Recognition of Exogenous and Endogenous Nitroxyl in Living Cells via a Two-Photon Fluorescent Probe

Article abstract of DOI:10.1021/acs.analchem.7b05172

Nitroxyl (HNO), one electron reduced and protonated form of nitric oxide (NO), plays vital in various biological functions and pharmacological activities, such as mediating β-agonist dobutamine, inhibiting the activity of enzyme and treating cardiovascula

Full text of DOI:10.1021/acs.analchem.7b05172

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Recognition of Exogenous and Endogenous Nitroxyl
in Living Cells via a Two-photon Fluorescent Probe
Haidong Li, Qichao Yao, Feng Xu, Ning Xu, Xuechao Ma, Jiangli
Fan, Saran Long, Jianjun Du, Jingyun Wang, and Xiaojun Peng
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05172 • Publication Date (Web): 08 Mar 2018
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Analytical Chemistry
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Recognition of Exogenous and Endogenous Nitroxyl in Living Cells via a
Two-photon Fluorescent Probe
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Haidong Li,† Qichao Yao,† Feng Xu,† Ning Xu,† Xuechao Ma,† Jiangli Fan,† Saran Long,† Jianjun Du,† Jingyun
Wang,‡ and Xiaojun Peng*†
†State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, High-tech District, Da-
lian 116024, China. *Email: pengxj@dlut.edu.cn. Tel/Fax: +86 -411- 84986306.
‡Department School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road,
High-tech District, Dalian 116024, China.
ABSTRACT: Nitroxyl (HNO), one electron reduced and protonated form of nitric oxide (NO), plays vital in various bio-
logical functions and pharmacological activities, such as mediating β-agonist dobutamine, inhibiting the activity of en-
zyme and treating cardiovascular diseases. However, the accurate mechanism of HNO in living cells is not thoroughly
understood due to lacking of effective methods. In this work, a novel two-photon fluorescent probe TP-HNO was de-
signed and synthesized based on 6-hydroxyl-quinonline-2-benzothiazole derivatives through introducing
2-(diphenylphosphino)benzoate as the ideal HNO recognition unit, which demonstrated the merits of outstanding selec-
tivity, excellent sensitivity (DL 0.19 μM) and rapid response (20 min). In addition, owing to the high cell permeability and
low biotoxicity of probe TP-HNO, it was successfully used for the qualitative and bioimaging of exogenous and endoge-
nous nitroxyl concentration fluctuations in living cells via a two-photon laser confocal fluorescence microscopy, respec-
tively, which is of importance for revealing the biological of HNO in the further.
operation and well-defined spatiotemporal resolution,
INTRODUCTION
small-molecule fluorescent probes have been attracted
Nitroxyl (HNO), one electron reduced and protonated
tremendous attention.15-23 Especially, compared with others
form of nitric oxide (NO), can be generated endogenously
analytical techniques such as colorimetric method,24 mass
in living cells under the catalysis of nitric oxide synthase in
spectrometry,25 electron paramagnetic resonance,26 high
appropriate conditions.1, It plays the unique roles in
performance liquid chromatography27 and electrochemical
various biological functions and pharmacological
analysis,28 it is more suitable for in-suit recognition of
activities,2-4 which distinct from the birth of NO.5-7 For
HNO in vivo without tedious sample pretreatments, time
instance, increasing numerous evidences have found that
consuming and invasiveness characteristics.29-38 Toward
HNO is additive to the β-agonist dobutamine,8 and could
this end, some judiciously fluorescent probe have been
inhibit the activity of enzyme through reacting with thiols
designed to the detection of HNO in vitro or vivo
in aldehyde dehydrogenase,9 and treat cardiovascular
depending on Cu(II)-based39-42 or reaction-based
diseases by upregulating the calcitonin gene-related
2
method43-51
. For examples, Lippard and cooperators
peptide
(CGRP).10,11
Besides,
through
activating
fabricated some ingenious copper(II)-based probe
CuDHX1 to visualize HNO in mammalian cells for
exploring the physiological processes.52 Tan and Zhang’s
group reported probe P-CM for the quantitative detection
of nitroxyl in bovine serum with satisfactory results.53
Based on coumarin fluorophore, by introducing
triphenylphosphonium salt as the mitochondria
localization group, Kim and coworkers developed probe
Mito-1 for the sensing of HNO in mitochondria
overcoming fluorescence interference form biothoils in
cellular milieus.44
voltage-dependent K+ channel, HNO also could regulate
the relaxation of resistant-like arteries in mammalian
vascular systems.12-14 In recent years, the investigation
physiological function of HNO has gained more and more
attention. However, the specific molecular mechanism of
nitroxyl is not fully understood due to lacking of effective
methods. Therefore, it is highly needed to develop the
efficient method for monitoring HNO in biological
system.
