amounts of bisulfite with these compounds were
about 2.5, 1.5 and 1.5 equivalents (Fig. 3, S9 and S10, inset a),
respectively.
Generally speaking, the benzo[e]indolium derivatives involving con-
jugated structures displayed very weak emissions. However, after
adding enough amounts of HSO−3 , the intensity of the maximum emis-
sion peak of 1 increased at 465 nm (Fig. 3, inset b), while 2 displayed
an increasing emission at 466 nm along with the decreasing emission
at 558 nm (Fig. S9, inset b). And 3 showed an increasing emission at
470 nm (Fig. S10, inset b). These almost similarly increasing emission
peaks indicated that their conjugated structures were destroyed after
adding HSO−3 , and the fluorescence of 2,3,3-trimethylbenzo[e]indole
was recovered [15–21].
3.3. The Interactions Between Metal Ions and 1-SO3H, 2-SO3H and 3-SO3H
According to the results of titration experiments (Fig. 3, S9 and S10),
the compounds 1-SO3H, 2-SO3H and 3-SO3H were prepared in situ by
the additions of appropriate amounts of bisulfite ions into solutions of
these three compounds, respectively. Firstly, in consideration of possi-
ble hydrolysis of common metal ions including Hg2+ and Cu2+, explor-
ing the effect of various pH values on these ensembles was
indispensable. So we focused on the influence of Hg2+ and Cu2+ on ab-
sorption changes of these ensembles at varying pH (2.49–10.02) values.
As shown in Fig. 4, these three compounds 1, 2 and 3 are stable in a pH
range of 2.49–10.02. After mixing with bisulfite, the corresponding
Fig. 3. Absorption changes of 1 (10 μM) with increasing amounts of bisulfite at room
temperature. Inset: a) titration curve of 1 with HSO−3 ; b) The fluorescent spectra before
and after the addition of bisulfite at room temperature (λex = 380 nm, slit: 10/10 nm).
Fig. 4. Absorption intensity changes of 1 (10 μM), 2 (10 μM) and 3 (10 μM) in the absence
or presence of appropriate amounts of HSO−3 , Cu2+ or Hg2+ under different pH conditions
at room temperature, respectively.

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Then, the interactions of some common metal ions on these in situ
absorption intensity increased at 366 and 420 nm, and the solution
color changed from colorless to pale yellow (Fig. S11, c). As result, for
the compounds 1, 2 and 3, the reactions with bisulfite led to decreasing
absorption intensities between 350–600 nm. Nevertheless, the subse-
quent additions of Hg2 + and Cu2 + showed almost inverse absorption
changes. It is should be noted that the results were quite different
from the direct additions of metal ions into CH3CN/H2O or CH3CN/
HEPES solutions of these compounds, which were further confirmed
by the fluorescence spectra changes of these bisulfite adducts. It is men-
tioned above that in the range of 350–700 nm, the interactions of metal
ions on fluorescence of compounds 1−3 in buffer solutions could be
negligible (Fig. S8). However, when they reacted with HSO−3 , the arising
generated systems were studied by UV–visible absorption spectra. As
shown in Fig. S11, these common metal ions have no obvious effects
on absorption spectra of the bisulfite adducts of 1, 2, and 3 except that
Hg2+ and Cu2+ can cause dramatic changes. It can be seen that after
adding Hg2+ and Cu2+ into the buffer solution of 1-SO3H, the absorp-
tion band centered at 476 nm arose, resulting in the solution color
changing from colorless to orange (Fig. S11, a). Moreover, after adding
Hg2+ and Cu2+ into the solution of 2-SO3H, the intensities of the ab-
sorption bands centered at 384 and 432 nm are also increasing, along
with the solution color changing from colorless to yellow (Fig. S11, b).
Upon the addition of Hg2 + and Cu2 + into the solution of 3-SO3H, the
Fig. 5. Absorption responses of 1-SO3H (10 μM), 2-SO3H (10 μM) and 3-SO3H (10 μM) upon addition of Cu2+ (a, c, e) and Hg2+ (b, d, f) in CH3CN/HEPES buffer, respectively.

