Synthesis and bioapplication of a highly selective and sensitive fluorescent probe for HOCl based on a phenothiazine-dicyanoisophorone conjugate with large Stokes shift

Article abstract of DOI:10.1039/c8nj00279g

Herein, a fluorescent probe, Dcp-EPtz, for HOCl based on a strong push-pull phenothiazine-dicyanoisophorone conjugate was prepared. The probe exhibits a very large Stokes shift of over 170 nm. Dcp-EPtz turns out to be a fast-responsive (<10 s) fluorescent

Full text of DOI:10.1039/c8nj00279g

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Synthesis and bioapplication of a highly selective and sensitive
fluorescent probe for HOCl based on a phenothiazine-
dicyanoisophorone conjugate with large Stokes shift
Received 00th January 20xx,
Accepted 00th January 20xx
Dasheng Zheng,a Xiaoying Qiu,a Chang Liu,a Xiaojie Jiao,a Song He,a Liancheng Zhaoa,b and Xianshun
Zeng*a,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
A fluorescent probe Dcp-EPtz for HOCl based on a strong push-pull phenothiazine-dicyanoisophorone conjugate has been
prepared. The probe exhibits a very large Stokes shift over 170 nm. Dcp-EPtz turns out to be a fast-responsive (< 10 s)
fluorescent probe for HOCl with high selectivity and sensitivity. It shows a good linear relationship of the enhanced
fluorescence intensity at 617 nm versus concentration of hypochlorous acid in the range of 0-10 µM, and with the
detection limit of 3.9 × 10-8 M. Meanwhile, the probe Dcp-EPtz exhibits the ability for endogenous HOCl monitoring in
living cells.
the visual investigation of the metabolism processes of HOCl
with high spatial and temporal resolution in living cells.7
Nowadays, many probes for HOCl/OCl- have been developed
Introduction
Reactive oxygen species (ROS) and reactive nitrogen (RNS)
are important intermediates in biological processes. However,
elevated ROS levels may give rise to the disorder of
metabolism and the dysfunction of organ system, and lead to a
series of diseases, such as aging, immunity, chronic
inflammatory, atherosclerosis, cardiovascular, and cancers.1
Among a variety of biologically important ROS, hypochlorous
acid (HOCl, a weak acid with pKa = 7.63) is a powerful fungicide
in the innate immune system.2 The endogenous HOCl is
biologically synthesized to resist the microbial invasion in the
phagolysosome of neutrophil by the oxidation of chloride ion
with hydrogen peroxide in the presence of myeloperoxidase
(MPO).3,4 However, an excessive production of endogenous
HOCl in human body can result in tissue injury and various
diseases.5 Thus, the development of highly sensitive and
selective methods for visual monitoring endogenous HOCl in
the vivo is needed urgently.
by taking full advantage of the strong oxidizing ability of
hypochlorite species.7-9
It is well known that sulfur-containing compounds are very
susceptible to oxidation from various oxidizing agents under
different conditions.10 Based on such a feature of sulfur-
containing compounds, a number of sulfur- and heavier
chalcogen(Se, or Te)-containing probes have been designed
and prepared recently for selective detection of HOCl.11 In
2007, Nagano and coworkers11a demonstrated
a spiro-
thioether of rhodamine analogue (HySOx) for detection of HOCl
with high selectivity. The probe HySO0 showed excellent
brightness and high on/off ratios by the oxidation of S atom
within the spiro-thioether, followed by the ring-opening
reaction of the nonfluorescent spiro-thioether form HySO0 to
the ring-opened fluorescent form HySO3H. Since then, a few of
spiro-thioxanthene derivatives have been developed for
detection of HOCl.11 On the other hand, the lone pair electrons
of chalcogen atom played an important role in the modulation
In the past decades, the detection techniques, including
chromatographic, electrochemical, colorimetric, fluorometric,
and probe-based fluorescence imaging methods, had been
established for the determination of HOCl under different
environments.6 Among the reported techniques, fluorescence
imaging techniques exhibit many advantages over others for
of the photoinduced electron transfer (PET) processes,12
a
number of OFF/ON fluorescent probes have also been
prepared for determination of HOCl/OCl via a PET process
from the electron-rich chalcogen donor to the fluorophores.13
Nevertheless, many of documented probes suffered from
small Stokes Shift, a short-wavelength emission band, and
complicated multi-step chemical synthesis, which limited their
applications in bioimaging.