Owing to its high selectivity, excellent sensitivity, simple
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suppliers and were used without further purification
Page 2 of 9
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unless otherwise stated. Solvents used were purified via
standard methods. Twice-distilled purified water used in
all experiments was from Milli-Q systems. 1H NMR and 13C
NMR spectra were recorded on a VARIAN INOVA-400 (or a
Bruker Avance II 400 MHz or 500 MHz) spectrometer.
Chemical shifts (δ) were reported as ppm (in CDCl3 or
DMSO-d6, with TMS as the internal standard). Flash
column chromatography was performed using silica gel
(100-200 mesh) obtained from Qingdao Ocean Chemicals.
Angeli's salt (Na2N2O3, AS) was purchased from Cayman
Chemical. Absorption spectra were recorded on a CARY 60
UV-Vis spectrophotometer in 10×10 mm quartz cell.
Fluorescence spectra were performed on a VAEIAN CARY
Scheme 1. Recognition mechanism of probe TP-HNO to-
wards HNO
Chen and Yu et al. constructed a versatile fluorescence
probe Cyto-JN based on azadipyrromethane fluorophore
(Aza-BODIPY), which was applied for the detection and
imaging intracellular HNO in living cells and mices.54
Zhang et al. designed and synthesized a fluorescent
chemodosimter HNO-HBT for the rapid detection of
HNO in living cells.48 Unfortunately, we found that most of
reported fluorescent probes suffered from short excitation
wavelength, interference from autofluorescence and
photobleaching with one-photon irradiation. On the
contrast, using near-infrared (NIR, 650-900 nm,
coincident with the biological optical window) light as the
excitation source, two-photon fluorescence probes having
the prominent advantages, such as reducing photodamage,
lowering self-absorption and increasing tissue penetration
depth, had drawn more and more attentions in medical
and biological research.37,55-57 So far, there are few excellent
performances two-photon fluorescent probe developed for
the determination of HNO in vivo. Therefore, the
aforementioned considerations inspire us to propose and
synthesize a novel fluorescent probe for monitoring and
bioimaging of HNO in complicated biological system
under two-photon microscopy.
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Eclipse
fluorescence
spectrophotometer
(Serial
No.MY15210003) in 10×10 mm quartz cell. Excitation and
emission slit widths were modified to adjust the
fluorescence intensity to
a
suitable range. Mass
spectrometric data were achieved with HP1100LC/MSD MS
and an LC/Q-TOF-MS instruments. All pH measurements
were performed using a Model PHS-3C meter. The
fluorescence quantum yields for compounds with Absolute
PL Quantum Yield Spectrometer (HAMAMATSU C11347).
Instruments used in cell imaging tests were carried out on
Olympus FV1000-IX81 confocal microscopy (Olympus,
Japan). Slight pH variations in the solutions were achieved
by adding the minimum volumes of HCl or NaOH (1 M).
