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Sulfonyl-semicarbazide group as a recognition site: A novel ratiometric fluorescent probe for hypochlorous acid and imaging in living cells
Journal Pre-proof Sulfonyl-semicarbazide group as a recognition site: A novel ratiometric fluorescent probe for hypochlorous acid and imaging in living cells Wei Ye, Qingna Bian, Yuelin Huang, Qingshuang Lin, Xinqi Zhan, Hong Zheng PII:
S0143-7208(19)31877-7
DOI:
https://doi.org/10.1016/j.dyepig.2019.107987
Reference:
DYPI 107987
To appear in:
Dyes and Pigments
Received Date: 8 August 2019 Revised Date:
19 October 2019
Accepted Date: 21 October 2019
Please cite this article as: Ye W, Bian Q, Huang Y, Lin Q, Zhan X, Zheng H, Sulfonyl-semicarbazide group as a recognition site: A novel ratiometric fluorescent probe for hypochlorous acid and imaging in living cells, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107987. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
1
Sulfonyl-Semicarbazide group as a recognition site: a novel ratiometric
2
fluorescent probe for hypochlorous acid and imaging in living Cells
3
Wei Ye a, Qingna Bian a, Yuelin Huang a, Qingshuang Linb, Xinqi Zhan b*, Hong
4
Zheng a *
5
a
6
MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Xiamen
7
University, Xiamen 361005, China
8
b
9
361102, China
Department of Chemistry, College of Chemistry and Chemical Engineering, and the
Basic Medicine Department, School of Medicine, Xiamen University, Xiamen,
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
∗ Corresponding author. Tel: +86 592 2186401; Fax: +86 592 2186731;
33
E-mail address: [email protected] (X.Q.Zhan); [email protected] (H. Zheng).
1
34 35
Abstract:
36
In this work a sulfonyl-semicarbazide moiety was developed initially to be a
37
selective recognition site for the design of HClO fluorescent probe. The
38
1,8-naphthalimide fluorophore platform was chosen to construct the probe by making
39
use of its intramolecular charge transfer (ICT) nature. By modifying the
40
electron-donating 4-amino group on the 1,8-naphthalimide molecular with the more
41
electron-deficient sulfonyl-semicarbazide group, probe 1 was synthesized and showed
42
ratiometric fluorescent responses towards HClO exclusively. This was ascribed to the
43
specific cleavage of HClO towards the substituent resulting in the release of
44
4-amino-1,8-naphthalimide. Also, the probe showed excellent performance in high
45
sensitivity and good selectivity towards HClO over other reactive oxygen species and
46
a wide variety of coexist species in biological pH condition. The probe was
47
successfully applied to detect hypochlorous acid in real water samples and image
48
HClO in living U-373MG cells.
49
Keywords: 4-Amino-1,8-naphthalimide fluorophore; Sulfonyl-semicarbazide;
50
Hypochlorous acid; Fluorescent ratiometry
51 52
1. Introduction
53
Hypochlorous acid (HClO), an important reactive oxygen species (ROS) produced
54
by the myeloperoxidase (MPO)-H2O2-Cl- system of phagocytes, plays a vital role in
55
the ability of these cells to kill a wide range of pathogens. However, the generation of
56
the potent oxidant also places the host at considerable risk since HClO has the
57
potential to damage host tissue through the same processes used in the destruction of
58
invading microorganisms. It was reported that abnormal amount of HClO may cause
59
various diseases such as atherosclerosis, rheumatoid arthritis and even cancers [1-3]. 2
60
A surge of research into HClO biology in order to elucidate the exact functions in
61
pathologic processes has accelerated the development of fluorescent probes for HClO
62
detection that are specific and sensitive under physiological conditions in recent years,
63
and several reviews have been reported in the field [4-7].
64
As a key element for fluorescent probes, recognition moieties that could selective
65
and sensitive response to HClO are indispensable for fluorescent probe designing.
66
Nowadays, a variety of recognition moiety sites for HClO sensing were proposed
67
which engineered in various fluorescent architectures via different sensing
68
mechanisms. There are some catalogs of recognition groups for HClO such as: (1)
69
Chalcogenide (S, Se, Te), of which sulphur [8,9], selenium [10-12] and tellurium [12]
70
atoms could be oxidized or chloridized easily with HClO. (2) Recognition group of
71
2-ethylamine
72
monothio-bishydrazide
73
N,N-dimethylthiocarbamate[25,26] (3) C ﹦ C unsaturated bond [27-29]. (4)
74
p-Methoxyphenol [30,31] or p-alkoxyaniline groups [32,33]. (5) Borane groups
75
[34,35] and others [36,37]. Although great advances have been gained in this area,
76
there are still rooms for searching new recognition groups for HClO sensing. In this
77
work, we propose a new moiety, namely sulfonyl-semicarbazide, for HClO sensing.
78
We chose 1,8-naphthalimide fluorophore as a platform to construct the probe not only
79
because of its excellent properties such as good photo stability, high fluorescence
80
quantum yields and large Stokes’ shift, but also because of its intramolecular charge
81
transfer (ICT) nature which is more easy to acquire a desirable ratiometric fluorescent
thiourea
derivatives [18,19],
[13,14],
thiohydrazide
3
dihydrazide [21-23],
oxime
[15-17], [24],
82
response. As we all know, compared with the intensity-based fluorescent probes
83
which may be affected by the variations in environment, concentration and excitation
84
intensity, ratiometric fluorescent probes can effectively alleviate these problems since
85
they recorded ratio signals of two emission intensities at different wavelength
86
affording a built-in correction. Up to date a variety of ratiometric fluorescent probes
87
for HClO sensing have been reported [25, 29, 38] and reviewed [39]. Previously our
88
group also developed a probe with the excitation wavelength and the dual-emission
89
wavelengths located at NIR region [14].
90
We herein present rationally designed probe 1 by modifying the electron-donating
91
4-amino group on the 1,8-naphthalimide molecular with the more electron-deficient
92
sulfonyl-semicarbazide group
93
from colorless with absorption band at λmax = 370 nm to yellow with that at λmax =
94
435 nm in the presence of HClO, accompanied by a change of fluorescent emission
95
peak at 488 nm to 542 nm. The ratiometric fluorescent behavior could be rationalized
96
by the HClO-induced transformation of sulfonyl-semicarbazide moiety to 4-amino
97
group which affected the characteristics of intramolecular charge transfer (ICT) of the
98
1,8-naphthalimide fluorophore.
(Scheme 1). 1 displayed remarkable color change
99
100 4
101
Scheme 1. Modified as sulfonyl-semicarbazide and its reaction with HClO.
102 103
2. Experimental
104
2.1. Instrumentation
105
NMR spectra were recorded on Bruker AV400 NMR. Mass spectra were measured
106
with a JEOL JMS-T100LC mass spectrometer (ESI+). HR-MS spectra were measured
107
with a Bruker En Apex ultra 7.0T FT-MS mass spectrometer. Fluorescence spectra
108
were performed on a Hitachi F7000 fluorescence spectrometer (Tokyo, Japan).
109
Absorption spectra were recorded on a HITACHI U3900 spectrophotometer.
110
Fluorescence images were captured using a Olympus Fluoriew 1000 laser-scanning
111
confocal microscope.
