Aggregated-induced emission phenothiazine probe for selective ratiometric response of hypochlorite over other reactive oxygen species

Article abstract of DOI:10.1016/j.dyepig.2016.01.010

In order to perform a ratiometric fluorescent sensor to recognize hypochlorite over other reaction oxidation species, a simple phenothiazine probe (QC1) with double dioxaborolane moieties was designed and synthesized. QC1 provided blue emission in aqueous and solid state. Double dioxaborolane and quaternary phosphonium salt was introduced into the molecule to prevent aggregation. The sulphur atom at the centre of phenothiazine was to respond to the hypochlorite at room temperature over other ROS because of its stronger oxidized ability. QC1 gave good linear fitting in the ratiometric mode under both absorption and emission titration experiments. Moreover, QC1 showed lower detection limited that reached 0.95 μM in absorption titration and 0.41 μM in fluorescent titration process.

Full text of DOI:10.1016/j.dyepig.2016.01.010

Accepted Manuscript
Aggregated-induced Emission Phenothiazine Probe for Selective Ratiometric
Response of Hypochlorite Over Other Reactive Oxygen Species
Linlin Wang, Yue Hu, Yi Qu, Jingli Xu, Jian Cao
PII:
S0143-7208(16)00021-8
10.1016/j.dyepig.2016.01.010
DYPI 5063
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 23 November 2015
Revised Date: 7 January 2016
Accepted Date: 8 January 2016
Please cite this article as: Wang L, Hu Y, Qu Y, Xu J, Cao J, Aggregated-induced Emission
Phenothiazine Probe for Selective Ratiometric Response of Hypochlorite Over Other Reactive Oxygen
Species, Dyes and Pigments (2016), doi: 10.1016/j.dyepig.2016.01.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Aggregated-induced Emission Phenothiazine Probe for Selective Ratiometric
Response of Hypochlorite Over Other Reactive Oxygen Species
Linlin Wang, Yue Hu, Yi Qu*, Jingli Xu and Jian Cao*
School of Chemistry and Chemical Engineering, Shanghai University of Engineering
Science, 333 Longteng Road, Shanghai, 201620, PR China. Fax: 86-21-67791214.
E-mail: caoj@sues.edu.cn; quyi@fudan.edu.cn
Abstract
In order to perform a ratiometric fluorescent sensor to recognize hypochlorite over
other reaction oxidation species, a simple phenothiazine probe (QC1) with double
dioxaborolane moieties was designed and synthesized. QC1 provided blue emission
in aqueous and solid state. Double dioxaborolane and quaternary phosphonium salt
was introduced into the molecule to prevent aggregation. The sulphur atom at the
centre of phenothiazine was to respond to the hypochlorite at room temperature over
other ROS because of its stronger oxidized ability. QC1 gave good linear fitting in the
ratiometric mode under both absorption and emission titration experiments. Moreover,
QC1 showed lower detection limited that reached 0.95 ꢀM in absorption titration and
0.41 ꢀM in fluorescent titration process.
Keywords: fluorescent probe, ratiometric, phenothiazine, aggregated-induced
emission, hypochlorite
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1. Introduction
Reactive oxygen species (ROS) plays an indispensable role in a wide variety of
biological processes [1-4]. For example, recent studies reported that cancer cells
constantly generate high levels of intracellular ROS, due to oncogenic transformation
[5]. Among the various ROS, hypochlorous acid (HClO) is a powerful microbicidal
agent in the innate immune system for its strong oxidizing property. The endogenous
HClO released by the neutrophil plays a vital role in killing a wide range of pathogens
[6, 7]. As a result, excessive generation of HClO always implicates in many human
diseases, and can be found as inflammation-associated tissue injury [8-12]. Moreover,
it is considered that HClO contributes to cardiovascular disease, and cell apoptosis
through calcium dependent calpain activation. Therefore, there is urgent need to sense
the various biological effects of HClO with lower detection limit, good selectivity and
in real time [13, 14].
With high sensitivity and adjustable structure, many small molecular fluorescent
sensors were reported, such as cyanide dyes, fluorescein, rhodamine, BODIPY,
naphthalimide, Dicyanomethylene-4H-pyran, and so on [15-27]. There are still some
challenges to optimize the small molecular fluorescent sensors for HClO: 1) some
commercial dyes show poor stability for oxidants. For example, the cyanide dyes are
always bleached by ROS [28]; 2) the ratiometric response probe with different
fluorescent channels is still not common [29-32].
To solve such problems, it is needed to develop stabile fluorophores and the
useful redox reactions that can output the ratiometric signals for sensing HClO