The test system were performed in PBS buffer solution
(0.01 M, pH 7.4 and 25 μM CTAB) in vitro. All the
interferential reagents were prepared based on published
literature.23, 35, 37
Determination of the detection limit
Hence, based on the excellent two-photon absorption
cross section of 6-hydroxyl-quinonline-2-benzothiazole
The detection limit (DL) was calculation based on the
fluorescence titration of probe TP-HNO (10 μM) in the
presence of AS reagent (0-20 μM). The fluorescence inten-
sity of probe TP-HNO was measured and standard devia-
tion of the blank measurement was achieved. The detec-
tion limit was calculated with the following equation:
55, 58-60
derivatives,
we have developed a novel two-photon
fluorescent probe TP-HNO through introducing
2-(diphenylphosphino)benzoate as an the ideal HNO
recognition moiety. When the reagent (HNO donor) was
added into PBS buffer solution, the fluorescence emission
intensity of around 550 nm demonstrated an obvious
enhancement within 20 min. Probe TP-HNO showed the
exclusive selectivity of HNO overcoming interferences
from biological reducants including glutathione. The
sensing mechanism was verified by the High Resolving
Mass Spectrum (HRMS). Furthermore, the detection limit
of probe was calculated as low as 0.19 μM based on the
principle of 3σ/k, which showed that it was excellent
sensitivity towards HNO in vitro. More importantly, owing
to its high biocompatibility, low biotoxicity and good
stability under physiological conditions, probe TP-HNO
was successfully applied to recognize and image exogenous
and endogenous nitroxyl in living cells through
two-photon fluorescence microscopy.
Detection limit = 3σ/k
Where σ is the standard deviation of the blank meas-
urement, k is the slope between the fluorescence intensity
(F545 nm) versus various the different concentrations of AS
reagent.
Determination of the quantum yield
The test method was operated according to the previous
literature.38
Cell incubation
HeLa cells and RAW 264.7 cells were purchased from
Institute of Basic Medical Sciences (IBMS) of the Chinese
Academy of Medical Sciences. Cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen)
supplemented with 10 % fetal bovine serum (Invitrogen).
The cells were seeded in confocal culture dishes and then
incubated for 24 h at 37 ºC under a humidified atmosphere
EXPERIMENTAL SECTION
General information and materials
All reagents used were obtained from commercial
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Analytical Chemistry
The synthesis of 2. Compound 1 (318 mg, 2 mM) and
containing 5% CO2.
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K2CO3 (358.8 mg, 2.6 mM) were dissolved in 6 mL dry di-
methyl formamide (DMF) solution. The reaction system
Cytotoxicity assays
Measurement of cell viability was tested by reducing of
o
was stirred at 80 C for 30 min under N2 protection, then,
MTT
(3-(4,
5)-dimethylthiahiazo
(-2-yl)-3,
benzyl chloride (304 mg, 2.4 mM) was added via drop by
drop strategy until the end. Afterwards, the mixture was
continued to refluxing for 24 h. After cooling to room
temperature, the mixture was extracted with ethyl acetate
three times. The organic phase was dried with anhydrous
Na2SO4 for overnight and vacuum filter. Next, the crude
product was purified through silica gel column chroma-
5-diphenytetrazoliumromide) to formazan crystals using
mitochondrial dehydrogenases. MCF-7 cells were seeded in
96-well microplates at a density of 1 × 105 cells/mL in 100
μL medium containing 10 % FBS buffer. After 24 h of cell
attachment, the plates were then washed with 100 μL/well
by PBS. The cells were then cultured in medium with 0, 1, 5,
10 and 20 μM of probe TP-HNO for 24 h, respectively. Cells
in culture medium without probe TP-HNO were used as
the control. Six replicate wells were used for each control
and test concentration. 10 μL of MTT (5 mg/mL) prepared
in PBS was added to each well and the plates were incu-
bated at 37 ºC for another 4 h in a 5% CO2 humidified in-
cubator. The medium was then carefully removed, and the
purple crystals were lysed in 200 μL DMSO. Optical densi-
ty was determined on a microplate reader (Thermo Fisher
Scientific) at 570 nm with subtraction of the absorbance of
the cell-free blank volume at 630 nm. Cell viability was
expressed as a percent of the control culture value, and it
was calculated using the following equation:
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tography to obtain 2.61 g white product 2 (Yield 89%). H
NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 8.4 Hz, 1H), 7.84
(d, J = 8.8 Hz, 1H), 7.52 (d, J = 7.4 Hz, 2H), 7.40 (dd, J =
22.0, 9.4 Hz, 6H), 5.22 (s, 2H), 2.61 (s, 3H).