a.Tianjin Key Laboratory for Photoelectric Materials and Devices, School of
Materials Science and Engineering, Tianjin University of Technology, Tianjin
300384, China. E-mail: xshzeng@tjut.edu.cn.
With the above concerns in mind, we reported herein the
synthesis and biological applications of a new fluorescent
probe Dcp-EPtz for HOCl based on phenothiazine-
b.School of Materials Science and Engineering, Harbin Institute of Technology,
Harbin 150001, China.
Electronic supplementary information (ESI) available. See DOI:
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Scheme 1 Synthesis of the probe Dcp-EPtz. Reagents and conditions: a) C2H5Br, NaH,
dry DMF, 0 oC, 12 h; b) dry DMF, POCl3, C2H4Cl2, 48 h; c) benzaldehyde, piperidine,
CH3CN, 40 oC, 12 h.
dicyanoisophorone conjugate (Scheme 1). After the reaction
with HOCl in aqueous media, the oxidation product of Dcp-
EPtz shows the absorption and emission maxima at 447 nm
and 618 nm, respectively. The probe features a high degree of
selectivity and sensitivity for HOCl. At the same time, it also
exhibits favourable properties as the fluorescent probe for
HOCl in biological applications, including response in real time
(< 10 s), very large Stokes shift (> 170 nm), and pH-
independence of fluorescence signals under physiological
conditions (pH 5.0-8.0). The probe can effectively discriminate
HClO from other reactive oxygen species (ROS) and reactive
nitrogen (RNS) in living cells by means of fluorescence
microscopy.
Fig. 1 (a) UV-Vis titration spectra of the probe Dcp-EPtz (10 μM) and (b) fluorescent
titration spectra of Dcp-EPtz (5 μM) in the presence of different concentrations of
NaOCl in PBS (10 mM, pH = 7.4): ethanol = 1:1 (v/v). λex = 475 nm, Slit = 5 nm, 5 nm.
M-1 cm-1) were observed. Two isosbestic points at 395 nm and
485 nm were observed, indicating the production of a new
compound by the reaction of the probe Dcp-EPtz and OCl-. The
absorbencies of the probe at 447 nm and 495 nm linearly
increased and decreased up to a 1.8:1 [OCl-]/[Dcp-EPtz] ratio,
respectively. The absorbance at 447 nm keeps constant in the
presence of more than 2.0 equivalents of OCl-. Under
excitation at 475 nm, the probe Dcp-EPtz emitted a very weak
Results and discussion
Preparation of the probe Dcp-EPtz
As shown in Scheme 1, the preparation of the fluorescent probe
(Dcp-EPtz) is simple and convenient. According to a modified
literature procedure, 10-ethyl-phenothiazine (EPtz) was prepared as
white solid in 90% yield by the reaction of phenothiazine with
bromoethane in anhydrous DMF in the presence of sodium
hydride.14c Then, the reaction of EPtz with POCl3 and anhydrous
DMF afforded the key intermediate 10-ethylphenothiazine-3-
carbaldehyde (2) as yellow solid in 83% yield. Subsequently, the
probe Dcp-EPtz was synthesized as a red powder at a yield of 52%
by the reaction of 2 with 2-(3,5,5-trimethylcyclohex-2-enylidene)-
malononitrile 1 in dry acetonitrile in the presence of piperidine as a
catalyst. The chemical structure of the probe Dcp-EPtz was
confirmed by 1H, 13C NMR (Figure S1-S2).
