A benzothiazole-based fluorescent probe for selective detection of H2S in living cells and mouse hippocampal tissues

Article abstract of DOI:10.1016/j.dyepig.2016.11.038

This study reports a benzothiazole-based fluorescent probe EPS-HS for real-time detection of H₂S. The probe is characterized in a large red-shift, good selectivity, high sensitivity and favorable biocompatibility. We measured the detection limi

Full text of DOI:10.1016/j.dyepig.2016.11.038

Accepted Manuscript
A benzothiazole-based fluorescent probe for selective detection of H S in living cells
2
and mouse hippocampal tissues
Yi Liu, Yan Ding, Jie Huang, Xiaofei Zhang, Tongyong Fang, Yuyun Zhang, Xian
Zheng, Xia Yang
PII:
S0143-7208(16)30712-4
10.1016/j.dyepig.2016.11.038
DYPI 5607
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 13 September 2016
Revised Date: 22 October 2016
Accepted Date: 20 November 2016
Please cite this article as: Liu Y, Ding Y, Huang J, Zhang X, Fang T, Zhang Y, Zheng X, Yang X,
A benzothiazole-based fluorescent probe for selective detection of H S in living cells and mouse
2
hippocampal tissues, Dyes and Pigments (2016), doi: 10.1016/j.dyepig.2016.11.038.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
1
2
A benzothiazole-based fluorescent probe for selective detection of H2S
in living cells and mouse hippocampal tissues
3
4
5
6
7
8
Yi Liu,#*a Yan Ding,*a Jie Huang,a Xiaofei Zhang,a Tongyong Fang,a Yuyun Zhang,a
Xian Zheng,a and Xia Yanga
a
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of
Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China.
# Fax: +86-0516-83262136, E-mail: cbpeliuyinew@163.com.
9
10
11
12
13
†Electronic Supplementary Information (ESI) available: Synthesis, additional
methods, additional plots of UV and fluorescence, NMR and HR-MS analysis data,
additional other supporting data.
14
15
16
*These authors contributed equally to this paper.
1

ACCEPTED MANUSCRIPT
17 Abstract
18
19
20
21
22
23
24
25
26
This study reports a benzothiazole-based fluorescent probe EPS-HS for real-time
detection of H2S. The probe is characterized in a large red-shift, good selectivity, high
sensitivity and favorable biocompatibility. We measured the detection limit of
EPS-HS in PBS buffer and fetal bovine serum. Moreover, in HT22 living cell imaging
studies, EPS-HS showed good cell permeability and H2S could be detected within the
cells. Furthermore, in situ visualization of H2S was performed on the hippocampal
slices of normal mice with EPS-HS. We were also able to estimate the concentration
of sulfide in mouse hippocampus tissues. The probe will be applied for better
treatment of neurological deficits caused by damage to the hippocampus.
27
28
Key words: hydrogen sulfide, probe, fluorescence, hippocampus
2

ACCEPTED MANUSCRIPT
29 1. Introduction
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Hydrogen sulfide (H2S), an colorless gas with the characteristic foul odor of
rotten eggs, has been considered as a toxic pollutant for more than three hundred
years1. Nevertheless, recent studies indicate H2S as the third gaseous signal molecule
which is almost as important as nitric oxide (NO) and carbon monoxide (CO) in
biological systems2,3,4. H2S may show either protective or toxic effects on cells at
different concentrations5-8. Normally, in mammalian cells, H2S is endogenously
produced by enzymes such as cystathionine β-synthetase (CBS), cystathionine γ-lyase
(CSE), 3-mercaptopyruvate sulfurtransferase (3-MST) and cysteine lyase (CL) in
cysteine-related sources 9,10. However, accumulating evidence has confirmed that
abnormal H2S concentrations are related to various diseases, including Alzheimer’s
disease11, diabetes12 and tumor13, and found within many cell lines such as porcine
oocytes14 and MCF-7 cells15.
Interestingly, several studies also provide evidence supporting the excellent role
of H2S in neuromodulation16,17. For example, H2S can act as a neuroprotectant, protect
neurons from oxidative stress, and produce potential therapeutic effects against
neurodegenerative disorders18-20. Furthermore, researchers show that H2S can inhibit
the apoptosis of hippocampal neurons and reduce the damage of hippocampal
neurons21-24. Hence, more attention have been drawn on the possible role that H2S
might play in neurological deficits caused by damage to the hippocampus. Thus, a
highly selective and sensitive analytical measurement for better understanding how
this gasotransmitter contributes to convoluted biological processes is directly
3

ACCEPTED MANUSCRIPT
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
requested25, which can accurately and reliably determine the biologically relevant
concentrations of H2S in the nervous system.
Traditional analytical techniques for H2S detection include the methylene blue
method, the monobromobimane method, gas chromatography and the sulfide ion
selective electrode method26-29. In contrast, fluorescent probes have been widely
applied for detecting the concentrations of H2S in living biological systems due to
their high selectivity and sensitivity, short response time, non-invasive detection, and
real-time imaging30,31. Unfortunately, the short excitation wavelength light (ca.
350-550 nm) limits its application in deep-tissue imaging because of the shallow
penetration depth. Recently, two-photon fluorescence probes, which can be excited by
two-photon absorption, provided an opportunity to overcome the problems originated
from the single-photon fluorescence technology compared with cellular imaging, the
organic imaging also has iconic significance owning to its more complex structures
and research value32-35. Most recently, Chen et al.34 and Liu et al.36 reported the
detection and imaging of H2S in cardiac and liver tissues. These remind us that
histological-section researching is a breakthrough in vivo detection for H2S.
In the current study, an easily synthesized benzothiazole-based fluorescent probe
EPS-HS was designed. Its original group has stable chemical properties and
fluorescence spectrum is not affected by environmental factors37. Therefore, first,
density functional theory (DFT) calculations were performed to optimize the structure
of EPS-HS at the B3LYP/6-31G* level using a suite of Gaussian 09 programs38,39
.
Then the structure of the target compound was fully characterized by the standard
4

