Design, synthesis, crystal structure and cytotoxicity studies of colorimetric fluorescent “OFF-ON” probes for rapid detection of hydrogen sulfide based on Cu(II) complex

Article abstract of DOI:10.1016/j.inoche.2018.10.030

Four colorimetric fluorescent probes based on copper(II) complexes were synthesized and the molecular structures were characterized by X-ray diffraction, ESI-HRMS and elemental analysis. When exposed to HS^?, the synthesized probes showed signif

Full text of DOI:10.1016/j.inoche.2018.10.030

Accepted Manuscript
Design, synthesis, crystal structure and cytotoxicity studies of
colorimetric fluorescent “OFF-ON” probes for rapid detection of
hydrogen sulfide based on Cu(II) complex
Xuefang Shang, Xuejing Yue, Yanmei Chen, Congshu Li, Hongli
Chen, Tianyun Wang
PII:
S1387-7003(18)30876-1
DOI:
Reference:
https://doi.org/10.1016/j.inoche.2018.10.030
INOCHE 7164
To appear in:
Inorganic Chemistry Communications
Received date:
Revised date:
Accepted date:
21 September 2018
31 October 2018
31 October 2018
Please cite this article as: Xuefang Shang, Xuejing Yue, Yanmei Chen, Congshu Li, Hongli
Chen, Tianyun Wang , Design, synthesis, crystal structure and cytotoxicity studies of
colorimetric fluorescent “OFF-ON” probes for rapid detection of hydrogen sulfide based
on Cu(II) complex. Inoche (2018), https://doi.org/10.1016/j.inoche.2018.10.030
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ACCEPTED MANUSCRIPT
Design, synthesis, crystal structure and cytotoxicity studies of
colorimetric fluorescent “OFF-ON” probes for rapid detection of
hydrogen sulfide based on Cu(Ⅱ) complex
Xuefang Shanga*, Xuejing Yueb, Yanmei Chena, Congshu Lia, Hongli Chenc, Tianyun
Wangd
aKey Laboratory of Medical Molecular Probes, Department of Chemistry, School of Basic
Medical Sciences, Xinxiang Medical University, Xinxiang, Henan 453003, China
bClinical skills training center，Xinxiang Medical University，Xinxiang，China，453003, China
cSchool of Life Science and Technology, Xinxiang Medical University, Xinxiang, Henan 453003,
China
dDepartment of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang,
Henan 453003, China
*Corresponding author: Tel +86-373-3029128, Fax +86-373-3029959
E-mail: xuefangshang@126.com
Abstract: Four colorimetric fluorescent probes based on copper(II) complexes were
synthesized and the molecular structures were characterized by X-ray diffraction,
ESI-HRMS and elemental analysis. When exposed to HS–, the synthesized probes
showed significant color changes from yellow to red and remarkable increase of
fluorescence intensity (over 80-fold). What’s more, the interaction of host-guest could
be completed in 2 minutes. However, no clear color and fluorescence intensity
–
changes were observed in the presence of other anions (AcO–, H2PO4 , F–, Cl–, Br–, I–,
2-
2-
2-
SO4 , SO3 , CO3 , Cys and GSH). The synthesized colorimetric fluorescent probes
could detect HS– rapidly and conveniently. Cytotoxicity studies indicated that the
synthesized fluorescent probes were low cytotoxicity in Hela cells and may be used to
detect H2S level in vivo.
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Key words: hydrogen sulfide; fluorescent probe; colorimetric detection; cytotoxicity;
copper complex crystal
1. Introduction
It is well known that hydrogen sulfide (H2S) is the rotten egg smell and considered
merely as a toxin with no beneficial physiological significance in the past years. With
the initial observation by Kimura’s group, H2S is an important signaling molecule as
the biological relevance [1]. Followed by carbon monoxide(CO) and nitric oxide(NO),
H2S is identified as the third member of the gasotransmitter family [2-6]. Some
literatures reported that H2S usually appeared in various physiological processes,
including relaxation of vascular smooth muscles, mediation of neurotransmission,
regulation of inflammation and O2 sensing, and also could protect against
ischemia/reperfusion injury [7-10]. Besides, the abnormal level of H2S was linked to
some diseases such as Huntington’s disease (HD), Down’s syndrome, diabetes and
liver cirrhosis [11-15]. Therefore, the detection research on H2S, intriguing gas,
showed the potential therapeutic implication in the biological and pathological
process [16]. However, the above aspect is still in its infancy. The quantified detection
of H2S in living cells is crucial in order to understand its biological and pathological
roles.