112 113 114
2.2. Materials Doubly
distilled
water
was
used
throughout
the
experiments.
3,
115
3-Dimethylglutaricacid was purchased from Alfa Aesar. H2O2 (aq. 30%) and NaOCl
116
(aq. 6%) were purchased from Shanghai Reagent Co. of China. A stock solution of
117
NaClO was standardized at 292 nm using an extinction coefficient 350 M−1 cm−1 at
118
pH 12 [40]. Nitric oxide (NO) was prepared from l-hydroxy-2-oxo-3- (3-aminopropyl)
119
-3- methyl-l-triazene, which was synthesized according to the literature [41]. ROO•
120
was generated from 2,2-Azobis(2-amidinopropane)dihydrochloride (100 µM) which
121
was firstly prepared in deionizer water and then added appropriate amount into the
122
probe testing solutions at 37 °C for 1 h [41]. Superoxide was generated from KO2 in 5
123
aqueous solution [42]. Hydroxyl radical was generated by Fenton reaction in which
124
ferrous chloride (100 µM) was added in the presence of 10 eq. of H2O2 [42]. Singlet
125
oxygen was generated from 3-(1,4-Dihydro-1,4-epidioxy-1-naphthyl)propionic acid
126
(100 µM) which was firstly prepared in deionizer water and then added appropriate
127
amount into the probe testing solutions at 37 °C for 1 h [42]. Peroxynitrite solution
128
was synthesized according to the report [43] and the concentration was determined
129
with an extinction coefficient of 1670 ± 50 cm−1 (mol/L)−1 at 302 nm. Glutathione
130
(GSH), cysteine (Cys) and trypan blue were purchased from J & K Science Ltd.
131
Lipopolysaccharides (LPS) and phorbol myristate acetate (PMA) were purchased
132
from Sigma-Aldrich. Solutions for testing foreign anions and cations were prepared
133
by dissolving their corresponding sodium salts and nitrates in water, respectively.
134
Reactions were monitored by TLC with visual observation of the dye spots. Products
135
were purified by column chromatography.
136 137 138
2.3. Synthesis of probe 1 The solution of para-toluensulfonyl chloride (42 mg, 1.2 eq) in acetonitrile was
139
added dropwise to the acetonitrile solution containing compound 2[44] (60 mg, 1.0 eq)
140
and pyridine (297 mg, 20.0 eq). The mixture was refluxed under N2 atmosphere
141
overnight. When the reaction was completed, the acetonitrile was removed under
142
reduced pressure. The residue was extracted with ethyl acetate, and the organic layer
143
was collected, washed with water and then dried over sodium sulfate. The organic
144
solution was filtered and evaporated under reduced pressure, and the residue was
6
145
purified by silica gel chromatography using ethyl acetate: petroleum ether (1/4 = v / v)
146
as an eluent to get a yellow powder in 52.4% yield.
147
1
148
8.57 (m, 1H), 8.50 (m, 1H), 8.40 (d, J = 6.8Hz, 1H), 8.18 (d, J = 6.4Hz, 1H), 7.83 (m,
149
1H), 7.78 (d, J = 6.8Hz, 2H), 7.41(d, J = 6.4Hz, 2H), 4.02 (t, J = 6.0Hz, 2H), 2.38 (s,
150
3H), 1.60 (m, 2H), 1.34(m, 2H), 0.92 (t, J = 5.6Hz, 3H).
151
13
152
141.657, 136.048, 134.207, 132.369, 131.185, 130.236, 130.073, 128.810, 128.225,
153
127.530, 126.703, 122.711, 116.341, 39.610, 30.155, 21.504, 20.264, 14.182.
154
FT-MS: calcd. for C24H24N4O5S, 479.13891 (M-), found, 479.13802.
H NMR (d6-DMSO, 400MHz) δ (ppm): δ 9.84 (s, 1H), 9.49 (s, 1H), 9.09 (s, 1H),
C NMR (d6-DMSO, 100MHz) δ (ppm):163.947, 163.382, 154.660, 143.979,
155 156
2.4 Preparation process of compound 3 by reaction of 1 with NaClO
157
A pH 6.0 aqueous solution of NaClO (1.2 eq) was added dropwise to the acetone
158
solution containing probe 1 (30 mg, 1.0 eq) at room temperature. The mixture was
159
stirred overnight. The solution was removed under reduced pressure, and the residue
160
was purified by silica gel chromatography to get 3 in ca.61.3% yield.
161
1
162
1H), 8.18 (d, J = 8.4 Hz, 1H), 7.65 (m, 1H),7.44 (s, 2H), 6.84 (d, J = 8.4 Hz, 1H), 4.00
163
(t, J = 7.2 Hz, 2H), 1.65–1.51(m, 2H), 1.40–1.27 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).
164
13
165
130.14, 129.73, 124.44, 122.27, 119.83, 108.62, 108.04, 39.36, 30.31, 20.31, 14.22.
166
FT-MS: calcd. for C16H15N2O2,(3-H) +, 267.11335, found, 267.11335
H NMR (d6-DMSO,400 MHz): δ (ppm) 8.60 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 7.3 Hz,
C NMR (d6-DMSO, 100 MHz): δ (ppm) 164.23, 163.37, 153.14, 134.39, 131.44,
167 168 169 170
2.5 General procedure for the measurement of absorption and fluorescence spectra Probe 1 was dissolved in 1,4-dioxane to make a 1.0×10-3M stock solution. NaClO was dissolved in water to make a 1.0×10-3 M stock solution for total HClO.
7
171
To 5-mL glass tubes containing 0.40 mL of 100 mM 3, 3-dimethylglutaric
172
acid-NaOH buffer solution (pH 7.0), 1.0 mL acetone and various amount of stock
173
solution of total hypochlorous acid were added, respectively. Then the solution was
174
diluted with redistilled water to the scale and mixed thoroughly, and 50.0 µL of the
175
stock solution of probe 1 was added to the solution finally and mixed well. The
176
absorption and fluorescence sensing of HClO were run after 2 minutes.
177 178
2.6 Cytotoxicity assay
179
For each dishes, 180 µL of the cells was added onto a 96-well plate, followed by
180
addition of 20 µL of increasing concentrations of the probe 1 (95% PBS and 5%
181
acetone) and the final concentrations of the probe were kept from 0.1µM to 100µM
182
(n=3). The incubation was at 370C in an atmosphere of 5% CO2 in a CO2 incubator
183
for 4h, followed by trypan blue assays. Untreated assays in PBS-acetone (V/V : 95 / 5)
184
(n=3) was also conducted at the same conditions.
185 186
2.7 Cell imaging
187
Cell cultures: U-373MG cells were cultured in DMEM (Dulbecco’s modified
188
Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) in an atmosphere
189
of 5% CO2 and 95% air at 37 0C.
190
Imaging of exogenous HOCl in cells: Before imaging, the cells were seeded on
191
two groups of 20 mm glass-bottomed dishes and incubated with probe 1 (10 µM
192
containing 5% acetone as a cosolvent in PBS) for 30 min at 37℃. Rinsed with PBS
193
two times, the second group was incubated with NaOCl (50 µM) for 30 min at 37 0C.