ACCEPTED MANUSCRIPT
13
73
74
75
76
1H-NMR, C-NMR, and high resolution mass spectrometry. Finally, the biological
application of EPS-HS was discussed using HT22 living cells and the hippocampus of
normal mice.
77 2. Experimental
78
79
2.1. Synthesis of EPS-HS
The synthesis process of EPS-HS is showed in Route 1. A mixture of Compound
80
81
82
83
84
85
86
87
88
89
90
91
1 which was synthesized by means of the reference paper40,41 (0.227 g, 1.0 mmol), 2,
4-dinitrofluorobenzene (0.186 g, 1.0 mmol), triethylamine (0.303 g, 3.0 mmol) in 10
mL dry DMF (dimethyl formamide) were stirred at N2 atmosphere, and the solution
was stirred at 80°C for 6 - 8 h. The reaction mixture was cooled to room temperature
and poured into a mixture of ice and water (100 mL) before a light yellow solid was
precipitated. The raw product was filtered and recrystallized from EtOAc to provide a
light yellow solid in 85 % yield. m.p. 128.2°C -129.3°C. TLC (silica, hexane : DCM,
2:1 v/v); Rf = 0.4; 1H NMR (400 MHz, DMSO-d6), δ (ppm): 8.93 (d, J = 2.8 Hz, 1H),
8.49 (dd, J1 = 9.2 Hz, J2 = 2.8 Hz, 1H), 8.22-8.24 (m, 2H), 8.17 (d, J = 8.0 Hz, 1H),
8.08 (d, J = 8.0 Hz, 1H), 7.55-7.59 (m, 1H), 7.40-7.51 (m, 4H). 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 121.1, 121.2, 122.4, 122.9, 123.4, 126.1, 127.2, 130.1, 130.2,
130.9, 135.1, 140.4, 142.6, 154.0, 154.4, 156.8, 166.5. HRMS (ESI+): (M + H)+ calcd.
92
93
94
for EPS-HS (C19H12N3O5S 394.0498; found 394.0489.
)
2.2 Characterization
Thin layer chromatography was performed on silica gel 60 F254 plates (250 ꢀm)
95
and column chromatography was conducted over silica gel (300-400 mesh).
5

ACCEPTED MANUSCRIPT
96
97
Visualization of the developed chromatogram was accomplished by a UV lamp.
UV-Vis absorption spectra were recorded on a Shanghai MAPADA UV-3100PC
UV-Visible spectrophotometer. All fluorescence measurements were recorded on a
Hitachi F4600 Fluorescence Spectrophotometer. The pH measurements were
performed on a Mettler-Toledo Delta 320 pH meter. All fluorescence imaging
experiments were conducted under a FV1000 confocal laser scanning microscope
(Olympus, Japan). All the solvents were of analytic grade. The stock solution of
Na2S 9H2O (≥98.0%) was prepared in doubly distilled water, which was freshly
prepared each time before use. All fluorescence measurements were carried out at
room temperature on a Hitachi Fluorescence Spectrophotometer F-4600. The samples
were excited at 285 nm with the excitation and emission slit widths set at 5.0 nm. The
emission spectrum was scanned from 350 nm to 560 nm at 1200 nm/min. The
photomultiplier voltage was set at 900 V.
98
99
100
101
102
103
104
105
106
107
108
109
110 3. Results and discussion
111
112
113
114
115
116
117
3.1 Computational calculations
DFT calculations were conducted to achieve the preferential conformation of
EPS-HS and its parent nucleus (Compound 1) (Figures 1A and 1B) and to investigate
the mechanism by which EPS-HS reacted with H2S (Figure 1C). Results showed that
the photoexcitation of EPS-HS from S0 to S1 states mainly involved electron
transitions from the highest occupied molecular orbital (HOMO) to the lowest
occupied molecular orbital (LUMO). The HOMO of EPS-HS was mainly located at
6

ACCEPTED MANUSCRIPT
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
the benzothiazolyl part, but the LUMO were mostly located at the nitrobenzene part.
It can be seen in the frontier molecular orbital diagram that the HOMO did not
overlap with the LUMO.
Based on the frontier molecular orbital theory, the computational results of
EPS-HS indicate that the increased electric density area is mainly within the
nitrobenzene part. The ether linkage becomes the target of nucleophilic reagent (H2S).
There was no obvious difference as to average energy between the LUMO and the
HOMO.
3.2 Fluorescence and absorption spectroscopy
The changes in the fluorescence and absorption spectroscopy of EPS-HS (10 ꢀM)
was tested at 37°C in 20 mM PBS buffer (pH 7.4) using Na2S (100 ꢀM) as a sulfide
source. Results showed that the maximum absorption peak of EPS-HS was shifted
which was similar to Compound 1 (Figure 2A). Meanwhile, a robust increase in
fluorescence intensity (> 40-fold) was found with the maximum emission peak at 470
nm when excited at 285 nm, which was completed within 20 min (Figure 2B). These
data indicated that our experimental results were consistent with the frontier orbital
theory.
Next, we examined the sensitivity of EPS-HS for H2S using various
concentrations of Na2S (0 - 300 ꢀM) (Figure 3A). Upon exposure to H2S, the probe
was cleaved to release fluorophores. The fluorescence intensity was increased about 0
- 40 folds. Then, EPS-HS was further reacted with different concentrations of Na2S (0
- 60 ꢀM), showing a linear relationship of emission intensity versus sulfide
7