So far, many traditional methods have been reported for the detection of H2S
including electrochemistry, chemiluminescence, chromatography, and methylene blue
assay [17-19]. The above methods are generally limited because of their invasive and
destructive nature to the living organisms. On the other hand, small fluorescent probes
have attracted the most attention for sensing and visualizing analytes in living cells
because fluorescent probes exhibited many advantages such as high sensitivity and
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good cell permeability [20-36]. A few small molecular fluorescent turn-on probes
[37-41] and ratiometric probes [42, 43] have been successfully developed based on
the H2S-induced specific reactions, such as displacement method [44-46], reduction of
azide [47], nucleophilic reaction [48]. And some of the above methods have been
used for intracellular H2S imaging [49] which can provide real-time, easy-to-use,
nondestructive detection in live cells or tissues.
Based on the above considerations, we designed and synthesized a series of
copper(Ⅱ) complex (Scheme 1) to study the anion binding ability, especially for HS–
detection. In addition, bromine group and naphthylamine acted as colorimetric group
and fluorometric group respectively. Fortunately, the crystals of the copper complexes
were also obtained. As expected, the synthesized complexes showed the strong
binding ability for HS– (a commonly employed H2S donor) among the tested anions
−
2-
2-
2-
(HS–, AcO−, H2PO4 , F−, Cl−, Br− , I−, SO4 , SO3 , CO3 , Cys and GSH).
2. Material and methods
2.1 Experiment
Unless otherwise specified, all materials and solvents were of analytical grade.
Sodium hydrosulfide hydrate, all anions in the form of tetrabutylammonium salts
(such
as
(n-C4H9)4NCl,
(n-C4H9)4NBr,
(n-C4H9)4NI,
(n-C4H9)4NAcO,
(n-C4H9)4NH2PO4, NaHS, Na2SO4, Na2SO3, Na2CO3, Cys and GSH) were purchased
from Aladdin Chemistry Co. Ltd (Shanghai, China). All anions were stored in a
desiccator under vacuum. Dimethyl sulfoxide (DMSO) was distilled in vacuum after
being dried with CaH2. 1H NMR spectra were recorded using a Bruker AscendTM 400
spectrometer with chemical shifts reported as ppm with TMS as internal standard.
ESI-HRMS was performed using a Bruker Microtof-QIII spectrophotometer. UV-vis
titration experiments were carried using a Shimadzu UV2600 Spectrophotometer at
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298 K. Fluorescence spectra were measured using an Eclipse fluorescence
spectrophotometer (Agilent, USA). The binding constant, Ks, was obtained by
non-linear least squares calculation method for data fitting.
For cytotoxicity test, the living Hela cells were seeded into a 96-well plate at a
density of 5 × 104 cells/well. After the cells form monolayer, the culture media was
replaced by the complex solution (2.5-80 g·mL-1). After incubated for 24 h, complex
solution was then replaced with fresh media after several washing steps, and 20 μL
MTT (5 mg·mL-1) was added to each well and incubated for a further 4 h in
humidified atmosphere. After that, the culture media was removed and the solvent
(DMSO, 150 μL) was then added. Finally, the absorbance of cells was detected at
490 nm using the microplate reader (Thermo Multiscan MK3, Thermo Fisher
Scientific, MA, USA) with the plain cell culture media as the control. Cell viability
was expressed by the ratio between the absorbance of the cells incubated with
complex and that of blank culture media.
2.2 Synthesis
2.2.1 Bis(3,5-dibromosalicylidene-aniline) copper(II) (compound 1)
3, 5-dibromosalicylidene-aniline was synthesized according to the literature [50]. It
was obtained by refluxing the ethanol solution (40 mL) of 3, 5-dibromosalicyl-
aldehyde (10 mmol, 2.8 g) and phenylamine (10 mmol, 930 mg) for 4 h and the
yellow precipitate was recrystalled by ethanol, washed with water, and dried under
1
vacuum. Yield: 85%. m.p. 102.5-104.0 ℃. H NMR (400 MHz, CDCl3) δ 14.53 (s,
1H, -OH), 8.57 (s, 1H, -CH=N-), 7.78 (d, J = 2.3 Hz, 1H, ph-H), 7.52 (d, J = 2.3 Hz,
1H, ph-H), 7.48 (dd, J = 10.6, 4.9 Hz, 2H, ph-H), 7.40 – 7.30 (m, 3H, ph-H) (S1).