fluorescence (
Φ = 0.00082) at ca. 617 nm. With the increase of
OCl- concentration, the fluorescent intensity of the probe at
617 nm increased significantly, and the fluorescence intensity
reached the maximum after the addition of 3.6 equivalents of
OCl-. The quantum yield (
Φ) increased to 0.1068 after reaching
the fluorescence emission maximum. It is interesting to note
that a good linearity between the concentration of OCl- in the
range of 0-10 µM and the fluorescent intensity at 617 nm were
observed. From the OCl--dependent fluorescence changes at
617 nm, the detection limit of the probe towards OCl- was
estimated to be 3.9 × 10-8 M (detection limit = 3
σbi/m) (Fig.
S3).15 This result suggests that Dcp-EPtz can be used as a highly
sensitive fluorescence probe for the quantitative detection of
OCl-. On the other hand, a very large Stokes shift (with a peak-
to-peak Stokes shift over 170 nm) of Dcp-EPtz suggests that it
can eliminate excitation light backscattering effect for
fluorescence imaging.16
Sensing properties of the probe Dcp-EPtz toward HOCl
The spectroscopic properties of Dcp-EPtz and those in the
presence of an incremental amount of NaOCl were measured
in PBS (10 mM, pH = 7.4): ethanol = 1:1 (v/v). As shown in Fig.
1a, the absorption bands at 375 nm (
and 495 nm ( = 3.06
104 M-1 cm-1) were observed for the
ε = 2.33 ×
104 M-1 cm-1)
Then, we evaluated the responses of the probe Dcp-EPtz to
other species by UV-Vis absorption and fluorescence spectra in
PBS (10 mM, pH = 7.4) : ethanol = 1 : 1 (v/v) (Fig. 2). As can be
seen from Fig. 2a, the addition of 10 equivalents of various
ε
×
free probe Dcp-EPtz (10 µM), which can be assigned to the π-
π* transitions of the Ptz moiety and the Dcp-EPtz conjugate,
respectively. Upon addition of NaOCl (0 - 2.5 equiv.), the
obvious decrease in the absorption spectra of Dcp-EPtz at 495
1
-
ROS, such as O2 , ClO4 , H2O2, O2.-, .OH, ONOO-, TBHP, and NO,
and some anionic species, including AcO-, Br-, Cl-, CO32-, F-,
nm and an increase in the new band at 447 nm (ε = 4.92 ×
104
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Fig. 3 Timed-dependent fluorescence intensity of Dcp-EPtz (10 μM) upon the addition
of 10 equiv. OCl- in PBS (10 mM, pH = 7.4): ethanol = 1 : 1 (v/v). λex = 475 nm, λem = 617
nm, Slit = 5 nm, 5 nm.
Fig. 2 The absorption (a) and fluorescence emission (b) spectra of the probe Dcp-EPtz
upon the addition of 10.0 equiv. other anions and ROS: AcO-, Br-, Cl-, OCl-, ClO4-, CO32-, F-
-
2-
, H2O2, H2PO4-, HCO3-, HPO42-, HSO4-, I-, NO, NO2 , NO3-, O2-, ·OH, ONOO-, PO43-, S2-, SO3
,
SO42-, TBHP, and O21 in PBS (10 mM, pH = 7.4): ethanol = 1 : 1 (v/v). λex = 475 nm, Slit = 5
nm, 5 nm.
-
-
-
-
2-
H2PO4 , HCO3 , HPO42-, HSO4 , I-, NO3 , PO43-, S2-, SO32- and SO4
Scheme 2 Proposed sensing mechanism of the probe Dcp-EPtz towards NaOCl.
has no marked effect on the UV-Vis absorption profiles of the
probe Dcp-EPtz (10 µM) at 495 nm. However, the addition of
OCl- elicited an obvious blueshift of the absorption
maximumfrom 495 nm to 447 nm, along with an immediate
color change from red to yellow. The observations indicate
that Dcp-EPtz can serve as a colorimetric and 'naked-eye'
probe for the determination of OCl-. Under excitation at 475
nm, the addition of 10 equivalents of various ROS and anionic
species has no obvious turn-on effect on the fluorescence
properties of Dcp-EPtz (5 µM) (Fig. 2b). Nevertheless, the
addition of OCl- resulted immediately a significant fluorescence
enhancement at 617 nm (with an enhancement factor of 130-
fold), suggesting that Dcp-EPtz can be used as a fluorescent
probe for selective determination of OCl-.