ACCEPTED MANUSCRIPT
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
concentration in PBS buffer and fetal bovine serum (Figure S1, ESI†).
The fluorescence intensity of EPS-HS (10 ꢀM) in PBS and fetal bovine serum
was assessed with the presence of different concentrations of Na2S for 20 min (Figure
S1, Supporting Information). But, the fluorescence response in serum was lower than
that observed in PBS buffer, which may attribute to the fast metabolism of sulfide in
plasma. The fast responses and excellent linear relationship provided a real-time
quantitative method for detecting sulfide in biological samples. The detection limit
was calculated to be 108 nM in PBS butter and 154 nM in fetal bovine serum. After
the addition of 100 ꢀM Na2S buffered solution, a significant fluorescence increase
was observed between 0 to 45 min after mixture, and the reaction was completed at
37°C within 20 min (Figure 3B). Further studies indicated that the emission intensity
reached the peak from pH 7.0 to 9.0 (Figure S3, Supporting Information), which
meets the requirement of real-time detection of H2S in the living body.
3.3 Selectivity of EPS-HS
Moreover, according to turn-on fluorescence responses, EPS-HS was found to be
more selective for sulfide than other biologically relevant thiols such as GSH, Cys and
Hcy et al.(Figure 4A), amino acids (Figure 4B) and other species (Figure S2,
Supporting Information) in PBS buffer. This may because that H2S is a small gas
molecule, its pKa1 is about 6.9, while pKa value of other thiol (such as GSH, Cys) in
cell is higher (about 8.5). The design principle of this type probe is based on thiol pKa
values to distinguish each other. The recognizing groups of probe EPS-HS we
designed is m-dinitrobenzene. The m-dinitrobenzene ether is used to protect tyrosine
8

ACCEPTED MANUSCRIPT
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
in synthesis of peptide chain. In weak alkali conditions, using thiol as sulfur agent can
remove the protection group. Therefore under the physiological conditions, the probe
EPS-HS could detect hydrogen sulfide selectively rather than thiols like GSH and
Hcy.
3.4 Application of EPS-HS in live-cell imaging
Then, the potential ability of EPS-HS to detect H2S was tested in HT22 cells
using confocal microscopy imaging (Figure 5). First of all, the cytotoxicity of
EPS-HS toward HT22 cells was examined by MTT assay. EPS-HS exhibited an IC50
of 58.99 ± 1.7 ꢀM, which demonstrated that EPS-HS was of low toxicity toward
cultured cell lines (Figure 5). Then, to assess the ability of EPS-HS for H2S
fluorescence imaging, HT22 cells were incubated with 10 ꢀM EPS-HS alone at 37°C
for 20 min (Figure 6, 1A) which is equivalent to the intracellular basal level of
endogenous H2S. As expected, incrementally stronger yellowish-green fluorescence
was detected in HT22 cells treated with 10 ꢀM EPS-HS and 10 ꢀM or 100 ꢀM Na2S
(Figure 6, 2B and 3B). These results indicate that EPS-HS is readily internalized into
living cells and act as a fluorescent probe to detect the concentration of H2S in living
cells (Figure 6C). Subsequently, cells were pretreated with ZnCl2 (an effcient
eliminator of H2S) and then incubated with EPS-HS (10 ꢀM) for 10 min. With the
addition of Na2S (100 ꢀM) and GSH (100 ꢀM) to the ZnCl2-pretreated cells, no
fluorescence intensity increases were observed (Figure 6, 4B). The results indicated
that the fluorescence change of EPS-HS in the cells arises from H2S.
3.5 Application of EPS-HS in mouse hippocampus imaging
9

ACCEPTED MANUSCRIPT
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
We further evaluated the visualization of H2S in KM mice hippocampal tissues
using EPS-HS. The mouse brains were rapidly taken and fixed in 4%
paraformaldehyde for 30 min. Then the tissues were embedded in ornithine
carbamoyltransferase (OCT), and serially sectioned at 7 ꢀm for fluorescent detection
analysis (Figure 7). As shown in Figure 7A, there was no background fluorescence
emission for hippocampal tissue slices after incubation with PBS working fluid. When
the slices pretreated with Na2S (10 ꢀM) (Figure 7B) and Na2S (100 ꢀM) (Figure 7C)
were incubated with EPS-HS (10 ꢀM) working fluid at 37°C for 20 min,
yellowish-green fluorescence signals were obviously observed. When the slices
pretreated with GSH (100 ꢀM) (Figure 7D) were incubated with EPS-HS (10 ꢀM)
working fluid at 37°C for 20 min, yellowish-green fluorescence signals were hardly
observed. These findings demonstrated that EPS-HS is capable of detecting H2S in the
tissues.
3.6 Detection of sulfide in mouse hippocampus
Finally, EPS-HS was added to measure sulfide concentrations in mouse
hippocampal tissues, where the spiked Na2S (X, X+0.2, X+0.4, X+0.6, X+0.8 ꢀM) were
used as internal standard. The spiked homogenate samples were subsequently
precipitated by DMSO to remove proteins. To the supernatant of the spiked
hippocampus homogenates, EPS-HS (10 ꢀM) was added. The mixture was incubated
in PBS buffer at 37°C for 20 min before analysis. We found that the average sulfide
concentration in fresh mouse hippocampus was 1.267 ± 0.020 ꢀmol/g protein (Table
1). Overall, these findings demonstrated that EPS-HS is suitable to detect sulfide in
10