ESI-MS (m/z): 354.0 (M-H)-.
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Bis(3,5-dibromosalicylidene-aniline) copper(II) (compound 1) was synthesized
according to the following procedure. Copper acetate hydrate (99 mg, 0.5 mmol) was
added to a stirred solution of 3, 5-dibromosalicylideneaniline (355 mg, 1 mmol) in
ethanol (15 mL). The mixture was stirred for 1 h at 55 ℃, and then stood overnight at
room temperature. Suitable brown single crystal for X-ray crystal structural analysis
was obtained, separated by filtration, washed with cyclohexane. Yield: 82%. m.p.
251.8 – 254.2 ℃. ESI-HRMS (m/z): 789.7123 (M+Na)+ (S5).
2.2.2 Bis(5-bromosalicylidene-aniline) copper(II) (compound 2)
The synthesis method was similar to the above procedure.
1
5-bromosalicylideneaniline Yield: 75%. m.p. 121.3 - 123.1 ℃. H NMR (400 MHz,
CDCl3) δ 13.31 (s, 1H, -OH), 8.58 (s, 1H, -CH=N-), 7.54 (d, J = 1.5 Hz, 1H, ph-H),
7.47 (t, J = 7.6 Hz, 3H, ph-H), 7.35 (d, J = 6.9 Hz, 1H, ph-H), 7.31 (s, 1H, ph-H), 7.29
(s, 1H, ph-H), 6.96 (d, J = 8.8 Hz, 1H, ph-H) (S2). ESI-MS (m/z): 274.0 (M-H)-.
Bis(5-bromosalicylideneaniline) copper(II) (compound 2). Yield: 82%. m.p.
282-284℃. ESI-HRMS (m/z): 633.9071 (M+Na)+ (S6).
2.2.3 Bis(3,5-dibromosalicylidene-α-Naphthylamine) copper(II) (compound 3)
The synthesis method was similar to the above procedure. 3, 5-dibromosalicylid-
ene-α-Naphthylamine Yield: 75%. mp. 151. 4 -153.2 ℃. 1H NMR (400 MHz, CDCl3)
δ 14.75 – 14.19 (m, 1H, -OH), 8.66 (s, 1H, -CH=N-), 8.28 – 8.23 (m, 1H, ph-H), 7.92
(dt, J = 5.2, 3.0 Hz, 1H, ph-H), 7.87 (d, J = 8.3 Hz, 1H, naphthyl-H), 7.83 (d, J = 2.3
Hz, 1H, ph-H), 7.62 – 7.57 (m, 3H, naphthyl-H), 7.57 – 7.50 (m, 1H, naphthyl-H),
7.24 (dd, J = 7.3, 0.9 Hz, 1H, naphthyl-H) (S3). ESI-MS (m/z): 404.0 (M-H)-.
2.2.4 Bis(5-bromosalicylidene-α-Naphthylamine) copper(II) (compound 4)
The synthesis method was similar to the above procedure.
5-bromosalicylidene-α-Naphthylamine Yield: 75%. m.p. 108 - 110 ℃. 1H NMR (400
5

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MHz, CDCl3) δ 13.44 (s, 1H, -OH), 8.66 (s, 1H, -CH=N-), 8.24 (dt, J = 6.8, 3.4 Hz,
1H, ph-H), 7.95 – 7.87 (m, 1H, ph-H), 7.84 (d, J = 8.3 Hz, 1H), 7.60 (s, 1H,
naphthyl-H), 7.58 (t, J = 2.9 Hz, 1H, naphthyl-H), 7.56 – 7.48 (m, 2H, naphthyl-H),
7.28 (s, 1H, ph-H), 7.20 (dd, J = 7.3, 0.7 Hz, 1H, naphthyl-H), 7.03 (d, J = 8.8 Hz, 1H,
naphthyl-H) (S4). ESI-MS (m/z): 324.0 (M-H)-.