194
Imaging of endogenous HOCl: Two groups of cells were seeded on 20mm
195
glass-bottomed dishes and incubated with probe 1 (10 µM containing 5% acetone as a
196
cosolvent in PBS) for 30 min at 37℃. Rinsed with PBS two times, the second group
197
was incubated with 2mg/mL PMA (phorbol 12-myristate 13-acetate) and 2mg/mL
198
LPS (lipopoly-saccharides) for 2 h at 37 0C. 8
199
3. Results and Discussion
200
3.1 Design and synthesis of probe 1
201
Modulation of the electron-donating 4-amino donor on a 1,8-naphthalimide affects
202
both ICT and emission color, so we changed this amino donor into a more
203
electron-withdrawing carbamate group, namely
204
result in ICT-induced blue shifts in emission maxima. We reasoned that when the
205
group was removed by HClO back to the initial amino donor it would provide a
206
switch for ratiometric detection of HClO. The synthetic procedures of probe 1 are
207
given in Scheme 2; probe 1 was synthesized from compound 2 [44] by one-step
208
procedure with a total yield of 52%, and the structure of probe 1 was confirmed by 1H
209
NMR, 13C NMR, and FT-MS spectroscopy (Figures S1, S2 and S3).
sulfonyl-semicarbazide, would
210 211
Scheme 2. Synthetic route for probe 1
212 213
3.2 Spectral characteristics
214
The absorption spectra of 1 as well as the solutions in presence of HOCl were
215
monitored firstly in buffer solutions of 3,3-dimethylglutaric acid-NaOH (pH 7.0,
216
10mM in Acetone/H2O = 20/80, v/v). As shown in Figure 1, the solution of 1 alone
217
(1.0×10-5 M) exhibited an absorption maximum at 370 nm. With the addition of HClO,
218
a new absorption band at 435 nm increased at the expense of the decrease of the band
219
at 370 nm with an isosbestic point at 412 nm which could be attributed to the
220
formation of a new species. Also, a large bathochromic shift of 65 nm in the
9
221
absorption behavior changes the color of the resultant solution from colorless to
222
yellow, which could be detected even by the naked.
223
The fluorescence characteristics of probe 1 were also studied. When excited at 412
224
nm the free probe 1 exhibited an emission peak at 488 nm. Upon consistent
225
introduction of HClO, the peak at 488 nm gradually decreased accompanied by a new
226
peak at 542 nm emerged increasingly with a clear iso-emission point at 513 nm. Thus
227
a ratiometric fluorescence response was observed.
228 229
Figure 1. Absorption spectra of 1 with HOCl at pH 7.0 of 0.01 M
230
3,3-dimethylglutaric acid-NaOH buffer solution (Acetone / H2O = 20/80, v/v). [1] =
231
10.0 µmol/L, [total HOCl] = 10.0 µmol/L.
232
10
233 234
Figure 2. Fluorescence titration of probe 1 (10 µM) at pH 7.0 of 0.01 M
235
3,3-dimethylglutaric acid-NaOH buffer solution (Acetone/H2O = 2/8, v/v) in the
236
presence of different amounts of total HOCl. Excitation was performed at 412 nm.
237 238
3.3 Effect of pH dependency of the ratiometric signaling behavior
239
As showed in Figure 3, the fluorescent ratio signals of probe 1 itself, F542/F488,
240
remain stable and relatively small over a pH range of 2.0-7.4. In the presence of
241
HOCl, negligible variation of the fluorescent ratios was observed when pH within
242
5.0-8.0. Thus buffer solutions of 3,3-dimethylglutaric acid/NaOH at pH 7.0 were
243
chosen as for determination in this work.
11
244 245
Figure 3. pH-dependent fluorescence intensity ratio changes of probe 1 only and the
246
probe 1 with total HClO. [1] = 10 µM, [total HOCl] = 10 µM. pH 7.0 of 0.01 M
247
3,3-dimethylglutaric acid/NaOH buffer solution (Acetone/H2O = 2/8, v/v). Excitation
248
was performed at 412 nm.
249 250
3.4 Time-dependent fluorescent intensity ratio changes of probe 1
251
Further, the fluorescence intensity ratios (F542/F488) were recorded as a function of
252
reaction time. It can be observed that the ratio signals exhibited no noticeable changes
253
in case of free probe 1, while reached their maximum within 2 min in presence of four
254
tested total HClO concentrations (Figure 4). Thus an incubation time of 2 min was
255
used in this work.
12
256 257
Figure 4. Kinetics of the ratiometric fluorescent responses of 1 toward various
258
concentrations of total HOCl at pH 7.0 of 0.01 M 3,3-dimethylglutaric acid-NaOH
259
buffer solution (Acetone/H2O = 20/80, v/v). [1] = 10µM; [total HOCl] = 0, 1.0, 4.0,
260
7.0, 10.0µM, respectively (from black bottom to purple top).
261
performed at 412 nm.
Excitation was
262 263
3.5 Selectivity of the reaction of 1 with HClO
264
As the absorption spectra of probe 1 changed significantly upon reaction with
265
HClO, we firstly used the absorbance ratio at two wavelengths (A435/370 nm) to study
266
the selectivity of HClO toward ROSs. Important ROSs, such as H2O2, O21, NO, O2•−,
267
ONOO−, •OH, ROO•, and total HClO, were tested toward a solution of 1.0 equiv. of
268
1 (10 µM) at the concentration of 10 µM respectively. HClO was found to greatly
269
enhance the absorbance ratio (A435/370) of probe 1 exclusively with no noticeable
270
response of other ROSs (Figure 5) .
13
271 272
Figure 5. Ratiometric absorbance responses of 1 (10 µM) with 1.0 equiv of ROSs at
273
pH 7.0 of 0.01M 3,3-dimethylglutaric acid-NaOH buffer solution (Acetone/H2O =
274
20/80, v/v). [1] = 10.0 µM. ROSs adding (from left to right at horizontal ordinate): (1)
275
none, (2) total HOCl, (3) H2O2, (4) O2·-, (5) ONOO- , (6) •OH, (7) NO, (8) 1O2 and (9)
276
ROO•. [ROS] = 10.0 µM, respectively.
277 278
Accordingly,
the
ratiometric
fluorescent
responses
of
1
toward
the
279
above-mentioned ROSs were also tested independently. As showed in Figure 6, the
280
solution of 1 alone exhibits nearly no change in the fluorescence ratio (F542 nm / F488 nm )
281
upon the addition of the ROSs except HClO. Remarkable increase of the ratio value
282
was obtained exclusively in presence of HClO, indicating that our probe showed
283
selective response towards HClO over other ROSs.
284
Moreover, a wide variety of other interference species, such as ionic species and
285
representative biothiols, including Ca2+, Co2+, Ni2+, Fe3+, Zn2+, Cu2+, F-, Cl-, I-, NO3-,
286
SO42-, CH3COO-, IO3-, PO43-, glutathione (GSH) and cysteine (Cys), were also tested
287
respectively to show negligible interference towards HClO sensing of probe 1 (Figure
288
S4). In addition, the coexistence effects of the ions and biothiols on ratiometric
289
fluorescence response of HClO were also evaluated and the results showed that 10.0
290
times excess of different coexist species caused almost no interference behavior onto
291
the detection (Figure S5). 14
292
293 294
Figure 6. Ratiometric fluorescent responses of 1 (10 µM) with 1.0 equiv of ROSs at
295
pH 7.0 of 0.01M 3,3-dimethylglutaric acid-NaOH buffer solution (Acetone/H2O =
296
20/80, v/v). [1] = 10.0 µM. ROSs adding (from left to right at horizontal ordinate): (1)
297
none, (2) total HOCl, (3) H2O2, (4) O2·-, (5) ONOO- , (6) •OH, (7) NO, (8) 1O2 and (9)
298
ROO•. [ROS] = 10.0 µM, respectively.