ACCEPTED MANUSCRIPT
206
207
208
209
real biological samples in a rapid manner. Importantly, hydrogen sulfide is recognized
as a neuromodulator as well as neuroprotectant in the brain. Then, we repeated the
measurement with probe NAP-142. The concentration of sulfide was determined to be
1.242 ± 0.047 ꢀmol g-1 protein, which was consistent with the results from EPS-HS .
210 4. Conclusions
211
212
213
214
215
In summary, EPS-HS was designed as a benzothiazole - based H2S fluorescent
probe, which shows a significant emission increase in the fast response to sulfide
within the biologically relevant pH range. EPS-HS produces a turn-on fluorescence
signal for responding H2S. It can be applied for parallel measurement of sulfide
concentrations in HT22 cells and mouse hippocampus.
216 Acknowledgements
217
218
219
220
221
This study was financially supported by the National Natural Science Foundation
of China (81671069 to Y. L.), the Postdoctoral Science Foundation of China
(2012M521125 to L. Y) and the Open Program of Key Laboratory of Nuclear
Medicine, Ministry of Health and Jiangsu Key Laboratory of Molecular Nuclear
Medicine (KF201503).
222 Notes and references
223
224
225
226
227
228
229
230
231
232
1
Liu, C. et al. Capture and Visualization of Hydrogen Sulfide by a Fluorescent Probe.
Angewandte Chemie International Edition 50, 10327-10329 (2011).
Wang, X. et al. A near-infrared ratiometric fluorescent probe for rapid and highly sensitive
imaging of endogenous hydrogen sulfide in living cells. Chemical Science 4, 2551-2556
(2013).
2
3
4
Li, L., Rose, P. & Moore, P. K. Hydrogen Sulfide and Cell Signaling. Annual Review of
Pharmacology & Toxicology 51, 169-187 (2011).
Han, Y., Qin, J., Chang, X., Yang, Z. & Du, J. Hydrogen sulfide and carbon monoxide are in
synergy with each other in the pathogenesis of recurrent febrile seizures. Cellular &
Molecular Neurobiology 26, 101-107 (2006).
11

ACCEPTED MANUSCRIPT
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
5
6
Shim, H. S. & Longo, V. A Protein Restriction-Dependent Sulfur Code for Longevity. Cell 160,
15–17 (2015).
Yi, L. et al. A Dual-Response Fluorescent Probe Reveals the H2 O2 -Induced H2 S Biogenesis
through a Cystathionine beta-Synthase Pathway. Chemistry (Weinheim an der Bergstrasse,
Germany) 21, 15167-15172, doi:10.1002/chem.201502832 (2015).
7
8
Reiffenstein, R. J., Hulbert, W. C. & Roth, S. H. Toxicology of hydrogen sulfide. Annual
Review of Pharmacology & Toxicology 32, 109-134 (1992).
Szabo, C. et al. Tumor-derived hydrogen sulfide, produced by cystathionine-{beta}-synthase,
stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer [Pharmacology].
(2013).
9
Ruth, P., Elddie, R. M. & Juan, L. G. Hydrogen sulfide and hemeproteins: knowledge and
mysteries. Antioxidants & Redox Signaling 15, 393-404 (2011).
10
Shibuya, N. et al. 3-Mercaptopyruvate Sulfurtransferase Produces Hydrogen Sulfide and
Bound Sulfane Sulfur in the Brain | Abstract. Mary Ann Liebert Inc Huguenot Street Floor
New Rochelle Ny Usa.
11
Eto, K., Asada, T., Arima, K., Makifuchi, T. & Kimura, H. Brain hydrogen sulfide is severely
decreased in Alzheimer's disease. Biochemical
Communications 293, 1485–1488 (2002).
&
Biophysical Research
12
13
14
15
16
17
18
19
Wu, L. et al. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker
diabetic rats. Laboratory Investigation 89, 59-67 (2008).
Emanuela, P. et al. Hydrogen sulfide promotes calcium signals and migration in tumor-derived
endothelial cells. Free Radical Biology & Medicine 51, 1765-1773 (2011).
Nevoral, J. et al. Endogenously produced hydrogen sulfide is involved in porcine oocyte
maturation in vitro. Nitric Oxide 51, 24-35 (2015).
Li, H. D. et al. A fluorescent probe for H2S in vivo with fast response and high sensitivity.
Chemical Communications 51, 16225-16228 (2015).
Lu, M. et al. Hydrogen sulfide regulates intracellular pH in rat primary cultured glia cells.
Neuroscience Research 66, 92-98 (2010).
ZHOU, Cheng-fang, TANG & Xiao-qing. Hydrogen sulfide and nervous system regulation.
Chinese Medical Journal 124, 3576-3582 (2011).
Schreier, S. M. et al. Hydrogen Sulfide Scavenges the Cytotoxic Lipid Oxidation Product
4-HNE. Neurotoxicity Research 17, 249-256 (2010).
Tiong, C. X., Lu, M. & Bian, J. S. Protective effect of hydrogen sulphide against
6-OHDA-induced cell injury in SH-SY5Y cells involves PKC/PI3K/Akt pathway. British
Journal of Pharmacology 168, 2011–2012 (2013).
20
Kida, K. et al. Inhaled hydrogen sulfide prevents neurodegeneration and movement disorder
in a mouse model of Parkinson's disease. British Journal of Industrial Relations 50, 590-592
(2011).
21
22
Lefer, D. J. A new gaseous signaling molecule emerges: cardioprotective role of hydrogen
sulfide. Proceedings of the National Academy of Sciences 104, 17907-17908 (2007).
Zanardo, R. C. et al. Hydrogen sulfide is an endogenous modulator of leukocyte-mediated
inflammation. Faseb Journal Official Publication of the Federation of American Societies for
Experimental Biology 20, 2118-2120 (2006).
23
JW, E. et al. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by
12