Bis(5-bromosalicylidene-α-Naphthylamine) copper(II) (compound 4). Yield:
75%. ESI-HRMS (m/z): 733.9186 (M+Na)+ (S7).
Scheme 1 Synthesis route for Copper(Ⅱ) Complexes
3. Results and discussion
3.1 X-ray crystal structure
Single crystals of synthesized complexes (1-4) were all obtained at room
temperature from the ethanol solvent by slow evaporation. In order to confirm the
chemical structure of the complex, single crystal X-ray analysis was used to
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determine the structure of the copper complex. Parameters in CIF format are available
from Cambridge Crystallographic Date Center (CCDC 1432286 1432284
1448106 and1432281). The molecular structures of the copper complexes with the
atoms numbering scheme were given in Fig. 1. The detailed summary of the crystal
data was given in Table 1. Selected bond lengths and bond angles were listed in Table
2.
2
1
3
4
Fig.1 The ORTEP view of copper complexes (1-4)
The bond angles were all fit with the ideal value (180°). The crystallographic
data revealed that the metal center was four-coordinated system by two oxygen and
nitrogen atoms in two Schiff base ligands. The bond (Cu–O) was
in trans- configuration and the distance (Cu–O) was shorter than the distance (Cu–N).
The Schiff base lost a proton from the hydroxyl group and acted as a single charged
bidentate ligand coordinating to copper(II). The ligands coordinated to the Cu(II)
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center in trans- orientation with respect to each other. The geometry around the metal
center was a distorted square-planar, with P21/c space group. All these parameters
were in close agreement with those reported for square planar copper(II) compounds
[51-53].
Table 1. Crystal data and structure refinement for complexes
Compound
1
2
3
4
Empirical formula
C26H16Br4
CuN2O2
771.59
C26H18Br2
CuN2O2
613.78
C34H20Br4 C34H22Br2
CuN2O2
871.70
153(2)
CuN2O2
713.9
Formula weight
T (K)
296(2)
296(2)
296(2)
λ (Å)
0.71073 Å
monoclinic
P21/c
0.71073
monoclinic
P21/n
0.71073
tetragonal
P21c
0.71073
monoclinic
P21/c
Crystal system
Space group
Crystal color
Brown
Brown
Brown
Brown
Crystal size mm × mm 0.370 x 0.251 x 0.261 x 0.219
0.21 x 0.10 x 0.20 x 0.16 x
0.10 0.06
11.1806(12) 11.628(2)
10.8307(11) 9.7474(19)
13.2203(13) 13.809(3)
× mm
a (Å)
0.202
10.3880(11)
x 0.086
9.7702(5)
b (Å)
9.5412(9)
13.0905(12)
90
9.8852(5)
13.2482(6)
90
c (Å)
α (°)
90
90
β (°)
103.789(3)
90
110.7396(16)
90
112.718(3)
90
114.33(3)
90
γ (°)
V (Å3)
μ (mm−1)
Dcalc (g/mL)
Z
1260.1(2)
7.237
1196.60(10)
4.278
1476.7(3)
6.188
1.960(1)
2
1426.1(5)
3.603
1.662
2
2.034
1.704
2
2
F(0 0 0)
742
606
846
710
θ range for data
collection (°)
Index ranges
3.108 to 27.53
3.036 to 27.54 3.16 to 25.00 3.24 to 24.50
−13
−12
−17
h
k
13
12
16
−12
−11
−17
h
k
12
12
17
−13
−12
−15
h
k
13 −13
12 −11
15 −16 l
h
k
13
11
16
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
⩽
l
l
l
⩽ ⩽
⩽ ⩽
⩽ ⩽ ⩽ ⩽
Maximum and
minimum transmission 0.018
0.053 and
0.692 and
0.341
0.5823 and
0.3593
0.8183 and
0.5313
Refinement method Full-matrix
Full-matrix
Full-matrix
Full-matrix
least-squares
least-squares
least-squares least-squares
2
2
2
2
on F