299 300
3.6 Analytical figures and Application of the method in real water samples
301
According to the optimized conditions, the calibration curves were constructed. The
302
linear range for total HClO showed to be (0.1–6.0)×10-6 mol/L with the correlation
303
coefficient R2 = 0.9966 (Figure 7). The detection limit, based on the definition by
304
IUPAC [45] (CDL =3Sb/m), was found to be 6.1×10-8 mol L-1 from eleven blank
305
solutions.
306
Freshly collected lake and tap water samples in our school were analyzed by the
307
proposed method. Tap water samples were used with no pre-treatment, the lake water
308
samples were filtered firstly to remove minute sediments. Different water samples
309
were collected at different times and tested under the optimized condition
310
subsequently. The water samples spiked with standard NaClO at different
311
concentration levels were also analyzed by proposed method. The results were
312
summarized in Table S1 with satisfactory recovery, indicating that the present 15
313
fluorescent probe can be applicable for the determination of total HClO in real water
314
samples.
315 316
Figure 7. The linear range of ratiometric fluorescent response of 1 vs. the
317
concentrations of total HClO at pH 7.0 of 0.01M 3,3-dimethylglutaric acid-NaOH
318
buffer solution (Acetone/H2O = 20/80, v/v).[1] = 10.0 µM.
319 320
3.7 Reaction Mechanism
321
It was reported that dibenzoylhydrazine can be selectively oxidized by HClO to a
322
reactive intermedia, dibenzoyl diimide, and further undergo hydrolysis in aqueous
323
solvents [17]. We hypothesized that reaction of probe 1 with HClO may conduct a
324
similar process. The possible mechanism was supposed as follow (Scheme 3):
16
325
Scheme 3 The proposed mechanism of probe 1 with HClO
326 327 328
To verify the reaction mechanism supposed, we first take the reaction solution of 1
329
with HOCl for FT-MS experiment. As expected, product 3 was confirmed by its
330
high-resolution mass spectra (HR-MS): calcd. for C16H15N2O2, 267.11335(3-H) +,
331
found,
332
4-amino-1,8-naphthalimide fluorophore was produced after the reaction of 1 with
333
HOCl. Furthermore, the 1H and
334
HOCl to be 4-amino-1,8-naphthalimide fluorophore 3 (Figure S7 and S8).
335
Significantly, the proton signals of δ 9.84 (s, 1H), 9.49 (s, 1H) and 9.09 (s, 1H) on
336
p-TsNHNHCONH group in probe 1 are disappeared in product 3; moreover, the
337
proton signals of 7.78 (d, J = 6.8Hz, 2H), 7.41(d, J = 6.4Hz, 2H) and 2.38 (s, 3H) on
338
CH3C6H4 group in probe 1 are also disappeared in product 3; finally, the proton
267.11335
(Figure
S6).
13
The
result
preliminarily
proved
that
C NMR data confirmed that the product of 1 with
17
339
signals of 7.44 (s, 2H) on 4-NH2-1,8-naphthalamide fluorophore (the compound 3),
340
are appeared. These results confirmed the reliability of the reaction mechanism we
341
envisaged in Scheme 3. Thus the ratiometric fluorescent behavior of 1 towards HClO
342
could be rationalized by the HClO-induced transformation of electron-deficient
343
sulfonyl-semicarbazide moiety to electron-donating amino group on 4-position on the
344
1,8-naphthalimide fluorophore which affected the characteristics of intramolecular
345
charge transfer (ICT).
346 347
3.8 Cell Cytotoxicity assay
348
The cell livability was expressed as percent (mean for three assays) relative to
349
untreated cells as shown in Fig. 8. It could be seen that the probe was almost nontoxic
350
to cells when its concentration was below 100 µM. Therefore, probe 1 was applied for
351
fluorescence imaging of HOCl in cells.
352
353 354
Figure 8. Cytotoxicity study of probe 1 for cells.
355 356 18
357
3.9 Cell imaging of probe 1
358
Firstly, we applied probe 1 to imaging exogenous HOCl in living U-373MG cells.
359
As exhibited in Figure 9, the living U-373MG cells incubated with 1 for 0.5 h at 37℃
360
provided a strong blue fluorescence in the short wavelength emission window (Figure
361
9a) while less green fluorescence in the long wavelength emission window (Figure
362
9b). The living U-373MG cells treated firstly with NaOCl and then with 1 gave a
363
marked decrease in the blue emission (Figure 9e) and a significant increase in the
364
green emission (Figure 9f), consistent with the HOCl-induced ratiometric fluorescent
365
response.
366 367
Figure 9. Confocal fluorescence imaging without (up) and with (down) exogenous
368
NaClO.
369
collected at long wavelength emission window; (c, g) merged images of (a+b) and
370
(e+f); (d, h) bright field image.
(a, e) images collected at short wavelength emission window; (b, f) images
371 372
We then proceeded to investigate the feasibility of the probe for imaging
373
endogenous HOCl in living U-373MG cells. The cells which incubated only with the
374
probe displayed strong fluorescence in the blue channel (Fig. S9a) and almost no
375
fluorescence in the green channel (Fig. S9b). However, a marked enhancement in the
376
green channel (Fig. S9d) was observed in the cells stimulated by lipopolysaccharides
377
(LPS, 2mg/mL) and phorbol myristate acetate (PMA, 2mg/mL) together. The
378
remarkably increased green fluorescence signal is mainly due to the reaction between
379
the probes and endogenous HOCl, indicating that the probe was cell membrane
380
permeable and could be employed for fluorescence imaging of HOCl in living cells. 19
381 382
Conclusion
383
In summary, a naphthalimide-based fluorescent probe has been successfully
384
developed for the rapid ratiometric fluorescence detection of hypochlorous acid based
385
on the specific HOCl-promoted oxidation-hydrolysis of sulfonyl-semicarbazide
386
group. The experimental results clearly indicate that probe 1 can be used as a
387
chemodosimeter for HOCl with good selectivity and high sensitivity. In addition,
388
confocal fluorescence microscopy imaging using U-373MG cells showed that the new
389
probe could be used as a promising fluorescent probe for real water samples and
390
imaging HOCl in living cells.
391 392
Conflicts of interest
393
There are no conflicts to declare.
394 395
Acknowledgments
396
This work was financially supported by the National Natural Science Foundation
397
of China (No. 21435003), Program for Changjiang Scholars and Innovative Research
398
Team in University (PCSIRT, No.IRT13036).
399 400
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25
Highlights
•An ICT-based ratiometric fluorescence probe for HClO was developed with the skeleton of 4-amino-1, 8-naphthalimide fluorophore.