ACCEPTED MANUSCRIPT
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
preservation of mitochondrial function. Proceedings of the National Academy of Sciences of
the United States of America 104, 15560-15565 (2007).
24
Y, K., Y, K., H, K. & I, N. L-cysteine inhibits insulin release from the pancreatic beta-cell:
possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitter.
Diabetes 55, 1391-1397 (2006).
25
26
27
28
Chem., A. Visualization of In Vivo Hydrogen Sulfide Production by a Novel Bioluminescence
Probe in Cancer Cells and Nude Mice. Analytical Chemistry (2015).
Ishigami, M. et al. A source of hydrogen sulfide and a mechanism of its release in the brain.
Antioxidants & Redox Signaling 11, 205-214 (2009).
Doeller, J. E. et al. Polarographic measurement of hydrogen sulfide production and
consumption by mammalian tissues. Analytical Biochemistry 341, 40-51 (2005).
Lawrence, N. S. et al. The Electrochemical Analog of the Methylene Blue Reaction: A Novel
Amperometric Approach to the Detection of Hydrogen Sulfide. Electroanalysis 12, 1453-1460
(2000).
29
30
31
32
33
Zhou, X., Lee, S., Xu, Z. & Yoon, J. Recent Progress on the Development of Chemosensors
for Gases. Chemical Reviews 115, 7944-8000 (2015).
Zhang, H. et al. An iminocoumarin benzothiazole-based fluorescent probe for imaging
hydrogen sulfide in living cells. Talanta 135, 2310-2313 (2015).
Zhang, C. et al. A Redox-Nucleophilic Dual-Reactable Probe for Highly Selective and
Sensitive Detection of H2S: Synthesis, Spectra and Bioimaging. Scientific Reports 6 (2016).
Sajal Kumar, D., Su, L. C., Young, Y. S., Hee, H. J. & Bong Rae, C. A small molecule
two-photon probe for hydrogen sulfide in live tissues. Cheminform 48, 8395-8397 (2012).
Sung Keun, B. et al. A ratiometric two-photon fluorescent probe reveals reduction in
mitochondrial H2S production in Parkinson's disease gene knockout astrocytes. Journal of the
American Chemical Society 135, 9915-9923 (2013).
34
35
Chen, B. et al. Fluorescent probe for highly selective and sensitive detection of hydrogen
sulfide in living cells and cardiac tissues. Analyst 138, 946-951 (2013).
Gryko, D. T. et al. Dipolar Dyes with Pyrrolo[2,3‐b]quinoxaline Skeleton Containing a
Cyano Group and a Bridged Tertiary Amino Group ‐ Synthesis, Solvatofluorochromism
and Bioimaging. Chemistry An Asian Journal 11 (2016).
36
37
38
Liu, Y., Meng, F., He, L., Liu, K. & Lin, W. A dual-site two-photon fluorescent probe for
visualizing lysosomes and tracking lysosomal hydrogen sulfide with two different sets of
fluorescence signals in the living cells and mouse liver tissues. Chemical Communications 52,
7016-7019 (2016).
Riswan Ahamed, M. A., Azarudeen, R. S. & Kani, N. M. Antimicrobial Applications of
Transition Metal Complexes of Benzothiazole Based Terpolymer: Synthesis, Characterization,
and Effect on Bacterial and Fungal Strains. Bioinorganic Chemistry & Applications 2014,
764085-764085 (2014).
Frisch, M. et al. Gaussian 09, revision A. 02; Gaussian, Inc. Wallingford, CT 19, 227-238
(2009).
39
40
Frisch, M. et al.
(Gaussian, Inc. Wallingford, CT, 2009).
Liu, S. D., Zhang, L. W. & Liu, X. A highly sensitive and selective fluorescent probe for Fe3+
based on 2-(2-hydroxyphenyl)benzothiazole. New Journal of Chemistry 37, 821-826 (2013).
Pratap, U. R., Mali, J. R., Jawale, D. V. & Mane, R. A. Bakers’ yeast catalyzed synthesis of
41
13

ACCEPTED MANUSCRIPT
321
322
323
324
benzothiazoles in an organic medium. Tetrahedron Letters 50, 1352-1354 (2009).
Ling, Z. et al. A colorimetric and ratiometric fluorescent probe for the imaging of endogenous
hydrogen sulphide in living cells and sulphide determination in mouse hippocampus. Organic
& Biomolecular Chemistry 12, 5115-5125 (2014).
42
325
14