Goodness-of-fit on F2 1.010
R1 and wR2 indices
[I > 2σ(I)]
R1 and wR2 indices
(all data)
Largest difference in
on F
on F
1.029
R1 = 0.0617, R1 = 0.0966,
on F
1.076
R1 = 0.0504,
1.293
R1 = 0.0456,
wR2 = 0.1260
R1 = 0.0523,
wR2 = 0.1351
1.579 and
wR2 = 0.1369 wR2=0.1431 wR2 =0.2163
R1 = 0.0782, R1 = 0.1128, R1 = 0.2123,
wR2 = 0.1607 wR2=0.1680 wR2 =0.2583
0.935 and
-0.723
0.972 and
-0.393
0.917 and
-0.432
peak and hole (e Å−3) -1.004
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Table 2. Selected bond lengths (Å) and angles (°) for the complexes (1-4)
Angles (°)
Bond length(Å)
1
2
3
4
1.896(2)
1.896(2)
2.014(3)
2.014(3)
180.00(15)
180.00(15)
91.83(11)
91.83(11)
88.17(11)
88.17(11)
1.882(2)
1.882(2)
1.992(3)
1.992(3)
180.0
1.891(4)
1.891(4)
2.001(7)
2.001(7)
180.0(1)
180.0(1)
91.3(2)
91.3(2)
88.7(2)
88.7(2)
1.861(7)
1.861(7)
1.996(9)
1.996(9)
180.0(1)
180.0(1)
90.4(3)
90.4(3)
89.6(3)
89.6(3)
Cu(1)-O(1)
Cu(1)-O(1)#1
Cu(1)-N(1)
Cu(1)-N(1)#1
O(1)#1-Cu(1)-O(1)
N(1)-Cu(1)-N(1)#1
O(1)-Cu(1)-N(1)
O(1)#1-Cu(1)-N(1)#1
O(1)#1-Cu(1)-N(1)
O(1)-Cu(1)-N(1)#1
180.0
91.63(11)
91.63(11)
88.37(11)
88.37(11)
Symmetry transformations used to generate equivalent atoms: #1 -x+2,-y,-z+2
3.2 UV-vis titration
The binding abilities of copper complexes with anions were investigated using
UV-vis absorption spectra in DMSO and DMSO-H2O (9:1, v/v) at 298 K. The UV-vis
spectral changes of copper complex 1 (8.0×10−5 mol·L-1 in DMSO) were shown in Fig.
2. It was clear that the maximal absorption band of complex 1 at 390 nm was shifted
to the long wavelength gradually with the increasing amount of HS–. The wavelength
shifted to 430 nm when 1.0 equiv. of HS– was added. Three clear isosbestic points at
312, 374 and 420 nm suggested that complex 1 interacted with HS− which host-guest
complex informed each other. When 8 equiv. of HS− was added, the maximal
absorption band shifted to the long wave direction at 530 nm. At the same time, one
isosbestic point at 476 nm appeared and the intensity of new absorbance peak
enhanced by 46 times. The reason for red-shift phenomenon (Δλ=140 nm) may be
link with the introduction of bromine, acting as colorimetric group. In addition, the
color change of complex 1 solution could be observed from yellow to red (Fig. 4)
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upon the addition of HS− anion. The above results indicated that the synthesized com-
a
b
c
d
Fig. 2 UV-vis spectral changes of complex 1 upon the addition of various anions.
–
[complex] = 8.0×10-5 mol·L-1 in DMSO: (a)HS–, (b)H2PO4 , (c)F–, (d)AcO–. Arrows
indicate the direction of increasing anions concentration.
plex 1 may be used as colorimetric probe for the HS− detection. For an excellent
fluorescent probe, high selectivity is very important. Analogous investigations were
–
carried out on other normal anions. The additions of H2PO4 , AcO– and F– to complex
1 induced a blue-shift from 400 nm to 360 nm which indicated complex 1 also
interacted with the above anions. While, the interacted mechanism of complex 1 with
–
H2PO4 , AcO– and F– was different from HS– due to the different wave-shift
phenomenon. The additions of Cys and induced the weak similar spectral changes.