Fig. S5, the fluorescence intensity of the probe showed no
significant fluorescence at any pH, indicating that the probe is
not sensitive to pH change. After addition of OCl-, remarkable
fluorescence enhancement of Dcp-EPtz could be observed,
and the probe displayed almost constant fluorescence
intensity in the range of pH 5.0-8.0, implying that Dcp-EPtz
would work well under physiological pH conditions.
ROS in natures are highly reactive and short-lived in
physiological environments. The probe with the real-time
responsive properties to ROS is of vital importance for
biological applications. To elucidate the real-time responsive
properties of Dcp-EPtz toward HOCl, we then investigated the
time-dependent fluorescence response of the probe in PBS (10
mM, pH = 7.4) : ethanol = 1 : 1 (v/v). As shown in Fig. 3
, Dcp-
At the same time, fluorescent responses of the probe to
OCl- in the presence of a background of relevant ROS and
EPtz was essentially nonfluorescent under a continuous
biologically relevant anions, including O2 , ClO4 , H2O2, O2 , .OH,
1
-
-
excitation at 475 nm (0 s to 1200 s). After the addition of
NaOCl (10 equiv.), the fluorescence intensity at 617 nm
increased immediately and reached the maximum within 10 s,
indicating that the probe exhibited a real-time response to OCl-.
Then, the fluorescence intensity at 617 nm fluctuated within
the first 360 s, and then decreased gradually from 360 s to
1200 s. As can be seen from Fig. 3, although the fluorescence
intensity fluctuated within the first 5 minutes, it still had a
sufficient turn-on fluorescence intensity for the detection of
HOCl. We speculated that the fluctuation of fluorescence
signals within the first 5 minutes should be attributed to the
structural conversions during the oxidation process (Scheme
2). As shown in Scheme 2, upon the addition of NaOCl the
-
-
2-
ONOO-, TBHP, NO, AcO-, Br-, Cl-, CO32-, F-, H2PO4 , HCO3 , HPO4
, HSO4 , I-, NO3 , PO43-, S2-, SO32- and SO42-, were evaluated (Fig.
S4). As shown in Fig. S4, the fluorescence intensities of the
probe Dcp-EPtz is slightly diminished or increased to a
different extent compared with the OCl- anions alone, but they
still showed sufficient 'turn-on' effect for the determination of
OCl-. It is corroborated that the probe Dcp-EPtz can determine
OCl- anions with satisfactory selectivity in the presence of most
other biological relevant ROS and anionic competitive species.
In addition, we investigated the spectral properties of Dcp-
EPtz in the presence and absence of OCl- in a series of buffers
with different pH values ranging from 4 to 10. As shown in
-
-
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probe Dcp-EPtz was immediately converted to the radical
In light of the above desirable optical properties including
cation (Dcp-EPtz+.) by OCl-. Then, the intermediate Dcp-EPtz+. far-red fluorescence emission, very large Stokes shift and real-
was converted to the bication species (Dcp-EPtz2+) during the time fluorescence response of Dcp-EPtz as the probe for HOCl,
oxidation process, followed by the water addition and it was then applied to detect HOCl in L929 cells, a murine
deprotonation processes to produce the fluorescent product fibrosarcoma cell line (Fig. 4). As illustrated in Fig. 4A, when
Dcp-EPtzO 17
To verify the detection mechanism of Dcp-EPtz the L929 cells were incubated with the probe Dcp-EPtz (0.5
.