The synthesis of 3. Compound 2 (373.5 mg, 1.5 mM) was
put into 30 mL 1, 4-dioxane in glass bottle. The mixture
was stirred at 60 oC for 20 min. Afterwards, SeO2 (233.1 mg,
2.1 mM) was added into reaction system by dropwise with-
in 30 min. When aforementioned process was finished, the
o
temperature was adjusted to 80 C for stirring 4 h. After
vacuum filter, the filtrate was obtained for needing further
purification. The crude product was purified through silica
gel column chromatography to obtain 250 mg yellow
Cells viability (%) = (ODdye - ODKdye)/ (ODcontrol - ODKcon-
trol) × 100
1
product 3 (Yield 63%). H NMR (400 MHz, DMSO-d6) δ
10.09 (s, 1H), 8.46 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 9.0 Hz,
1H), 7.97 (d, J = 8.4 Hz, 1H), 7.76 -7.26 (m, 7H), 5.31 (s, 2H).
Living cells imaging
HeLa cells and RAW264.7 cells were seeded in
The synthesis of 4. Compound 3 (263 mg, 1.0 mM) and
2-aminothiophenol (187.5 mg, 1.5 mM, 0.16 mL) were dis-
solved 4 mL dry dimethyl sulfoxide (DMSO). The reaction
system was stirred at 130 oC for 6 h with N2 protection. Af-
ter cooling to room temperature, 10 mL water was added
into glass bottle. Then, the mixture was extracted with
ethyl acetate three times. The organic phase was dried with
anhydrous Na2SO4 for overnight and vacuum filter. Next,
the crude product was purified through silica gel column
chromatography to obtain 239.2 mg greyish white product
4 (Yield 65%). 1H NMR (400 MHz, DMSO-d6) δ 8.48 (d, J =
8.6 Hz, 1H), 8.42 (d, J = 8.6 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H),
8.15 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 9.2 Hz, 1H), 7.64 (d, J =
2.6 Hz, 1H), 7.58 (ddd, J = 18.2, 11.3, 6.0 Hz, 5H), 7.45 (t, J =
7.4 Hz, 2H), 7.39 (d, J = 7.2 Hz, 1H), 5.31 (s, 2H).
galass-bottom
culture
dishes
at
approximately
concentration of 2×104 cells/mL and allowed to culture for
24 h at 37 ºC in a 5% CO2 humidified incubator. Under the
confocal fluorescence microscope (Olympus FV1000-IX81)
with a 60 × objective lens, probe TP-HNO was excited at
405 nm (one-photon) and 730 nm (two-photon),
whereafter, fluorescence emission from green channel (OP
model: 500-580 nm; TP model: 520-560 nm) was gathered.
Quantitative image analysis of the average fluorescence
intensity of cells, determined from analysis of 9 regions of
interest (ROIs) across cells.
The synthesis of probe TP-HNO
The synthesis of 1. Aminophenol (2.18 g, 20 mM) was
dissolved in 15 mL hydrochloric acid solution (6 M). The
mixture was stirred at 100 oC for refluxing under N2
protection. Afterwards, the crotonaldehyde (2.24 g, 32 mM,
2.64 mL) was gradually added through needle tube within
45 min. Then, the reaction system was continued to
refluxing overnight. After cooling to room temperature
and adjusting the pH to neutral, the mixture was extracted
with ethyl acetate three times. The organic phase was dried
with anhydrous Na2SO4 for overnight, and vacuum filter.