2-
2-
2-
However, the additions of Cl–, Br–, I–, SO4 , SO3 , CO3 and GSH did not induce
any spectral responses which indicated that complex 1 showed almost no binding
abilities toward these anions or the binding abilities were very weak and could be
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ignored. UV-vis absorption spectra changes of complex 3 were similar to that of 1
which the intensity of absorption peak at 400 nm decreased gradually and a new
–
absorption peak at 520 nm increased upon the additions of HS–, H2PO4 , AcO– and F–
anions (S8). In addition, the interaction process accompanied with color changes. The
addition of Cys also induced similar spectral changes which the responses were
weaker than HS–. The above results indicated that complex 3 also interacted with HS–,
–
H2PO4 , AcO–, F–, Cys and GSH as different binding ability and also no binding
2-
2-
ability for Cl–, Br–, I–, SO4 , SO32- and CO3 .
Similarly, the UV-vis spectra of complex 2 also changed after the addition of HS–,
–
H2PO4 , AcO–, F– which indicated complex 2 interacted with the above anions (Fig. 3).
The spectral responses induced by Cys and GSH were very weak. Interestingly, the
interaction of complex 2 with NaHS didn’t cause the visible color changes and
red-shift phenomenon in spectra changes. The UV-vis spectral changes of complex 4
were similar to that of complex 2 (S9).
In order to research the real-life application of synthesized complexes, we also
carried out the UV-vis spectral changes in the mixed solvents containing water.
Experimental results suggested similar spectral changes of synthesized complexes
were also monitored in DMSO-H2O (9:1, v/v) solution (S10, S11, S12, S13),
compared with pure DMSO solution. It was found that HS– also could lead to the
color changes of complex (1, 3) in DMSO-H2O (9:1, v/v) from yellow to red, while
other tetrabutylammonium salts could not induce the obvious color changes of the
complex solution (Fig. 4). Thus, two complexes (1, 3) can detect HS– selectively by
the naked-eye in aqueous solution which may be applicated in real-life samples.
11

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a
b
c
d
Fig. 3 UV-vis spectral changes of complex 2 upon the addition of various anions.
–
[complex] = 8.0×10-5 mol·L-1 in DMSO: (a)HS–, (b)H2PO4 , (c)F–, (d)AcO–. Arrows
indicate the direction of increasing anions concentration.
–
Blank(1)
HS–
H2PO4
F–
AcO–
Cl–
Br–
I–
–
Blank(3)
HS–
H2PO4
F–
AcO–
Cl–
Br–
I–
Fig. 4 Photographic images of two complexes (8.0×10-5 mol·L-1) and the additions of
different anions (8.0 equiv. of complex) in DMSO-H2O (9:1, v/v).
12

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Furthermore, the detection limit of complex 4 in pure DMSO solution for HS− was
also investigated through UV-vis titration (S14). The complex 4 (2.0×10−5 mol·L-1)
and the concentration of HS− (0-2.5×10−4 mol·L-1) showed a good linear relationship
by UV-vis intensity which indicated the quantitative detection of HS−. The addition of
HS− (1.3×10−4 mol·L-1) to the complex 4, the corresponding UV-vis intensity
increased 1.3 times. The above results exhibited the detection limit for HS− was
1.3×10−4 mol·L-1. Also, the time-dependent experiment was conducted (S15). The
results indicated that the equilibrium between complex 4 and HS− completed in two
minutes which showed HS− could be detected quickly.
3.3 Fluorescent response
The fluorescence properties of the Cu(Ⅱ) complex (4) were investigated in
DMSO-H2O (9:1, v/v). The free probe displayed weak fluorescence intensity upon
excitation at 337 nm. As shown in Fig. 5, with the increasing concentration of HS–,
the fluorescence emission intensity was gradually strengthened at about 430 nm
because the added HS– could coordinate with Cu2+ and the free ligand was released.
For all quantitative analytical methods, a linear calibration curve is always desired
because it allows easy calculation. As shown in Fig. 5(b), a linear relationship
(y=2.495x-118.56, R2=0.9982, SD=17, N=24) existed between the fluorescence
intensity and the concentration (NaHS) in the range of 50-450 M, indicating that the
complex 4 was potentially useful for quantitative analysis of H2S. The fluorescence
titration of complex 4 with various anions was conducted to examine the selectivity.