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329
fluorescence centered at about 470 nm was then greatly quenched by
Hg2+ and Cu2+ (see Fig. S12). It can be found that these fluorescence
data matched the absorption changes well.
indicated that the sensitivity of these ensembles to Hg2+/Cu2+ is hardly
affected by these commonly coexistent ions. Therefore, these ensembles
of compounds 1−3 with bisulfite can act as a selective and sensitive
probe for Hg2+ and Cu2+ in the natural environments.
To better understand these changes, the interactions of the two
metal ions (Hg2+/Cu2+) on the in situ generated systems of 1, 2 and 3
with bisulfite were monitored by the absorption spectra and fluores-
cence spectra in details. As shown in Fig. 5, with the increasing amounts
Subsequently, Hg2+/Cu2+ was added to the solutions of these en-
sembles in the presence of metal ions including Li+, K+, Na+, Ca2+
Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Cu2+, Hg2+, Ag+, Pb2+, Co2+, Ni2+
,
,
Al3+. As shown in Fig. S13, prominent absorption and fluorescence
changes could be observed only after adding Hg2+/Cu2+. The results
Fig. 6. The fluorescence response of 1-SO3H, 2-SO3H and 3-SO3H (10 μM) upon addition of Cu2+ (a, c, e) and Hg2+ (b, d, f) in CH3CN/HEPES buffer at room temperature (The excitation
wavelengths used for compound 1, 2 and 3 were 380, 371 and 320 nm, respectively. Slit: 10/10 nm).

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Fig. 7. The 1HNMR spectra of 1 in DMSO-d6/D2O (5:1, v/v) at room temperature.
of Cu2+ and Hg2+, the absorption intensities of 1-SO3H are increasing
gradually at 476 nm and the intensities of absorption bands centered
at 318 and 262 nm decreased steadily, respectively. This changeable
tendency in absorption spectra was just the opposite the interaction
between 1 and HSO−3 (Fig. 5). For 1-SO3H, when the interactions with
Cu2+ and Hg2+ reached at equilibrium, the absorption intensity at
476 nm could be restored to 94% and 96% of 1, respectively. It is obvious
that compound 1 could provide much more coordination atoms than
compounds 2 and 3. However, upon the addition of Cu2+ and Hg2+, al-
most similar changed trends in absorptions were observed for 2-SO3H
and 3-SO3H, respectively. For 2-SO3H, the intensities of absorption
band centered at 432 and 384 nm were increasing, which were inverse
to the absorption changes after the addition of bisulfite to the solution
of 2. When reaching to the balance, Cu2+ and Hg2+ could lead to the ab-
sorption intensities at 432 nm reaching at 85% and 80% of 2, respec-
tively. Moreover, the absorption intensities of 3-SO3H increased
gradually at 420 and 366 nm. Finally, the absorption spectrum of 3-
SO3H with Cu2+ was almost coincident with that of 3 between 350
and 700 nm. The absorption changes between 3-SO3H and Hg2+ was
similar to that of Cu2+ in the range of 350 to 700 nm. However, the in-
tensity of the absorption at 420 nm can only be restored to 74% of 3. By
contrast, it can be seen that the recovery percentage of the absorption
intensity caused by Cu2+ is higher than that caused by Hg2+ as to 2-
SO3H and 3-SO3H, due to the stronger thermodynamic affinity with O
for Cu2+ [45]. However, for 1-SO3H, the little higher recovery percent-
age of the absorption intensity was observed for Hg2+, which can be at-
tributed to the involvement of the sulfur atom and is in agreement with
the HSAB theory [44].
in HEPES buffer. As results, the bond of C−SO3H was broken to make
a regeneration of the conjugation property of the vinyl with benzo[e]
indolium group.