ACCEPTED MANUSCRIPT
326
327
Article---Figure Legends
Figure 1. Optimized, low-energy conformations of the benzothiazole rings using DFT
328
(B3LYP/6-31G*) calculations: (A) Compound 1. (B) EPS-HS. (C) the structure of EPS-HS on
329
330
331
the HOMO and LUMO.
Route 1. The synthetic route of EPS-HS.
Figure 2. Fluorescence spectra (A) and absorption spectra (B) of EPS-HS (10
(10 M) and EPS-HS (10 M) + Na2S (100 M) in PBS buffer (20 mM, pH = 7.4, 5% DMSO).
Figure 3. Fluorescence spectra of EPS-HS (10 M) in PBS buffer (20 mM, pH 7.4, 5% DMSO) at
37°C for 20 min. Excitation: 285 nm, emission: 350 - 560 nm. (A) Incubated with different
concentrations of Na2S (0, 1.0, 2.0, 5.0, 10, 20, 30, 40, 50, 60, 80, 100, 150, 200 and 300 M) for
20 min. (B) Incubated with 100 M Na2S after 0, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 45 min.
Excitation: 285 nm, emission: 350 - 560 nm. Data are presented as the mean ± SD (n = 3).
ꢀM), Compound 1
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Figure 4. Fluorescence responses of EPS-HS (10 ꢀM) towards Na2S (100 ꢀM) and various
biothiols after 20 min of treatment. (A) (1) Na2S (0 ꢀM); (2) Na2S (100 ꢀM); (3) Hcy (100 ꢀM);
(4) GSH (100 ꢀM); (5) Cys (100 ꢀM); (6) Hcy (1 mM); (7) GSH (1 mM); (8) Cys (1 mM); (9)
Na2S (100 ꢀM)+Hcy (100 ꢀM); (10) Na2S (100 ꢀM) +Hcy (1 mM); (11) Na2S (100 ꢀM) + GSH
(100 ꢀM); (12) Na2S (100 ꢀM) + GSH (1 mM); (13) Na2S (100 ꢀM) + Cys (100 ꢀM); and (14)
Na2S (100 ꢀM) + Cys (1 mM). (B) Fluorescence responses of EPS-HS (10 ꢀM) towards Na2S
(100 ꢀM) and amino acids. Data are presented as the mean ± SD (n = 3).
Figure 5. (A) The inhibitory effect of EPS-HS on the cell growth of HT22 cells after treatment for
24 h. (B) The viability of HT22 cells after exposure to EPS-HS (10.0 ꢀM) for different times. Data
are presented as the mean ± SD (n = 3).
15

ACCEPTED MANUSCRIPT
348
349
350
351
352
353
354
355
356
357
358
359
360
Figure 6. Confocal fluorescence images in living cells. HT22 cells were incubated with EPS-HS
alone (10 ꢀM) for 20 min (1A and 1B). The cells were exposed to EPS-HS (10 ꢀM) followed by
Na2S (10 ꢀM) at 37oC for 20 min (2A). The cells were exposed to EPS-HS (10 ꢀM) followed by
Na2S (100 ꢀM) at 37oC for 20 min (3A). Cells were pretreated with 1 mM ZnCl2 for 10 min, then
incubated with EPS-HS (10 ꢀM) for 10 min and then incubated with Na2S (100ꢀM) and GSH (100
ꢀM) at 37°C for 20 min (4A); Overlay of the bright field image and the green channel (2B, 3B and
4B). Fluorescence intensity per one cell. Fluorescence images were acquired by confocal
microscopy (C). Scale bars = 10 ꢀm. Data are presented as the mean ± SD (n = 3).
Figure 7. In situ visualization of hippocampal tissues of the brains with EPS-HS. An image of
frozen mouse hippocampal slices incubated with PBS working fluid (A); an image of frozen
mouse hippocampal slices incubated with EPS-HS (10 ꢀM) working fluid after pretreatment with
Na2S (10 ꢀM) (B) and Na2S (100 ꢀM) (C) at 37oC for 20 min; Slices pretreated with GSH (100
ꢀM) were incubated with EPS-HS (10 ꢀM) working fluid at 37°C for 20 min (D). Scale bars = 20
ꢀm. The mean fluorescence intensity was obtained with 6-8 slices from different mice.
361
362
363
Table 1. Measurement of sulfide concentrations in fresh mouse hippocampus tissues
.
16

ACCEPTED MANUSCRIPT
Electronic Supplementary Information for
A benzothiazole-based fluorescent probe for selective
detection of H2S in living cells and mouse hippocampal
tissues
364
365
366
367
368
369
Yi Liu,*#a Yan Ding,*a Jie Huang,a Xiaofei Zhang,a Tongyong Fang,a Yuyun Zhang,a
Xian Zheng,a and Xia Yanga
370
371
372
373
a Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of
374
Pharmacy, Xuzhou Medical University Xuzhou, 221004 Jiangsu, China.
# Fax: +86-0516-83262136, E-mail: cbpeliuyinew@163.com.
375
376
17