•A novel recognition mechanism that specific removing a sulfonyl-semicarbazide group by HClO was observed.
•The probe showed high selectivity and sensitivity toward HOCl and was applied in real water samples and imaging HClO in living U-373MG cells..
S0143-7208(19)31877-7
DOI:
https://doi.org/10.1016/j.dyepig.2019.107987
Reference:
DYPI 107987
To appear in:
Dyes and Pigments
Received Date: 8 August 2019 Revised Date:
19 October 2019
Accepted Date: 21 October 2019
Please cite this article as: Ye W, Bian Q, Huang Y, Lin Q, Zhan X, Zheng H, Sulfonyl-semicarbazide group as a recognition site: A novel ratiometric fluorescent probe for hypochlorous acid and imaging in living cells, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107987. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
1
Sulfonyl-Semicarbazide group as a recognition site: a novel ratiometric
2
fluorescent probe for hypochlorous acid and imaging in living Cells
3
Wei Ye a, Qingna Bian a, Yuelin Huang a, Qingshuang Linb, Xinqi Zhan b*, Hong
4
Zheng a *
5
a
6
MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Xiamen
7
University, Xiamen 361005, China
8
b
9
361102, China
Department of Chemistry, College of Chemistry and Chemical Engineering, and the
Basic Medicine Department, School of Medicine, Xiamen University, Xiamen,
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
∗ Corresponding author. Tel: +86 592 2186401; Fax: +86 592 2186731;
33
E-mail address: [email protected] (X.Q.Zhan); [email protected] (H. Zheng).
1
34 35
Abstract:
36
In this work a sulfonyl-semicarbazide moiety was developed initially to be a
37
selective recognition site for the design of HClO fluorescent probe. The
38
1,8-naphthalimide fluorophore platform was chosen to construct the probe by making
39
use of its intramolecular charge transfer (ICT) nature. By modifying the
40
electron-donating 4-amino group on the 1,8-naphthalimide molecular with the more
41
electron-deficient sulfonyl-semicarbazide group, probe 1 was synthesized and showed
42
ratiometric fluorescent responses towards HClO exclusively. This was ascribed to the
43
specific cleavage of HClO towards the substituent resulting in the release of
44
4-amino-1,8-naphthalimide. Also, the probe showed excellent performance in high
45
sensitivity and good selectivity towards HClO over other reactive oxygen species and
46
a wide variety of coexist species in biological pH condition. The probe was
47
successfully applied to detect hypochlorous acid in real water samples and image
48
HClO in living U-373MG cells.
49
Keywords: 4-Amino-1,8-naphthalimide fluorophore; Sulfonyl-semicarbazide;
50
Hypochlorous acid; Fluorescent ratiometry
51 52
1. Introduction
53
Hypochlorous acid (HClO), an important reactive oxygen species (ROS) produced
54
by the myeloperoxidase (MPO)-H2O2-Cl- system of phagocytes, plays a vital role in
55
the ability of these cells to kill a wide range of pathogens. However, the generation of
56
the potent oxidant also places the host at considerable risk since HClO has the
57
potential to damage host tissue through the same processes used in the destruction of
58
invading microorganisms. It was reported that abnormal amount of HClO may cause
59
various diseases such as atherosclerosis, rheumatoid arthritis and even cancers [1-3]. 2
60
A surge of research into HClO biology in order to elucidate the exact functions in
61
pathologic processes has accelerated the development of fluorescent probes for HClO
62
detection that are specific and sensitive under physiological conditions in recent years,
63
and several reviews have been reported in the field [4-7].
64
As a key element for fluorescent probes, recognition moieties that could selective
65
and sensitive response to HClO are indispensable for fluorescent probe designing.
66
Nowadays, a variety of recognition moiety sites for HClO sensing were proposed
67
which engineered in various fluorescent architectures via different sensing
68
mechanisms. There are some catalogs of recognition groups for HClO such as: (1)
69
Chalcogenide (S, Se, Te), of which sulphur [8,9], selenium [10-12] and tellurium [12]
70
atoms could be oxidized or chloridized easily with HClO. (2) Recognition group of
71
2-ethylamine
72
monothio-bishydrazide
73
N,N-dimethylthiocarbamate[25,26] (3) C ﹦ C unsaturated bond [27-29]. (4)
74
p-Methoxyphenol [30,31] or p-alkoxyaniline groups [32,33]. (5) Borane groups
75
[34,35] and others [36,37]. Although great advances have been gained in this area,
76
there are still rooms for searching new recognition groups for HClO sensing. In this
77
work, we propose a new moiety, namely sulfonyl-semicarbazide, for HClO sensing.
78
We chose 1,8-naphthalimide fluorophore as a platform to construct the probe not only
79
because of its excellent properties such as good photo stability, high fluorescence
80
quantum yields and large Stokes’ shift, but also because of its intramolecular charge
81
transfer (ICT) nature which is more easy to acquire a desirable ratiometric fluorescent
thiourea
derivatives [18,19],
[13,14],
thiohydrazide
3
dihydrazide [21-23],
oxime
[15-17], [24],
82
response. As we all know, compared with the intensity-based fluorescent probes
83
which may be affected by the variations in environment, concentration and excitation
84
intensity, ratiometric fluorescent probes can effectively alleviate these problems since
85
they recorded ratio signals of two emission intensities at different wavelength
86
affording a built-in correction. Up to date a variety of ratiometric fluorescent probes
87
for HClO sensing have been reported [25, 29, 38] and reviewed [39]. Previously our
88
group also developed a probe with the excitation wavelength and the dual-emission
89
wavelengths located at NIR region [14].
90
We herein present rationally designed probe 1 by modifying the electron-donating
91
4-amino group on the 1,8-naphthalimide molecular with the more electron-deficient
92
sulfonyl-semicarbazide group
93
from colorless with absorption band at λmax = 370 nm to yellow with that at λmax =
94
435 nm in the presence of HClO, accompanied by a change of fluorescent emission
95
peak at 488 nm to 542 nm. The ratiometric fluorescent behavior could be rationalized
96
by the HClO-induced transformation of sulfonyl-semicarbazide moiety to 4-amino
97
group which affected the characteristics of intramolecular charge transfer (ICT) of the
98
1,8-naphthalimide fluorophore.
(Scheme 1). 1 displayed remarkable color change
99
100 4
101
Scheme 1. Modified as sulfonyl-semicarbazide and its reaction with HClO.
102 103
2. Experimental
104
2.1. Instrumentation
105
NMR spectra were recorded on Bruker AV400 NMR. Mass spectra were measured
106
with a JEOL JMS-T100LC mass spectrometer (ESI+). HR-MS spectra were measured
107
with a Bruker En Apex ultra 7.0T FT-MS mass spectrometer. Fluorescence spectra
108
were performed on a Hitachi F7000 fluorescence spectrometer (Tokyo, Japan).
109
Absorption spectra were recorded on a HITACHI U3900 spectrophotometer.
110
Fluorescence images were captured using a Olympus Fluoriew 1000 laser-scanning
111
confocal microscope.
112 113 114
2.2. Materials Doubly
distilled
water
was
used
throughout
the
experiments.