Next, the crude product was purified through silica gel
column chromatography to obtain 2.61 g product 1 (Yield
82%). 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.02 (d,
J = 8.4 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.26 (dd, J = 14.7,
5.4 Hz, 2H), 7.10 (s, 1H), 2.58 (s, 3H).
The synthesis of 5. Compound 4 (280 mg, 0.76 mM) was
fully dispersed in 7 mL dry dichloromethane (DCM) under
ice bath condition. After stirring about 15 min, BBr3-CH2Cl2
solution (1 M, 3.04 mL) was added via drop by drop until
the end. Then, the mixture was stirred for 3 h at room
temperature. Next, 10 mL water was added to quench reac-
tion and pH value of solution was adjusted to 7 with Na-
HCO3 solution. Then, the mixture was extracted with
CH2Cl2 three times. The organic phase was dried with an-
hydrous Na2SO4 for overnight and vacuum filter. Next, the
crude product was purified through silica gel column
chromatography to obtain 90 mg pale yellow product 5
(Yield 43%). 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H),
8.38 (d, J = 8.7 Hz, 1H), 8.34 (d, J = 8.6 Hz, 1H), 8.19 (d, J =
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7.7 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H),
Page 4 of 9
dure. The intermediates of each step were characterized
by nuclear magnetic resonance (1H NMR, Figure S1-5).
Besides, the target material TP-HNO was carefully con-
firmed by nuclear magnetic resonance (1H, 13C and 31P
NMR, Figure S6-8) and high resolution mass spectrum
(HRMS, Figure S9) in the Supporting Information section.
6-hydroxyl-quinonline-2-benzothiazole (intermediate 5)
was a vital fluorophore, which had been proved to possess
intrinsic two-photon absorption cross sections by Liu’s
group. 55, 58, 60 The maximum action two-photon absorp-
tion cross section (δmaxΦ) value of intermediate 5 was re-
ported up to 50 GM (1 GM = 10-50 cm4 s photon-1) at 730
nm,57 which demonstrated it was suitable for two-photon
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7.65-7.55 (m, 1H), 7.52 (dd, J = 11.0, 4.1 Hz, 1H), 7.43 (dd, J =
9.1, 2.7 Hz, 1H), 7.27 (d, J = 2.6 Hz, 1H).
The synthesis of probe TP-HNO. Compound 5 (90 mg,
0.32 mM), 2-(diphenylphosphanyl) benzoic acid (153 mg,
0.5 mM), EDCI (95.9 mg, 0.5 mM) and DMAP (4 mg, 0.03
mM) were dissolved in 8 mL dry CH2Cl2 under N2 protec-
tion. After stirring for overnight, 5 mL water was added
into this reaction system. Then, the mixture was extracted
with CH2Cl2 three times. The organic phase was dried with
anhydrous Na2SO4 for overnight and vacuum filter. Next,
the crude product was purified through silica gel column
chromatography to obtain 40 mg white product TP-HNO
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imaging
in
vivo.
Moreover,
(Yield 21%). H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J =
2-(diphenylphosphino)benzoate as the ideal HNO recog-
nition moiety was chosen to install on intermediate 5 for
constructing “potential” two-photon fluorescence probe
TP-HNO. When HNO encountered probe TP-HNO in
aqueous solution, it reacted with triphenylphosphine of
probe to get the corresponding phosphine oxide and
aza-ylide. Next, the aza-ylide went through a Staudinger
ligation to obtain an amide derivate and intermediate 5 as
the dual photon signal source. As the principle of probe
TP-HNO sensing HNO was outlined in Scheme 1. In order
to further verification the sensing mechanism of probe
TP-HNO for HNO, the high resolving mass spectrum
(HRMS) analysis was carried out. Obviously, one peak
centered at m/z 277.0439 (calcd. 277.0441 for C16H9N2OS-)
corresponding to deprotonation product [intermediate 5]-
was shown in Figure 1. Based on the above results, the
sensing mechanism exactly was depicted in Scheme 1.
8.6 Hz, 1H), 8.49 (d, J = 8.6 Hz, 1H), 8.33 (dd, J = 8.4, 4.0
Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.17 (dd, J = 8.5, 3.4 Hz,
2H), 7.76 (d, J = 2.2 Hz, 1H), 7.69-7.50 (m, 5H), 7.42 (d, J =
3.5 Hz, 6H), 7.32-7.19 (m, 4H), 6.97 (dd, J = 8.4, 4.1 Hz, 1H).
13C NMR (125 MHz, CDCl3) δ 169.60 ,165.09, 154.29, 151.11,
149.18, 145.86, 141.63, 141.41, 137.49, 136.70, 136.45, 134.50,
134.17, 134.01, 133.14, 132.76, 131.44, 131.06, 129.22, 128.88,
128.64, 128.39, 126.30, 125.90, 125.38, 123.77, 122.02, 118.77,
118.53. 31P NMR (162 MHz, CDCl3) δ -3.81 (s, 1H). HRMS:
m/z calcd for C35H23N2NaO2PS+ [M+Na]+: 589.1110, found:
589.1130.