To illustrate the interactions of these compounds with metal ions,
the interactions of Hg2+ on 1 and 1-SO3H were selected and studied
by 1H NMR spectra. As shown in Fig. 7, all of the 1H NMR signals of 1
shifted to up-field after HSO−3 was added into its solution. For instance,
the proton signal of the two methyl hydrogen at δ 1.99 ppm was shifted
up-field and divided into two single signals, which appeared at δ 1.84
and 1.58 ppm, respectively. It could be attributed to the formation of
nonequivalent hydrogen after formation of 1-SO3H. Moreover, the pro-
ton signal of CH2 connected with N+CH2CH3 was also upshifted from
4.72 to 3.68 ppm. And the characteristic proton linked to unsaturated
carbon atoms (δ 7.46–8.27 ppm) appeared at 5.21 ppm after the
HSO−3 was added. These evident up-field shifts can be attributed to
the nucleophilic attack of HSO−3 toward C-4 with positively charged in-
dole groups, which disturbed the π-conjugation and weakened its elec-
tron withdrawing character. Obviously, the absorption and fluorescence
changes of these three compounds in the presence of bisulfite ions were
in good agreements with the 1HNMR data. It should be mentioned that
the peaks at δ 5.21 and 3.68 ppm that attributed to characteristic signals
of 1-SO3H disappeared when excessive Hg2+ was added subsequently
into the solution of 1-SO3H, along with enhanced peaks occurring at δ
The fluorescent spectral changes of Hg2+ and Cu2+ on these in situ
generated systems were further investigated in details. As shown in
Fig. 6, with the increasing amounts of Hg2+ and Cu2+, the fluorescent
intensities gradually decreased at 465 nm, 466 and 470 nm for 1-
SO3H, 2-SO3H and 3-SO3H, respectively. All these fluorescent spectral
changes were converse to those changes which caused by the addition
of bisulfite to the solution of 1, 2 and 3, respectively. However, when
the reactions reached at equilibrium, the different fluorescent spectra
from those of 1, 2 and 3 were observed, respectively (see Fig. 6 inset).
Clearly, these spectra are different from the direct interactions between
these compounds with Cu2+ and Hg2+, which may be caused by the
presence of bisulfite ions in these mixed solutions. Taking the absorp-
tion spectra changes into consideration, it could be deduced that the
presence of Cu2+ and Hg2+ play important roles on C−SO3H groups
of these ensembles, which provided coordinating O atoms to chelate
with Hg2+ or Cu2+ and thus weakened the binding force of C−SO3H
Fig. 8. The energy levels of the highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO) of 1, 2, 3 with the HEPES, HSO−3 , and Cu2+ ions.

R. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 202 (2018) 324–332
331
4.68 and 1.96 ppm again. Obviously, the disappearance of signals at 5.21
and 3.68 ppm displayed that the fracture of C−SO3H may occur. On the
other hand, it was found that all of the hydrogen signals of 1 were also
shifted to the up-field after adding Hg2+. For example, the signal at δ
4.72 shifted to 4.69 ppm, and the signal at δ 1.99 shifted to 1.96 ppm.
These results indicated that the N and S atoms were involved in the co-
ordination with Hg2+, which caused the electron density decreasing
and led to upshifts of proton signals. It is noticeable that 1-SO3H with
Hg2+ and 1 + Hg2+ had almost similar signals at δ 1.96 and
4.69 ppm. Therefore, the interaction between 1-SO3H with Hg2+ could
be concluded that the coordination of Hg2+ with –SO3H resulted in
the fracture of C–SO3H to afford compound 1 in DMSO-predominated
solution, which further coordinated with N and S atoms.
In order to understand the interaction mechanisms of the
benzoindolium derivatives with HSO−3 and further reaction with Cu2+
/
Hg2+, we performed theoretical calculations to evaluate the binding
interactions between them. Because the experiments were carried out
in the HEPES buffer, the effects of HEPES anion are also taken into
account. The optimized geometries of 1-HEPES, 1-HEPES-HSO−3 , and
1-HEPES-HSO−3 -Cu2+ as well as the counterparts of 2 and 3 are shown
in Fig. S14. The frontier molecular orbitals are obtained as demonstrated
in Fig. 8. The calculated HOMO and LUMO distributions are shown in
Fig. S15. The energy gaps between the highest occupied molecular or-
bital (HOMO) and the lowest unoccupied molecular orbital (LUMO)
are 2.60, 2.50, 2.77 eV for 1, 2, 3 with HEPES, respectively. After addition
of HSO−3 , the HOMO−LUMO gaps increases by ca. 0.80–0.94 eV because
the formation of C–SO3H bond interrupts the conjugation between the
vinyl and benzoindolium groups. Furthermore, the HSO−3 is taken off
by Cu2+ ion coordinating with both HSO−3 and HEPES anion, which re-
covers the narrow HOMO−LUMO gaps (2.30, 2.47, 2.53 eV) and the
conjugation structures of 1, 2, and 3.