3,
115
3-Dimethylglutaricacid was purchased from Alfa Aesar. H2O2 (aq. 30%) and NaOCl
116
(aq. 6%) were purchased from Shanghai Reagent Co. of China. A stock solution of
117
NaClO was standardized at 292 nm using an extinction coefficient 350 M−1 cm−1 at
118
pH 12 [40]. Nitric oxide (NO) was prepared from l-hydroxy-2-oxo-3- (3-aminopropyl)
119
-3- methyl-l-triazene, which was synthesized according to the literature [41]. ROO•
120
was generated from 2,2-Azobis(2-amidinopropane)dihydrochloride (100 µM) which
121
was firstly prepared in deionizer water and then added appropriate amount into the
122
probe testing solutions at 37 °C for 1 h [41]. Superoxide was generated from KO2 in 5
123
aqueous solution [42]. Hydroxyl radical was generated by Fenton reaction in which
124
ferrous chloride (100 µM) was added in the presence of 10 eq. of H2O2 [42]. Singlet
125
oxygen was generated from 3-(1,4-Dihydro-1,4-epidioxy-1-naphthyl)propionic acid
126
(100 µM) which was firstly prepared in deionizer water and then added appropriate
127
amount into the probe testing solutions at 37 °C for 1 h [42]. Peroxynitrite solution
128
was synthesized according to the report [43] and the concentration was determined
129
with an extinction coefficient of 1670 ± 50 cm−1 (mol/L)−1 at 302 nm. Glutathione
130
(GSH), cysteine (Cys) and trypan blue were purchased from J & K Science Ltd.
131
Lipopolysaccharides (LPS) and phorbol myristate acetate (PMA) were purchased
132
from Sigma-Aldrich. Solutions for testing foreign anions and cations were prepared
133
by dissolving their corresponding sodium salts and nitrates in water, respectively.
134
Reactions were monitored by TLC with visual observation of the dye spots. Products
135
were purified by column chromatography.
136 137 138
2.3. Synthesis of probe 1 The solution of para-toluensulfonyl chloride (42 mg, 1.2 eq) in acetonitrile was
139
added dropwise to the acetonitrile solution containing compound 2[44] (60 mg, 1.0 eq)
140
and pyridine (297 mg, 20.0 eq). The mixture was refluxed under N2 atmosphere
141
overnight. When the reaction was completed, the acetonitrile was removed under
142
reduced pressure. The residue was extracted with ethyl acetate, and the organic layer
143
was collected, washed with water and then dried over sodium sulfate. The organic
144
solution was filtered and evaporated under reduced pressure, and the residue was
6
145
purified by silica gel chromatography using ethyl acetate: petroleum ether (1/4 = v / v)
146
as an eluent to get a yellow powder in 52.4% yield.
147
1
148
8.57 (m, 1H), 8.50 (m, 1H), 8.40 (d, J = 6.8Hz, 1H), 8.18 (d, J = 6.4Hz, 1H), 7.83 (m,
149
1H), 7.78 (d, J = 6.8Hz, 2H), 7.41(d, J = 6.4Hz, 2H), 4.02 (t, J = 6.0Hz, 2H), 2.38 (s,
150
3H), 1.60 (m, 2H), 1.34(m, 2H), 0.92 (t, J = 5.6Hz, 3H).
151
13
152
141.657, 136.048, 134.207, 132.369, 131.185, 130.236, 130.073, 128.810, 128.225,
153
127.530, 126.703, 122.711, 116.341, 39.610, 30.155, 21.504, 20.264, 14.182.
154
FT-MS: calcd. for C24H24N4O5S, 479.13891 (M-), found, 479.13802.
H NMR (d6-DMSO, 400MHz) δ (ppm): δ 9.84 (s, 1H), 9.49 (s, 1H), 9.09 (s, 1H),
C NMR (d6-DMSO, 100MHz) δ (ppm):163.947, 163.382, 154.660, 143.979,
155 156
2.4 Preparation process of compound 3 by reaction of 1 with NaClO
157
A pH 6.0 aqueous solution of NaClO (1.2 eq) was added dropwise to the acetone
158
solution containing probe 1 (30 mg, 1.0 eq) at room temperature. The mixture was
159
stirred overnight. The solution was removed under reduced pressure, and the residue
160
was purified by silica gel chromatography to get 3 in ca.61.3% yield.
161
1
162
1H), 8.18 (d, J = 8.4 Hz, 1H), 7.65 (m, 1H),7.44 (s, 2H), 6.84 (d, J = 8.4 Hz, 1H), 4.00
163
(t, J = 7.2 Hz, 2H), 1.65–1.51(m, 2H), 1.40–1.27 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).
164
13
165
130.14, 129.73, 124.44, 122.27, 119.83, 108.62, 108.04, 39.36, 30.31, 20.31, 14.22.
166
FT-MS: calcd. for C16H15N2O2,(3-H) +, 267.11335, found, 267.11335
H NMR (d6-DMSO,400 MHz): δ (ppm) 8.60 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 7.3 Hz,
C NMR (d6-DMSO, 100 MHz): δ (ppm) 164.23, 163.37, 153.14, 134.39, 131.44,
167 168 169 170
2.5 General procedure for the measurement of absorption and fluorescence spectra Probe 1 was dissolved in 1,4-dioxane to make a 1.0×10-3M stock solution. NaClO was dissolved in water to make a 1.0×10-3 M stock solution for total HClO.
7
171
To 5-mL glass tubes containing 0.40 mL of 100 mM 3, 3-dimethylglutaric
172
acid-NaOH buffer solution (pH 7.0), 1.0 mL acetone and various amount of stock
173
solution of total hypochlorous acid were added, respectively. Then the solution was
174
diluted with redistilled water to the scale and mixed thoroughly, and 50.0 µL of the
175
stock solution of probe 1 was added to the solution finally and mixed well. The
176
absorption and fluorescence sensing of HClO were run after 2 minutes.
177 178
2.6 Cytotoxicity assay
179
For each dishes, 180 µL of the cells was added onto a 96-well plate, followed by
180
addition of 20 µL of increasing concentrations of the probe 1 (95% PBS and 5%
181
acetone) and the final concentrations of the probe were kept from 0.1µM to 100µM
182
(n=3). The incubation was at 370C in an atmosphere of 5% CO2 in a CO2 incubator
183
for 4h, followed by trypan blue assays. Untreated assays in PBS-acetone (V/V : 95 / 5)
184
(n=3) was also conducted at the same conditions.
185 186
2.7 Cell imaging
187
Cell cultures: U-373MG cells were cultured in DMEM (Dulbecco’s modified
188
Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) in an atmosphere
189
of 5% CO2 and 95% air at 37 0C.
190
Imaging of exogenous HOCl in cells: Before imaging, the cells were seeded on
191
two groups of 20 mm glass-bottomed dishes and incubated with probe 1 (10 µM
192
containing 5% acetone as a cosolvent in PBS) for 30 min at 37℃. Rinsed with PBS
193
two times, the second group was incubated with NaOCl (50 µM) for 30 min at 37 0C.