Scheme 2. Synthetic procedures of probe TP-HNO
Figure 2. a) and b) Response time of probe TP-HNO to-
wards HNO; c) the solubility of probe TP-HNO; d) fluoro-
metric titration curve in PBS buffer solution (Insert: linearity
of F550 nm versus the different concentration of HNO). λex =363
nm, slit: 5/5 nm.
Figure 1. HRMS verification of probe TP-HNO for HNO
RESULTS AND DISCUSSION
Response speed and spectroscopic characteristics.
Initially, in order to evaluate the response speed of probe
to HNO, the time-dependent (0-30 min) fluorescence
responses of TP-HNO (5 μM) in the presence of 15 equiv
Design probe TP-HNO and recognition mechanism.
As shown in Scheme 2, the fluorescence probe TP-HNO
was successfully synthesized through a multistep proce-
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Analytical Chemistry
Angeli's salt (Na2N2O3, AS), a precursor of HNO, were
Moreover, competitive experiments were also performed to
estimate the availability of probe TP-HNO in complicated
systems. No obvious interference was obtained towards
aforementioned biological relevant species (Figure S12),
indicating the potential applications of probe TP-HNO for
detecting HNO in complex biological system. Whereafter,
the stability of probe was also studied through fluores-
cence assay measurements in PBS buffer solution (Figure
3b) and in living HeLa cells (Figure S13), which indicated
the possibility capability of this probe for long time imag-
ing in physiological environment.
1
2
3
4
5
6
7
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recorded every minute, as shown in Figure 2a. Upon addi-
tion of AS reagent in PBS buffer solution, the fluorescence
emission center around 550 nm gradually enhanced with
the excitation at 363 nm, signifying HNO triggered the
cleavage of
2-(diphenylphosphino)benzoate from
probe TP-HNO for releasing intermediate 5 signal part.
From the Figure 2b, the fluorescence intensity of 550 nm
wound not increase after 20 min, which indicated probe
TP-HNO had the potential for real-time monitoring of
HNO in biosystems. Next, we explored the fundamental
photophysical properties of probe TP-HNO. The UV-Vis
spectra and fluorescence spectra of TP-HNO in different
solvents were demonstrated in Figure S10a and Figure
S10b, respectively. In addition, the probe TP-HNO pos-
sessed a good solubility in testing system (Figure 2c and
Figure S11). Fluorescent titration experiments were carried
out to investigate the sensitivity of probe towards HNO.
As shown in Figure 2d, the fluorescence emission intensi-
ty of 550 nm was plotted versus the concentration of AS
reagent ranging from 0 to 20 μM, which exhibited an ex-
cellent linearity (R2=0.9809). Thus, the detection limit
was calculated as low as 0.19 μM based on the principle of
3σ/k. Such low limit of detection demonstrated that probe
TP-HNO was highly sensitive to HNO and could be acted
as an effective tool for realizing the quantitative detection
of HNO in vitro.
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Figure 4. a) The pH effects of probe TP-HNO in PBS buffer
solution and b) cytotoxicity assays of probe TP-HNO in living
MCF-7 cells.
pH effects and cytotoxicity assays. Subsequently, the
effects of pH on the recognition of probe TP-HNO (10 μM)
for HNO (15 equiv) were carried out in PBS buffer solution.