–
2-
2-
2-
The additions of AcO–, H2PO4 , F–, Cl–, Br–, I–, SO4 , SO3 , CO3 , Cys and GSH
(4.0×10−4 mol·L-1) produced a nominal change in the fluorescence spectra of complex
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4. All these results suggested that complex 4 was a practical probe for detection of
HS– with high selectivity.
a
b
Fig. 5 (a) Fluorescence response (λex, 337nm, slit widths: 5 nm/5 nm) of copper probe
4 (4.0×10−5 mol·L-1) upon the addition of NaHS (0 - 4.0×10−4 mol·L-1), arrows
indicate the increase direction of NaHS concentration. Spectra were acquired in
DMSO-H2O (9:1, v/v) after reaction of the probe with NaHS for 10 min. (b) The
linearity of increased fluorescence intensity with the concentration of added NaHS in
DMSO-H2O (9:1, v/v).
3.4 Binding constant
Cu(Ⅱ) complexes (1, 3) interacted with HS– as the ratio of 1:3 and complexes (2, 4)
interacted with HS– as the ratio of 1:1 according to the nonlinear fitting curves.
Binding constants of host-guest complexation were calculated according to the
equation (1) for 1:3 and equation (2) for 1:1 [54-56].
cGcH3
X  X
X  X
0


(1)
1 KScH3
1 Ksc
cGcH
0
(2)
where, cG and cH are the concentrations of guest and host, respectively. X is the
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absorbance intensity at certain concentration of host and guest. X0 is the absorbance
intensity of the host alone. Ks is the affinity constant for the host-guest complexation.
Δԑ is the change in molar extinction coefficient. The binding constants could be
obtained and listed in Table 3 based on the UV–vis data. Obviously, the anion binding
constants of Cu(Ⅱ) complex in DMSO/H2O (9:1, v/v) were lower than that in pure
DMSO due to the interference of H2O. The binding constants of AcO–, with
complexes (3, 4) containing naphthylamine cannot be calculated due to the weak
spectral changes. However complexes (1, 3) interacted with AcO– in pure DMSO, it
may be related to the steric inhibition of naphthylamine. From Table 3, Cu(Ⅱ)
complexes showed the strongest binding ability for HS– among anions tested. The
reason probably was the displacement reaction, in which the Cu2+ was captured by
HS– and the free ligand was released from the complex. The above results indicated
that the synthesized copper complexes could be used as chemosensors for the
detection of HS– in the sample of environment or pharmacy. Due to the spectral
2-
responses were very weak, the binding constants of complexes with Cl–, Br–, I–, SO4 ,
2-
2-
SO3 , CO3 could not be calculated which suggested the binding abilities were very
weak and could be ignored.
3.5 Mechanism
In order to verify the spectral sensing mechanism of copper complexes with NaHS,
further insight into the interaction of NaHS with complex was investigated by
performing HRMS after the addition of NaHS. As shown in Figure S16, after 3 equiv.
of NaHS was added, the ion peak of complex 1 ((M+Na)+: 789.7123) was vanished
and a new ion peak ((M-H) –: 353.8973) was observed which was the ion peak of free
ligand. And so the above results indicated the added HS– snatched copper cation and
the free ligand was released (Scheme 2). Obviously, the similar literatures (H2S probe
15

ACCEPTED MANUSCRIPT
involving metal cation) have been reported [57, 58], the red-shift phenomenon of abs-
Table 3 Binding constants of Cu(Ⅱ) complex with various anions
Anions
Ks (1)
Ks (2)
(2.36±0.08)×103a
(1.76±0.08)×103a
(5.05±0.05)×104a
(2.10±0.05)×104a
(3.27±0.08)×104a
(1.10±0.06)×104a
(1.7±0.05)×103a
ND
Ks (3)
Ks (4)
DMSO (2.45±0.01)×104b
aqueous (1.28±0.01)×104b
DMSO (2.71±0.09)×104a
aqueous (8.91±0.05)×103a
DMSO (1.23±0.07)×104a
aqueous (2.98±0.07)×103a
(1.74±0.05)×104b (2.88±0.02)×103a
(1.02±0.02)×104b (1.91±0.06)×103a
(5.02±0.01)×104a (2.14±0.01)×104a
(3.93±0.30)×104a (1.86±0.01)×104a
(1.04±0.05)×104a (1.60±0.09)×104a
(6.14±0.09)×103a (1.03±0.01)×104a
HS–
H2PO4
F–
–
DMSO
aqueous
DMSO
aqueous
DMSO
aqueous
(6.37±0.01)×103a
NDc
ND
ND
<10
<10
<10
<10
AcO–
Cys
ND
(4.72±0.08)×103a
(2.64±0.07)×103a
ND
ND
(3.18±0.09)×103a
(1.07±0.94)×102a
ND
(6.36±0.05)×102a
ND
GSH
<10
ND
ND
ND
aThe binding ratio of host-guest is 1:1.
bThe binding ratio of host-guest was 1:3.
cThe binding constant could not be determined.