ACCEPTED MANUSCRIPT
377
378
379
1. General information
2. Preparation of the test solutions
3. Routes and characterization of Compound 1
380
381
382
383
384
385
386
387
388
389
390
391
392
393
4. Evidence of mechanism detection
5. UV-Vis absorption spectrogram
6. Determination of the detection limit
7. MTT Assay
8. Fluorescence imaging in living HT22 cells
9. In situ fluorescent detection of H2S in the hippocampal tissues of the brains
Figure S1. EPS-HS probe incubated with different concentrations of Na2S
Figure S2. Selectivity of EPS-HS towards other species
Figure S3. Fluorescence spectra of EPS-HS with the presence of Na2S in buffer
solutions at different pH values
Figure S4. 1H NMR spectrum of Compound 1
Figure S5. HR-MS spectrum of EPS-HS
Figure S6. 1H NMR spectrum of EPS-HS
Figure S7. 13C NMR spectrum of EPS-HS
18

ACCEPTED MANUSCRIPT
394 1. Preparation of the test solutions
395
396
397
398
399
400
401
402
1.1 EPS-HS stock solution
EPS-HS (3.93 mg, 1 mmol) was dissolved into DMSO (10 mL) to get 1.0 mM
stock solution for general use.
1.2 Na2S stock solution
5 mg EDTA was dissolved in 10 mL DI H2O in a 25 mL volumetric flask. The
solution was purged vigorously with nitrogen gas for 15 min. Then 48 mg sodium
sulfide (Na2S·9H2O) was dissolved in the solution under nitrogen gas. The resulting
solution was 20 mM Na2S, which was then diluted to 1.0 - 2.0 mM stock solution for
general use.
403
404
405 2. Route and characterization of Compound 1
406
407
408
409
410
411
412
413
414
415
2-Aminothiophenol (0.625 g, 5 mmol) and 4-hydroxybenzaldehyde (0.611 g, 5
mmol) which was dissolved in 20 mL dry ethanol were added to a 250 mL
eggplant-shape flask equipped with a magnetic stirrer. Aq 37% HCl (15.0 mmol) and
aq 30% H2O2 (30 mmol) were added and the mixture was stirred for 1.5 h at room
temperature. A light yellow crystal was got by filtration. Then, the crude product was
purified by flash chromatography on silica gel obtaining at a yield of 92%. m.p.
188°C - 189.2°C. TLC (silica, hexane : EtOAc, 3:1 v/v): Rf = 0.3.
1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.54 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H),
7.93 (d, J = 8.0 Hz, 1H), 7.72 (dd, J1= 8.0 Hz, J2= 1.6 Hz, 1H), 7.51-7.55 (m, 1H),
7.39-7.46 (m, 2H), 7.13-7.15 (m, 1H), 6.97-7.01 (m, 1H). ESI-MS (m/z): 228.1 [M +
19

ACCEPTED MANUSCRIPT
416
417
H]+, calcd. for C13H9NOS = 227.04.
418 3. Evidence of mechanism detection
419
420
421
422
EPS-HS (78.6 mg, 0.2 mmol) was dissolved in DMSO (15 mL), followed by the
addition of Na2S - 9H2O (240 mg, 1.0 mmol) in PBS buffer (15mL, 20 mM, pH = 7.4).
The resultant mixture was stirred at room temperature for 3 h. Subsequently, EtOAc
(3 × 10 mL) was added into the solution for extraction. The thiolysis product was
characterized by HRMS and 1H NMR.
423
424
425 4. UV-Vis absorption spectrogram
426
427
428
429
Na2S is widely recognized as H2S donor. EPS-HS (10.0 ꢀM) was dissolved in
DMSO, which was then diluted in PBS buffer (2:3, v/v, 20 mM, pH 7.4) with 5%
DMSO. Before the addition of Na2S, EPS-HS (10 ꢀM) displayed an absorption peak
at 285 nm. After incubated with Na2S (100
ꢀM) in doubly distilled water for 20 min, a
430
431
new absorption peak was presented at 386 nm.
432 5. Determination of the detection limit
433
434
435
436
437
438
The detection limit was calculated based on the method reported in previous
literature. The fluorescence emission spectrum of EPS-HS without Na2S was
measured by 10 times and the standard deviation of blank measurement was obtained.
Then, the solution was treated with Na2S at concentrations from 0 to 60 ꢀM. A linear
regression curve was then achieved. The detection limit was calculated with the
following equation: Detection limit = 3σ/k, where σ is the standard deviation of blank
20

ACCEPTED MANUSCRIPT
measurements, and k is the slope between the fluorescence intensity ratios and Na2S
concentrations. The detection limit was 108 nM in PBS buffer and 154 nM in fetal
bovine serum, respectively.
439
440
441
442
443 6. MTT assay
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
The effect of EPS-HS on cell growth was measured using a colorimetric MTT
assay kit (Sigma-Aldrich). HT22 cells were seeded in 96-well plates at a density of
50,000 cells/well and then maintained in DMEM medium with 10% fetal bovine
serum/penicillin/streptomycin in a 5 % CO2 atmosphere at 37°C. The cells were
incubated with different concentrations of EPS-HS for 24 h, respectively. HT22 cells
in culture medium without EPS-HS were used as control. Then, 20 ꢀL of MTT dye
(3-[4, 5-dimethylthiazol-2-yl]- 2, 5-diphenyl tetrazolium bromide, 5 mg/mL in
phosphate buffered saline), was added to each well, and the plates were incubated at
37 °C for 4 h. Then, the remaining MTT solution was removed, and 150 ꢀL of DMSO
was added to each well to dissolve the formazan crystals. The plate was shaken for 10
min and the absorbance was measured at 570 nm on a microplate reader (ELX808IU,
Bio-tek Instruments Inc, USA). Each sample was performed in triplicate, and the
entire experiment was repeated three times. Calculation of IC50 values was done
according to Huber and Koella1.
459 7. Fluorescence imaging in living HT22 cells
460
HT22 cells were seeded in a 6-well plate in DMEM supplemented with 10 %
21