As shown in Figure 4a, probe TP-HNO demonstrated al-
most no changes in the fluorescence emission intensity of
550 nm in the range of pH 4.0-10.0, which indicated probe
TP-HNO would not be depended on the acidity of the
humoral environment. Upon addition 15 equiv HNO, the
fluorescence emission intensity of 550 nm displayed dis-
tinct enhancement in PBS buffer solution of pH 4.0-9.0,
clearly demonstrating the possibility capability of probe
TP-HNO for imaging HNO in physiological pH microen-
vironment. Prior to cell tests, in order to study the biologi-
Figure 3. a) The selectivity of TP-HNO (5 μM) towards
cal
cytotoxicity
of
probe
TP-HNO,
3-(4,
various analytes. Insert 1: blank; 100 equiv (2: K+; 3: Na+; 4:
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) assays in living human breast cancer cells (MCF-7
cells) with 0, 1, 5, 10 and 20 μM probe for 24 h were per-
formed, respectively. The results showed almost nontox-
icity of probe TP-HNO to cells at the concentration of 5
μM for 24 h under the experimental conditions (Figure
4b).
Ca2+; 5: Mg2+; 6: Zn2+; 7: NO3 ; 8: NO2 ; 9: F-; 10: HS-;) 50 equiv
(11: NaClO; 12: H2O2;) 100 equiv (13: Glutathione; 14: Cysteine;)
15: 250 equiv NO; 250 equiv (16: TBHP; 17: TBO•; 18: HO•; ) 19:
15 equiv HNO and b) the stability of time dependence of
TP-HNO (10 μM) with 15 equiv HNO (red line) or not (black
line) in PBS buffer solution. λex=363 nm, slit: 5/5 nm.
-
-
Selectivity and stability of probe TP-HNO. The spec-
ificity selectivity of fluorescent probe determines its avail-
ability in actual sample. Therefore, we investigated the
ability of probe TP-HNO to distinguish the HNO from
various biological relevant species through fluorescence
assay measurements in PBS buffer solution, such as ions
-
(K+; Na+; Ca2+; Mg2+; Zn2+; NO3-; NO2 ; F-; and HS-), nitric
oxide (NO), oxidizing substance (NaClO, H2O2, TBHP,
TBO•, HO• ), and common amino acids (Cysteine and
Glutathione). As depicted in Figure 3a, only adding AS
reagent could induce a significant fluorescence enhance-
ment at 550 nm, while others relative analytes exhibited no
negligible the fluorescence changes of probe TP-HNO.
Figure 5. Exogenous nitroxyl imaging in RAW 264.7 living
cells. One photon model: a) and f) bright channel; b) and g)
green channel; c) and h) merged channel, λex=405 nm. Two
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photon model: d) and i) green channel; e) and j) merged
of 9 regions of interest (ROIs) from different treated living
HeLa cells. λex-OP =405 nm, λex-TP=730 nm, scale bar = 20
μm.
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2
3
4
5
6
7
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channel, λex= 730 nm, scale bar= 20 μm.
Exogenous nitroxyl imaging in living cells. Given the
advantages of fluorescence probe TP-HNO, such as high
selectivity, excellent sensitivity, low cytotoxicity, rapid
response and working appropriately at the physiological
pH, which made it have the possibility of imaging at the
cellular level. So, cell imaging experiments were carried out
upon one-photon and two-photon excitation model. As
shown in Figure 5, RAW 264.7 cells were treated with 5 μM
Endogenous nitroxyl imaging in living cells.