towards HOCl/OCl-, the reaction product between Dcp-EPtz and µM) for 0.5 h in the culture medium and then rinsed by PBS,
NaOCl was characterized by the high-resolution mass spectra almost no fluorescence signal of the probe was present in L929
(HRMS) (Fig. S6). The peak at [Dcp-EPtzO + Na]+
= 462.1625 cells. In contrast, after incubation with NaOCl (1 μM) for 0.5 h,
(calc. m/z 462.1616) and a weak peak at [Dcp-EPtzO + H]+
440.1794 (calc. m/z 440.1791) corresponded to Dcp-EPtzO
=
clear fluorescence signals in red channel (580-645 nm) were
observed (Fig. 4B). Merged fluorescence and brightfield image
,
respectively, which provided strong evidence for the oxidation showed that the fluorescence signals of the probe were found
mechanism. In spite of a series of structural conversions during only in cell, demonstrating that Dcp-EPtz was suitable for
the oxidation processes, the probe Dcp-EPtz exhibited a very practical utilization as a fluorescent probe for visualizing HOCl
fast fluorescence response toward HOCl
.
in the living cell.
Subsequently, we examined the feasibility of the probe for
detecting endogenous HOCl produced by resting tissue
macrophages under mimic inflammatory conditions. In order
to give rise to an increase in endogenous HOCl levels, the living
murine RAW 264.7 macrophage cells were incubated with
lipopolysaccharide (LPS)18 and phorbol-12-myristate 13-
acetate (PMA).19 Then the probe Dcp-EPtz (0.5 µM) in PBS was
administrated and were incubated at 37 oC for another 20 min.
As shown in Figure 5, with LPS/PMA elicitation, significantly
enhanced fluorescence intensity was observed in the cells
(Figure 5B), indicated that the probe was activated by the
PMA-induced endogenous HOCl. However, no obvious
fluorescence was observed in the cells compared with the
control group (Figure 5A). The result indicated that the
fluorescence probe could detect endogenous HOCl produced
in stressed cells.
Fig. 4 Confocal fluorescence images of L929 cells incubated with the probe Dcp-EPtz
(0.5 µM). Cell images were obtained using an excitation wavelength of 488 nm and a
band-path (580 - 645 nm) emission filter. A) Fluorescence, brightfield and merged
images after 0.5 h incubation with the probe; B) Fluorescence, brightfield and merged
images after incubated with exogenous NaOCl (1 µM) for 0.5 h.
Experimental
Materials and equipments
Unless otherwise stated, all reagents and starting materials
were purchased from Innochem Chemical Company (Beijing,
China) or Tokyo Kasei Kogyo (TCI: Tokyo, Japan), AR grade, and
used without further purification. Dry solvents were purchased
from Alfa-Aesar, and used as received. For biological studies
dulbecco's modified eagle's medium (DMEM) trypsin-EDTA,
dimethylsulfoxide (DMSO) for cell culture were purchased
from Sigma Aldrich. Penicillin-Streptomycin, and fetal bovine
serum (FBS) were purchased from Invitrogen and Life
technologies. Melting points were recorded on a Boethius
Block apparatus and pH measurements were carried out on a
Mettler Toledo MP 220 pH meter. NMR spectra were
measured on a Bruker spectrometer at 400 (1H NMR) MHz and
100 (13C NMR) MHz. Chemical shifts (δ) were reported in ppm
down field from internal Me4Si (1H and 13C NMR). High
resolution mass spectra (HRMS) were acquired on an Agilent
6510 Q-TOF LC/MS instrument (Agilent Technologies, Palo
Alto, CA) equipped with an ESI source. All absorption spectra
Fig. 5 Confocal fluorescence images of RAW 264.7 cells incubated with the probe Dcp-
El-Ptz (0.5 µM). Cell images were obtained using an excitation wavelength of 488 nm
and a band-path (580 - 645 nm) emission filter. A) Fluorescence and brightfield image
after 30 min incubation with the probe; B) Fluorescence image of RAW 264.7 cells
pretreated with lipopolysaccharide (LPS; 1 µg/mL) for 0.5 h, phorbol-12-myristate 13-
acetate (PMA; 2.5 µg/mL) for 0.5 h, and then incubated with the probe for 0.5 h.