194
Imaging of endogenous HOCl: Two groups of cells were seeded on 20mm
195
glass-bottomed dishes and incubated with probe 1 (10 µM containing 5% acetone as a
196
cosolvent in PBS) for 30 min at 37℃. Rinsed with PBS two times, the second group
197
was incubated with 2mg/mL PMA (phorbol 12-myristate 13-acetate) and 2mg/mL
198
LPS (lipopoly-saccharides) for 2 h at 37 0C. 8
199
3. Results and Discussion
200
3.1 Design and synthesis of probe 1
201
Modulation of the electron-donating 4-amino donor on a 1,8-naphthalimide affects
202
both ICT and emission color, so we changed this amino donor into a more
203
electron-withdrawing carbamate group, namely
204
result in ICT-induced blue shifts in emission maxima. We reasoned that when the
205
group was removed by HClO back to the initial amino donor it would provide a
206
switch for ratiometric detection of HClO. The synthetic procedures of probe 1 are
207
given in Scheme 2; probe 1 was synthesized from compound 2 [44] by one-step
208
procedure with a total yield of 52%, and the structure of probe 1 was confirmed by 1H
209
NMR, 13C NMR, and FT-MS spectroscopy (Figures S1, S2 and S3).
sulfonyl-semicarbazide, would
210 211
Scheme 2. Synthetic route for probe 1
212 213
3.2 Spectral characteristics
214
The absorption spectra of 1 as well as the solutions in presence of HOCl were
215
monitored firstly in buffer solutions of 3,3-dimethylglutaric acid-NaOH (pH 7.0,
216
10mM in Acetone/H2O = 20/80, v/v). As shown in Figure 1, the solution of 1 alone
217
(1.0×10-5 M) exhibited an absorption maximum at 370 nm. With the addition of HClO,
218
a new absorption band at 435 nm increased at the expense of the decrease of the band
219
at 370 nm with an isosbestic point at 412 nm which could be attributed to the
220
formation of a new species. Also, a large bathochromic shift of 65 nm in the
9
221
absorption behavior changes the color of the resultant solution from colorless to
222
yellow, which could be detected even by the naked.
223
The fluorescence characteristics of probe 1 were also studied. When excited at 412
224
nm the free probe 1 exhibited an emission peak at 488 nm. Upon consistent
225
introduction of HClO, the peak at 488 nm gradually decreased accompanied by a new
226
peak at 542 nm emerged increasingly with a clear iso-emission point at 513 nm. Thus
227
a ratiometric fluorescence response was observed.
228 229
Figure 1. Absorption spectra of 1 with HOCl at pH 7.0 of 0.01 M
230
3,3-dimethylglutaric acid-NaOH buffer solution (Acetone / H2O = 20/80, v/v). [1] =
231
10.0 µmol/L, [total HOCl] = 10.0 µmol/L.
232
10
233 234
Figure 2. Fluorescence titration of probe 1 (10 µM) at pH 7.0 of 0.01 M
235
3,3-dimethylglutaric acid-NaOH buffer solution (Acetone/H2O = 2/8, v/v) in the
236
presence of different amounts of total HOCl. Excitation was performed at 412 nm.
237 238
3.3 Effect of pH dependency of the ratiometric signaling behavior
239
As showed in Figure 3, the fluorescent ratio signals of probe 1 itself, F542/F488,
240
remain stable and relatively small over a pH range of 2.0-7.4. In the presence of
241
HOCl, negligible variation of the fluorescent ratios was observed when pH within
242
5.0-8.0. Thus buffer solutions of 3,3-dimethylglutaric acid/NaOH at pH 7.0 were
243
chosen as for determination in this work.
11
244 245
Figure 3. pH-dependent fluorescence intensity ratio changes of probe 1 only and the
246
probe 1 with total HClO. [1] = 10 µM, [total HOCl] = 10 µM. pH 7.0 of 0.01 M
247
3,3-dimethylglutaric acid/NaOH buffer solution (Acetone/H2O = 2/8, v/v). Excitation
248
was performed at 412 nm.
249 250
3.4 Time-dependent fluorescent intensity ratio changes of probe 1
251
Further, the fluorescence intensity ratios (F542/F488) were recorded as a function of
252
reaction time. It can be observed that the ratio signals exhibited no noticeable changes
253
in case of free probe 1, while reached their maximum within 2 min in presence of four
254
tested total HClO concentrations (Figure 4). Thus an incubation time of 2 min was
255
used in this work.
12
256 257
Figure 4. Kinetics of the ratiometric fluorescent responses of 1 toward various
258
concentrations of total HOCl at pH 7.0 of 0.01 M 3,3-dimethylglutaric acid-NaOH
259
buffer solution (Acetone/H2O = 20/80, v/v). [1] = 10µM; [total HOCl] = 0, 1.0, 4.0,
260
7.0, 10.0µM, respectively (from black bottom to purple top).
261
performed at 412 nm.
Excitation was
262 263
3.5 Selectivity of the reaction of 1 with HClO
264
As the absorption spectra of probe 1 changed significantly upon reaction with
265
HClO, we firstly used the absorbance ratio at two wavelengths (A435/370 nm) to study
266
the selectivity of HClO toward ROSs. Important ROSs, such as H2O2, O21, NO, O2•−,
267
ONOO−, •OH, ROO•, and total HClO, were tested toward a solution of 1.0 equiv. of
268
1 (10 µM) at the concentration of 10 µM respectively. HClO was found to greatly
269
enhance the absorbance ratio (A435/370) of probe 1 exclusively with no noticeable
270
response of other ROSs (Figure 5) .
13
271 272
Figure 5. Ratiometric absorbance responses of 1 (10 µM) with 1.0 equiv of ROSs at
273
pH 7.0 of 0.01M 3,3-dimethylglutaric acid-NaOH buffer solution (Acetone/H2O =
274
20/80, v/v). [1] = 10.0 µM. ROSs adding (from left to right at horizontal ordinate): (1)
275
none, (2) total HOCl, (3) H2O2, (4) O2·-, (5) ONOO- , (6) •OH, (7) NO, (8) 1O2 and (9)
276
ROO•. [ROS] = 10.0 µM, respectively.
277 278
Accordingly,
the
ratiometric
fluorescent
responses
of
1
toward
the
279
above-mentioned ROSs were also tested independently. As showed in Figure 6, the
280
solution of 1 alone exhibits nearly no change in the fluorescence ratio (F542 nm / F488 nm )
281
upon the addition of the ROSs except HClO. Remarkable increase of the ratio value
282
was obtained exclusively in presence of HClO, indicating that our probe showed
283
selective response towards HClO over other ROSs.
284
Moreover, a wide variety of other interference species, such as ionic species and
285
representative biothiols, including Ca2+, Co2+, Ni2+, Fe3+, Zn2+, Cu2+, F-, Cl-, I-, NO3-,
286
SO42-, CH3COO-, IO3-, PO43-, glutathione (GSH) and cysteine (Cys), were also tested
287
respectively to show negligible interference towards HClO sensing of probe 1 (Figure
288
S4). In addition, the coexistence effects of the ions and biothiols on ratiometric
289
fluorescence response of HClO were also evaluated and the results showed that 10.0
290
times excess of different coexist species caused almost no interference behavior onto
291
the detection (Figure S5). 14
292
293 294
Figure 6. Ratiometric fluorescent responses of 1 (10 µM) with 1.0 equiv of ROSs at
295
pH 7.0 of 0.01M 3,3-dimethylglutaric acid-NaOH buffer solution (Acetone/H2O =
296
20/80, v/v). [1] = 10.0 µM. ROSs adding (from left to right at horizontal ordinate): (1)
297
none, (2) total HOCl, (3) H2O2, (4) O2·-, (5) ONOO- , (6) •OH, (7) NO, (8) 1O2 and (9)
298
ROO•. [ROS] = 10.0 µM, respectively.