ACCEPTED MANUSCRIPT
461
462
463
464
465
466
fetal bovine serum in an environment of 5 % CO2 at 37°C for 24 h. For a control
experiment, HT22 cells were incubated with EPS-HS (10 ꢀM) at 37°C for 60 min.
After washing with PBS three times to remove the remaining dyes, the cells were then
incubated with Na2S (10 ꢀM) or Na2S (100 ꢀM) for another 20 min. The fluorescence
imaging was carried out after washing with PBS buffer for three times.
467 8. In situ fluorescent detection of H2S in the hippocampal tissues of
468 the brains
469
470
471
472
473
474
475
476
477
The mouse brains were rapidly taken and fixed in 4 % paraformaldehyde. Then
the tissues were embedded in ornithine carbamoyltransferase (OCT), and serially
sectioned at 7 ꢀm using a Leica CM1950 cryostat (Leica Microsystems, Wetzlar,
Germany) for fluorescence detection. After dried for 1 h, the frozen sections were
washed for 3 times with 10 mM PBS for 3 min. The slices treated with Na2S (100 ꢀM)
were incubated with EPS-HS working fluid (10 ꢀM ) at 37°C for 20 min and
subsequently rinsed in PBS for 3 times for 3 min. The slices were mounted with 50 %
buffered glycerol before observation under a fluorescence microscope in time. The
mean fluorescence intensity was obtained using 6 - 8 slices from different mice.
22

ACCEPTED MANUSCRIPT
478
479
480
Figure Legends
Figure S1. EPS-HS probe (10 ꢀM) incubated with 0, 1.0, 2.0, 5.0, 10, 20, 30, 40, 50, and 60 ꢀM
Na2S at 37°C for 20 min in PBS buffer (A) and fetal bovine serum (B) (5 % DMSO). The insert: A
graph of the fluorescence intensity trend of EPS-HS (10 ꢀM) with the presence of different
concentrations of Na2S for 20 min in PBS buffer (20 mM, pH = 7.4, 5 % DMSO) and fetal bovine
serum (5 % DMSO), respectively. Data are presented as the mean ± SD (n = 3).
481
482
483
484
485
486
487
488
489
Figure S2. (A) Fluorescence spectra of EPS-HS (10 ꢀM) with Na2S (100 ꢀM) and biologically
relevant species in 20 mM PBS (pH 7.4) at 37°C for 20 min. (B) Fluorescence responses of
EPS-HS (10 ꢀM) towards Na2S (100 ꢀM) and biologically relevant species. (1) Blank, (2) Na2S,
-
(3) H2O2, (4) OCl-, (5) O2-, (6) -OH, (7) tBuOOH, (8) NO2 , (9) NO, (10) S-nitroso glutathione, (11)
KCl, (12) ZnSO4, (13) CaCl2, (14) MgCl2, (15) FeCl3, (16) NaCl, (17) NaH2PO4, (18) NADH, (19)
2-
2-
2-
2-
2-
Glucose, (20) S2O3 , (21)S2O5 ,(22) S2O4 , (23) SO3 , (24) SO4 , and (25) SCN-.
490
491
492
493
494
495
496
497
Figure S3. (A) Fluorescence spectra of EPS-HS (10 ꢀM) with Na2S (100 ꢀM) in buffer solutions
at different pH vlues (20 mM, pH 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, 8.2, 8.6, and 9.0, 5 % DMSO) at
37°C for 20 min. (B) Fluorescence responses of EPS-HS (10 ꢀM) with Na2S (100 ꢀM) in buffer
solutions at different pH values (20 mM, pH 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, 8.2, 8.6, and 9.0, 5 %
DMSO) at 37°C for 20 min.
Figure S4. 1H NMR spectrum of Compound 1.
Figure S5. HR-MS spectrum of EPS-HS.
Figure S6. 1H NMR spectrum of EPS-HS.
498
499
Figure S7. 13C NMR spectrum of EPS-HS.
23

ACCEPTED MANUSCRIPT
500
501
1
Huber, W. & Koella, J. C. A comparison of three methods of estimating EC 50 in studies of
drug resistance of malaria parasites. Acta Tropica 55, 257-261 (1993).
502
24

ACCEPTED MANUSCRIPT
Table 1.
Sample
H2Sα
H2S b
(µ mol g-1 protein)
(µ mol g-1 protein)
1
1.275
1.201
2
1.245
1.231
3
1.282
1.294
Mean ± SD
1.267 ± 0.020
1.242 ± 0.047
b
α Measurement of H2S concentrations in mouse hippocampus with EPS-HS; Measurement of
H2S concentrations in mouse hippocampus with NAP-1; data are presented as mean ± SD.

ACCEPTED MANUSCRIPT
1
2
Figures
Figure 1.
3
4
5
Route 1.
6
7
8

ACCEPTED MANUSCRIPT
9
Figure 2.
10
11
12
13
Figure 3.
14
15
16
17
18
19
20
21
22

ACCEPTED MANUSCRIPT
23
Figure 4.
24
25
26
27
Figure 5.
28
29
30
31
32
33
34
35
36

ACCEPTED MANUSCRIPT
37
38
Figure 6.
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53