Aqueous: the volume of DMSO/H2O was 9:1.
orbance and the fluorescence enhancement were most likely the reason that the added
NaHS could snatch the copper ions from the complex due to the much higher binding
constant between sulfide and copper ions. However other anions could only
coordinate with complex and form a five-coordinated complex according to
comprehensive analysis of UV-vis and fluorescent titration. Why did the complexes (1,
3) interact with HS– as the ratio of 1:3 and the complexes (2, 4) interact with HS– as
the ratio of 1:1? The binding ratio and binding constants also could illustrate it. As
showed in Table 3, the copper complex interacted with HS– as the ratio of 1:3 in
DMSO solution which indicated the copper atom was released from the complex.
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Scheme 2. The possible binding mode for the sensing of HS–.
3.6 Cytotoxicity test
For further biological application, the quantitative cytotoxicity study of the
complex 4 was conducted using the MTT assay. The MTT assay results were
collected from Hela cells with complex 4 (2.5 - 80 g·mL-1). Cell viability was
expressed by the ratio between the absorbance of the cells incubated with complex
and the cells incubated with blank culture media only. As shown in Fig. 6, the
complex 4 showed almost no cytotoxicity to Hela cell at the concentration (80
g·mL-1) indicating that the complex 4 showed the potential to detect H2S in cells.
Further in vivo studies are currently under way in our laboratory.
Fig. 6 Cell viability values (%) estimated by MTT proliferation test versus
concentrations of complex after 24 h incubation at 37°C. Hela cell was cultured in the
2.5-80 g·mL-1 complex solution at 37°C for 24 h (n=6).
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4. Conclusion
In summary, colorimetric fluorescent sensors have been designed and synthesized
effectively based on an inorganic-reaction. Two complexes (1, 3) exhibited naked-eye
detection for HS– at room temperature and could be used as colorimetric sensor.
Complex 4 displayed a remarkable fluorescence enhancement (over 80-fold) and
could be performed as fluorescent OFF–ON probe in DMSO and aqueous
solutions. Cytotoxicity studies by Hela cells indicated that complex 4 was low
cytotoxicity when the concentration was less than 80 g·mL-1. All these features
make the synthesized complexes suitable for the direct monitoring of biologically
important HS– in biological samples. The above method was advantage for the
simplicity analysis and low cost of the starting material in the present system. The
complex holds great potential for environmental analysis of HS-, S2-, H2S and their
use in bio-analysis is still challenging. Preparation of highly water soluble and highly
fluorescent copper complex is under investigation.
Acknowledgment
This work was supported by the Program for Science & Technology Innovation
Talents in Universities of Henan Province (15HASTIT039), Fund of Fluorescence
Probe and Biomedical Detection Research Team of Xinxiang City (CXTD16001),
Scientific and Technological Research Projects of Henan Province (182102311124).
Appendix A. Supplementary data
CCDC 1432286 1432284 1448106 1432281 contains the supplementary
crystallographic data for the copper complex 1, 2, 3, 4 reported in this paper. These
data
can
be
obtained
free
of
charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html,
or
from
the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)
18

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1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
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Graphical abstract
Four crystals based copper(II) complexes were obtained and the molecular structures
were all characterized by X-ray diffraction. We presented a simple, easily-prepared,
yet efficient, inorganic-reaction based sensors for HS-. An excellent linear relationship
and remarkable fluorescence enhancement (over 80-fold) were found in the
fluorescent titration of NaHS.
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Highlights:
Four crystals based on copper(II) complexes were obtained and characterized.
The colorimetric and fluorescence “OFF-ON” probes.
The fluorescent probes were low cytotoxicity Hela cell.
A rapid and convenient detection method for HS-.
27