were recorded on
a
Shimadzu UV-2550 UV/Vis
spectrophotometer with
1 cm quartz cell. Fluorescence
measurements were performed on
a
Hitachi F-4600
Fluorescent imaging of HClO in HeLa cells
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spectrofluorophotometer with a 1 cm quartz cell. Cells were column chromatography using petroleum ether/dichloromethane
o
imaged on a confocal microscope (Olympus FV1000-IX81).
(2:1; v/v) to give red solid in 52% yield (0.22 g); m.p.: 206 - 208 C;
HRMS: 424.1776 [M + H]+, calcd: 424.1769; 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.28 (s, 1H), 7.24 (s, 1H), 7.17-7.10 (m, 2H), 6.95-
Preparation of ROS and anions solutions
Stock solutions (10 mM) of the sodium salts of AcO-, Br-, Cl-, 6.79 (m, 6H), 3.95 (s, 2H), 2.58 (s, 2H), 2.43 (s, 2H), 1.43 (t, J = 6.9
ClO4 , CO32-, F-, H2PO4 , HCO3 , HPO42-, HSO4 , I-, NO2 , NO3 , PO43-, S2-, Hz, 3H), 1.07 (s, 6H). 13C NMR (100 MHz, CDCl3): δ (ppm) 169.1,
-
-
-
-
-
-
SO32-, and SO4 anions were prepared in distilled water. Stock 154.1, 136.0, 127.2, 122.89, 115.2, 115.0, 113.7, 112.9, 43.0, 39.3,
2-
solution of Dcp-EPtz (5 mM) was prepared in DMF. All ROS were 32.0, 28.0.
prepared before use as per the procedures reported in the
Cell imaging using the probe Dcp-EPtz
literature.9k Test solutions were prepared by placing appropriate
volume of the stock solution of Dcp-EPtz into a 5 mL vial, adding an
L929 cells, a murine fibrosarcoma cell line, were incubated in
appropriate aliquot of each anion or fresh ROS solution, and Dulbecco’s modification of Eagle’s medium (DMEM) with 10% fetal
diluting the solution to 3 mL with PBS (10 mM, pH = 7.4) : ethanol bovine serum (FBS) at 37 oC in a 5% CO2 atmosphere under sterile
= 1 : 1 (v/v).
environments. Cells were exponentially cultured as monolayer up
to ca. 80% confluence. Before staining with the probe, cells were
detached by treating with trypsin-EDTA (1×), and were seeded on
24 well chambered coverglass at a density of ca. 1 × 106 cells mL-1
for 24 h. The probe Dcp-EPtz dissolved in DMSO was added to the
culture medium (500 µL) at a desired final concentration. After
staining for 30 min, excess probes were removed by gentle rinsing
with phosphate buffered saline (PBS, pH 7.4) three times.
Fluorescence images were collected by sequentially line scanning
with an Olympus FV1000 confocal laser-scanning microscope. The
probe Dcp-EPtz was excited at 488 nm and their red emissions were
collected in the range of 580-645 nm.