We also simulate the UV–Vis absorption spectra by TDDFT calcula-
tions considering the water solvent effects (Fig. 9). The theoretical re-
sults agree well with those of the experiments in spite of tiny
overestimation of the absorption wavelength (20–30 nm) (data to be
confirmed, theory: 500, 315, 528 nm (1); 470, 277, 491 nm (2); 449,
269, 492 nm (3)) due to the systematic underestimation of excitation
energies by TDDFT methods [46]. Both the frontier orbital analysis and
the simulated absorption spectra confirmed the interaction mechanism
between benzoindolium derivatives and HSO−3 as well as further reac-
tion with Cu2+ in HEPES buffer, which was proposed and shown in
Scheme 2.
Finally, the detection limits and linear concentrations of these en-
sembles to Hg2+ and Cu2+ were then calculated (Fig. S16, S17) and
summarized in Table 1. It can be seen that 1 showed relative lower de-
tection limits to Hg2+ and Cu2+, which could be attributed to the much
more binding sites in the compound 1.
Fig. 9. Simulated UV–Vis absorption spectra of compounds 1, 2, 3 with the HEPES, HSO3−
and Cu2+ ion.
,
Scheme 2. The possible interaction mechanism of benzo[e]indolium derivatives with Cu2+/Hg2+ in the presence of bisulfite ions.

332
R. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 202 (2018) 324–332
Table 1
Sensing performance of 1, 2, 3 and their adducts with bisulfite.
Compoundsa
Added ions
Change of λabs, max/nm
Change of λem, max/nm
Φb/%
Linear rangec/μM
LODc/μM
A λabs, max
ΔI λem, max
A λabs, max
ΔI λem, max
1
1
–
–
–
0.95
3.0
0.71
0.34
2.1
5.0
2.2
1.7
1.9
44
–
–
HSO−3
Cu2+
Hg2+
–
476↓
476↑
476↑
–
432↓
432↑
432↑
–
465↑
465↓
465↓
–
466↑
466↓
466↓
–
0–10
0–35
0–20
–
0–10
0–35
0–25
–
0–10
0–8.0
0–50
–
1.0–8.0
2.0–60
0–25
–
1.8
4.9
2.9
0.17
0.33
1.3
1-SO3H
1-SO3H
2
2
HSO−3
Cu2+
Hg2+
–
1.5
7.1
7.5
0.21
2.6
0.67
2-SO3H
2-SO3H
3
3
HSO−3
Cu2+
Hg2+
420↓
420↑
420↑
470↑
470↓
470↓
0–10
0–35
0–30
0–6.0
0–50
0–40
5.4
21
28
0.19
2.8
1.9
3-SO3H
3-SO3H
2.3
3.0
a
CH3CN/HEPES buffer (pH 7.00, 1:8, v/v).
The quantum efficiency measurements were made in CH3CN/HEPES buffer (pH 7.00, 1:8, v/v) at 298 K.
Herein, ΔI is representative of emission intensity changes and A is absorption intensity.
b
c
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Hg2+ and Cu2+ have no obvious effects on the absorption and fluores-
cence spectra of benzo[e]indolium derivatives (1, 2 and 3) including
some donor atoms or not. However, after rapidly reacting with appro-
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−SO3H and made the fracture of C−SO3H possible, which resulted in
the recovery of absorption and fluorescence spectra to some extent.
DFT calculations reveal the interaction mechanisms between
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with Cu2+, which are in good agreements with the experiments.
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