Encouraged by the favorable property of probe TP-HNO
for the exogenous nitroxyl imaging in living cells, we tried
to study whether it possesses potential application for
monitoring endogenous nitroxyl. Only adding 5 μM probe
o
TP-HNO for 1 h in HeLa cells at 37 C in incubator, there
9
was weak fluorescent signal gathered from green channel
with one-photon excitation at 405 nm (Figure 6d2) and
two-photon excitation at 730 nm (Figure 6d4). Recently,
Doctorovich and coworkers found that intracellular HNO
could be formed through L-ascorbate reacting with NO in
biological media.61 Subsequently, the fluorescence
intensity from green channel gradually brighten when
HeLa cells were pretreated with 2.0 mM L-ascorbate
(NaASc) and 2.0 mM sodium nitroprusside (SNP, NO
donor) for another 2 h (Figure 6e2 and e4). And, with
more stimulation cells through SNP, more intracellular
HNO generated (Figure S14). By contrast, almost no
fluorescent emission intensity of green channel were
gathered (Figure 6a1-a5, 6b1-b5 and 6c1-5c5). Furthermore,
the fluorescence intensities of each experimental group
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o
probe TP-HNO for 30 min at 37 C in incubator, and no
fluorescence signals in green channel (Figure 5b and 5d)
were collected through one-photon (excitation with 405
nm) and two-photon (excitation with 730 nm) fluorescent
microscopy, respectively. On the contrast, upon addition 30
μM AS reagent for cultivating another 30 minutes, obvious
fluorescence signals in green channel (Figure 5g and 5i)
were gathered, clearly indicating that probe TP-HNO
could be used to monitoring exogenous nitroxyl imaging
in living cells with excitation 405 nm (OP) and 730 nm (TP)
via laser confocal fluorescence microscopy.
were extracted as averages of nine regions of interest (ROIs)
(Figure 5f), which demonstrated probe TP-HNO could be
competent for monitoring intracellular endogenous
nitroxyl changes in living HeLa cells via with two-photon
fluorescence microscope.
CONCLUSION
In conclusions, through rational design, we have pro-
posed and synthesized a versatile two-photon fluorescent
probe based on 6-hydroxyl-quinonline-2-benzothiazole
derivatives. Probe TP-HNO demonstrated rapid response,
high selectivity and excellent sensitivity for the recogni-
tion of HNO. The High Resolving Mass Spectrum (HRMS)
analysis was carried out to verify the sensing mechanism
of probe TP-HNO for HNO. In addition, probe TP-HNO
also displayed high biocompatibility and it could be ap-
plied to bioimaging of exogenous and endogenous nitrox-
yl in living cells through two-photon laser confocal fluo-
rescence microscopy. We anticipate that probe TP-HNO
as a vital tool could be used to insight into the pathology
and physiology biological effects of HNO in the further.
ASSOCIATED CONTENT
Supporting Information. 1H-NMR, 13C-NMR and
31P-NMR spectra, HRMS spectra, absorbance and emission
spectra and competitive experiments of probe TP-HNO
are available in Supporting Information. This material is
available free of charge on the ACS publications website.
Figure 6. Endogenous nitroxyl two-photon imaging in living
HeLa cells. a1-5) control group; b1-5) cells treated with 2 mM
NaASc; c1-5) cells treated with 2 mM SNP; d1-5) cells treated
with probe TP-HNO; e1-5) cells pre-treated with probe
TP-HNO and then incubated with 2 mM NaASc and 2 mM
SNP; f) green channel intensities were measured as averages
AUTHOR INFORMATION
Corresponding Author
*Email: pengxj@dlut.edu.cn. Fax/Tel: +86 -411- 84986306.
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Analytical Chemistry
Notes
Anal. Chem. 2017, 89, 8079-8083.
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(23) Li, H.; Yao, Q.; Fan, J.; Hu, C.; Xu, F.; Du, J.; Wang, J.; Peng,
X. Ind. Eng. Chem. Res. 2016, 55, 1477-1483.
The authors declare no competing financial interests.
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ACKNOWLEDGEMENTS
(24) Martí, M.A.; Bari, S.E.; Estrin, D.A.; Doctorovich, F. J.
Am. Chem. Soc. 2005, 127, 4680-4684.
This work was financially supported by National Natural
Science Foundation of China (21376039, 21421005, 21422601
and 21576037), NSFC-Liaoning United Fund (U1608222)
and National Basic Research Program of China
(2013CB733702).
(25) Cline, M.R.; Tu, C.; Silverman, D.N.; Toscano, J.P. Free
Radical Bio. Med. 2011, 50, 1274-1279.
(26) Samuni, U.; Samuni, Y.; Goldstein, S. J. Am. Chem. Soc.
2010, 132, 8428-8432.
(27) Reisz, J.A.; Zink, C.N.; King, S.B. J. Am. Chem. Soc. 2011,
133, 11675-11685.
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