Synthesis of 2-(3,5,5-trimethylcyclohex-2-enylidene)malononitrile
(1)
The intermediate 1 was prepared by adaption of relevant
procedure as described in literature.14a To be specified, to a solution
of isophorone (3.8 g, 27.6 mmol) and malononitrile (1.82 g, 27.6
mmol) in dry ethanol (150 mL), piperidine (23 mg, 0.267 mmol) was
added. The solution was stirred at 60 oC for 8 h. After cooling to
room temperature, the black solution was slowly poured into water
(200 mL). The product was extracted with dichloromethane (150 mL
× 3). The combined organic phase was dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure. The
residue was purified by column chromatography (SiO2, gradient:
petroleum ether/EtOAc from 100:0 to 90:10) in 90% (2.3 g) yield as
a white solid, m.p.: 78 - 80 oC. The spectral and optical data of
compound 1 perfectly matched those reported in literature.14a
Conclusions
In summary, we have successfully designed and
synthesized a novel fluorescent probe Dcp-EPtz by the reaction
of 2-(3,5,5-trimethylcyclohex-2-enylidene)malononitrile with
10-ethylphenothiazine-3-carbaldehyde. The probe Dcp-EPtz
shows a very high selectivity for HOCl over other ROS and
biologically relevant anions, and Dcp-EPtz also exhibits
favourable properties as the fluorescent probe for HOCl in
biological applications, such as a real-time response (< 10 s), a
very large Stokes shift (> 170 nm), and a constant 'turn-on'
fluorescence ratio in physiological conditions (pH 5.0-8.0). The
probe Dcp-EPtz exhibits the ability for endogenous HOCl
monitoring in living cells by means of fluorescence microscopy.
Thus, we anticipate that the probe can offer a new vehicle to
understanding the role of HOCl in the immune system.
Synthesis of 10-ethyl-10H-phenothiazine-3-carbaldehyde (2)
Prepared with a slight modification to what was reported in
relevant literature.14b In a 100 mL Schlenk flask, POCl3 (7 mL) was
added dropwise to dry DMF (5 mL) at 0 oC. After stirring the
reaction mixture for 30 minutes, the solution of EPtz (2.27 g) in dry
CH2Cl2 (25 ml) was added. The reaction mixture was heated and
refluxed for 48 h under nitrogen atmosphere. The reaction mixture
was quenched with dilute NaOH (aq) (50 mL) and extracted with
dichloromethane (50 mL × 3). The organic phase was dried with
anhydrous Na2SO4 and then the solvent was removed in vacuo. The
residue was purified by column chromatography using petroleum
ether/dichloromethane (1:1; v/v) to give yellow oil. Recrystallization
from petroleum ether/dichloromethane afforded a yellow solid in
83% yield (2.12 g). The spectral and optical data of compound 2
perfectly matched those reported in literature.14b
Acknowledgements
We gratefully acknowledge the Natural Science Foundation
of China (NNSFC 21272172), and the Natural Science
Foundation of Tianjin (12JCZDJC21000).
The preparation of Dcp-EPtz
Under an argon atmosphere, 1 (0.19 g, 1 mmol) and 2 (0.26 g, 1
mmol) were dissolved in dry acetonitrile (5 mL). Piperidine (0.0085
g, 0.01 mmol) was added and the solution was stirred at 40 oC till
starting material disappeared (detected by TLC). After evaporation
of acetonitrile under reduced pressure, H2O (20 mL) was added to
the residual mixtures, and then extracted with dichloromethane (20
mL × 3). The combined organic solution was dried over Na2SO4 and
then the solvent was removed in vacuo. The residue was purified by
Conflicts of interest
There are no conflicts to declare.
Notes and references
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Journal Name
1
2
D. I. Pattison, M. J. Davies, Chem. Res. Toxicol., 2001, 14
,
10 (a) J. A. Burkhard, C. Guerot, H. Knust, E. M. Carreira, Org.
Lett., 2012, 14, 66; (b) P. L. Robinson, C. N. Barry, J. W. Kelly,
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New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00279G
Synthesis and bioapplication of a highly selective and sensitive fluorescent probe
for HOCl based on a phenothiazine-dicyanoisophorone conjugate with large
Stokes shift
Dasheng Zheng, Xiaoying Qiu, Chang Liu, Xiaojie Jiao, Song He, Liancheng Zhao, Xianshun
Zeng
A fluorescent probe with large Stokes shift for endogeous HOCl monitoring in living cells has
been prepared.