299 300
3.6 Analytical figures and Application of the method in real water samples
301
According to the optimized conditions, the calibration curves were constructed. The
302
linear range for total HClO showed to be (0.1–6.0)×10-6 mol/L with the correlation
303
coefficient R2 = 0.9966 (Figure 7). The detection limit, based on the definition by
304
IUPAC [45] (CDL =3Sb/m), was found to be 6.1×10-8 mol L-1 from eleven blank
305
solutions.
306
Freshly collected lake and tap water samples in our school were analyzed by the
307
proposed method. Tap water samples were used with no pre-treatment, the lake water
308
samples were filtered firstly to remove minute sediments. Different water samples
309
were collected at different times and tested under the optimized condition
310
subsequently. The water samples spiked with standard NaClO at different
311
concentration levels were also analyzed by proposed method. The results were
312
summarized in Table S1 with satisfactory recovery, indicating that the present 15
313
fluorescent probe can be applicable for the determination of total HClO in real water
314
samples.
315 316
Figure 7. The linear range of ratiometric fluorescent response of 1 vs. the
317
concentrations of total HClO at pH 7.0 of 0.01M 3,3-dimethylglutaric acid-NaOH
318
buffer solution (Acetone/H2O = 20/80, v/v).[1] = 10.0 µM.
319 320
3.7 Reaction Mechanism
321
It was reported that dibenzoylhydrazine can be selectively oxidized by HClO to a
322
reactive intermedia, dibenzoyl diimide, and further undergo hydrolysis in aqueous
323
solvents [17]. We hypothesized that reaction of probe 1 with HClO may conduct a
324
similar process. The possible mechanism was supposed as follow (Scheme 3):
16
325
Scheme 3 The proposed mechanism of probe 1 with HClO
326 327 328
To verify the reaction mechanism supposed, we first take the reaction solution of 1
329
with HOCl for FT-MS experiment. As expected, product 3 was confirmed by its
330
high-resolution mass spectra (HR-MS): calcd. for C16H15N2O2, 267.11335(3-H) +,
331
found,
332
4-amino-1,8-naphthalimide fluorophore was produced after the reaction of 1 with
333
HOCl. Furthermore, the 1H and
334
HOCl to be 4-amino-1,8-naphthalimide fluorophore 3 (Figure S7 and S8).
335
Significantly, the proton signals of δ 9.84 (s, 1H), 9.49 (s, 1H) and 9.09 (s, 1H) on
336
p-TsNHNHCONH group in probe 1 are disappeared in product 3; moreover, the
337
proton signals of 7.78 (d, J = 6.8Hz, 2H), 7.41(d, J = 6.4Hz, 2H) and 2.38 (s, 3H) on
338
CH3C6H4 group in probe 1 are also disappeared in product 3; finally, the proton
267.11335
(Figure
S6).
13
The
result
preliminarily
proved
that
C NMR data confirmed that the product of 1 with
17
339
signals of 7.44 (s, 2H) on 4-NH2-1,8-naphthalamide fluorophore (the compound 3),
340
are appeared. These results confirmed the reliability of the reaction mechanism we
341
envisaged in Scheme 3. Thus the ratiometric fluorescent behavior of 1 towards HClO
342
could be rationalized by the HClO-induced transformation of electron-deficient
343
sulfonyl-semicarbazide moiety to electron-donating amino group on 4-position on the
344
1,8-naphthalimide fluorophore which affected the characteristics of intramolecular
345
charge transfer (ICT).
346 347
3.8 Cell Cytotoxicity assay
348
The cell livability was expressed as percent (mean for three assays) relative to
349
untreated cells as shown in Fig. 8. It could be seen that the probe was almost nontoxic
350
to cells when its concentration was below 100 µM. Therefore, probe 1 was applied for
351
fluorescence imaging of HOCl in cells.
352
353 354
Figure 8. Cytotoxicity study of probe 1 for cells.
355 356 18
357
3.9 Cell imaging of probe 1
358
Firstly, we applied probe 1 to imaging exogenous HOCl in living U-373MG cells.
359
As exhibited in Figure 9, the living U-373MG cells incubated with 1 for 0.5 h at 37℃
360
provided a strong blue fluorescence in the short wavelength emission window (Figure
361
9a) while less green fluorescence in the long wavelength emission window (Figure
362
9b). The living U-373MG cells treated firstly with NaOCl and then with 1 gave a
363
marked decrease in the blue emission (Figure 9e) and a significant increase in the
364
green emission (Figure 9f), consistent with the HOCl-induced ratiometric fluorescent
365
response.
366 367
Figure 9. Confocal fluorescence imaging without (up) and with (down) exogenous
368
NaClO.
369
collected at long wavelength emission window; (c, g) merged images of (a+b) and
370
(e+f); (d, h) bright field image.
(a, e) images collected at short wavelength emission window; (b, f) images
371 372
We then proceeded to investigate the feasibility of the probe for imaging
373
endogenous HOCl in living U-373MG cells. The cells which incubated only with the
374
probe displayed strong fluorescence in the blue channel (Fig. S9a) and almost no
375
fluorescence in the green channel (Fig. S9b). However, a marked enhancement in the
376
green channel (Fig. S9d) was observed in the cells stimulated by lipopolysaccharides
377
(LPS, 2mg/mL) and phorbol myristate acetate (PMA, 2mg/mL) together. The
378
remarkably increased green fluorescence signal is mainly due to the reaction between
379
the probes and endogenous HOCl, indicating that the probe was cell membrane
380
permeable and could be employed for fluorescence imaging of HOCl in living cells. 19
381 382
Conclusion
383
In summary, a naphthalimide-based fluorescent probe has been successfully
384
developed for the rapid ratiometric fluorescence detection of hypochlorous acid based
385
on the specific HOCl-promoted oxidation-hydrolysis of sulfonyl-semicarbazide
386
group. The experimental results clearly indicate that probe 1 can be used as a
387
chemodosimeter for HOCl with good selectivity and high sensitivity. In addition,
388
confocal fluorescence microscopy imaging using U-373MG cells showed that the new
389
probe could be used as a promising fluorescent probe for real water samples and
390
imaging HOCl in living cells.
391 392
Conflicts of interest
393
There are no conflicts to declare.
394 395
Acknowledgments
396
This work was financially supported by the National Natural Science Foundation
397
of China (No. 21435003), Program for Changjiang Scholars and Innovative Research
398
Team in University (PCSIRT, No.IRT13036).
399 400
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Highlights
•An ICT-based ratiometric fluorescence probe for HClO was developed with the skeleton of 4-amino-1, 8-naphthalimide fluorophore.
•A novel recognition mechanism that specific removing a sulfonyl-semicarbazide group by HClO was observed.
•The probe showed high selectivity and sensitivity toward HOCl and was applied in real water samples and imaging HClO in living U-373MG cells..