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beyond other ROS. In 1985, Brubaker et. al. reported a desulfurization technology
with NaClO [33]. As we know, one component of the organic sulphur in coal is
dibenzothiophene, an analogy of phenothiazine. Further research on the oxidation of
phenothiazine indicated the Chlorite can oxidize the dye to sulfones under stronger
oxidizing conditions such as the presence of fuming nitric acid and hydrogen peroxide
[34]. Based on this knowledge, we hypothesize that the modified phenothiazine can
only be oxidized by a strong oxidant, like ClO ꢀ , and we designed the
phenothiazine-based fluorescent sensor (QC1) to detect ClOꢀ. As shown in Scheme 1,
we introduced two dioxaborolane moieties to enhance the steric of phenothiazine that
can restrain π-π stacking of the rigidity dyes. The quaternary phosphonium salt
attached on the N atom of phenothiazine also showed steric hindrance effect for the
sensor.
<Scheme 1>
2. EXPERIMENTAL
2.1 Materials and instruments
All reagents and solvents were used as received without further purification.
Deionized water was used in the experiments throughout. Silica gel (100-200 mesh)
was used for column chromatography. Mass determination was made on a GC-TOF
MS spectrometry. NMR spectra were recorded on a Varian 400 MHz with chemical
shifts reported as ppm (in DMSO-d6 or CDCl3, TMS as internal standard).
Fluorescence measurements were performed on a FS-5 spectrophotometer (Edinburgh,
Britain) and the slit width was set as 1 nm for excitation and emission, respectively.
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Absorption spectrum was measured on a SHIMADZU UV-3600 spectrophotometer.
ꢀ
NO and ClO
were produced from the dissolution of NOC13
[1-Hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene][35] and NaClO in the
de-ion water, respectively. H2O2 was diluted from the stabilized 30% H2O2 solution.
Hydroxyl radical (·OH) was generated by reaction of 1mM Fe2+ with 200µM H2O2.
Tert-butoxy (t-BuOO·) was prepared from the reaction of 1mM Fe2+ with 200µM
ꢀ
ꢀ
TBHP. NaNO3 and NaNO2 were used as the source of NO3 and NO2 , respectively.
2.2 Synthesis of QC1
<Scheme 2>
2.2.1 Synthesis of compound 2
Compound 1 (180 mg, 0.5 mmol), bis(pinacolato)diboron (254 mg, 1.0 mmol),
Pd(dppf)Cl2 (36 mg, 0.05 mmol), and sodium acetate (287 mg, 3.5 mmol), were
added to a dry Schlenk tube. Then 5 mL of dry DMF was added via syringe and the
mixture was stirred under nitrogen at 80 °C for 2 hours. After cooling to room
temperature, the mixture was extracted with dichloromethane, washed with water, and
the solvent was removed under reduced pressure. The crude product was purified by
column chromatography (silica gel, petroleum ether/dichloromethane, 1/5, v/v) to
give a light yellow solid (215 mg, 70.0%). 1H NMR (400 MHz, CDCl3) δ (ppm)
7.50 - 7.65 (m, 4 H), 6.83 (d, J=8.03 Hz, 2 H), 3.90 (br. s., 2 H), 3.38 (t, J=6.78 Hz, 2
H), 1.76 - 1.88 (m, 4 H), 1.46 (br. s., 4 H), 1.34 (s, 24 H); 13C NMR (100MHz,
CDCl3) δ ppm 147.2, 134.0, 133.8, 124.3, 114.8, 83.7, 47.2, 33.7, 32.6, 29.7, 27.7,
25.9, 24.8.
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2.2.2 Synthesis of QC1
A solution of compound 2 (306 mg, 0.5 mmol) and triphenylphosphine (145 mg, 0.55
mmol) in dry toluene (15 mL) was refluxed under nitrogen. The reaction was
monitored by TLC. Upon completion, the solvent was removed under reduced
pressure. The residue was purified by recrystallization to afford QC1 (372 mg, 85%
yield) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.68 - 7.82 (m,
15 H), 7.54 - 7.63 (m, 2 H), 7.47 (s, 2 H), 6.75-6.90 (m, 2 H), 3.87 (br. s., 2 H), 3.53 -
3.66 (m, 2 H), 1.69 (br. s., 2 H), 1.59 (s, 4 H), 1.39-1.46 (m, 2 H), 1.32 (s, 24 H);
13C NMR (100MHz, CDCl3) δ (ppm) 147.28, 135.02, 134.21, 133.66, 133.56, 130.60,
130.48, 124.10, 118.64, 117.79, 115.20, 83.73, 46.84, 29.40, 25.90, 25.73, 24.84,
22.81, 22.30. HRMS(ESI): Found: [M-Br]ꢁ 796.3917; requires [M-Br]ꢁ 796.3927 .
2.2.3 Synthesis of compound 3
Sodium hypochlorite solution (7.0 %, 2 mL) was added dropwise to a solution of
phenothiazine (199 mg, 1 mmol) in acetic acid (10 mL) was stirred at 90 °C overnight.
After cooled down, the water was added and extracted with ethyl acetate. The
separated organic layer was and washed with water and brine, then dried over Na2SO4
and removed under reduced pressure. The crude product was purified by column
chromatography (silica gel, petroleum ether/ ethyl acetate, 1/4, v/v) to afford 180 mg
(78%) of 3 as a pale brown solid. 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.91 (s, 1
H), 7.92 (d, J = 8.0 Hz, 2 H), 7.65 (t, J = 7.8 Hz, 2 H), 7.35 (d, J = 8.3 Hz, 2 H), 7.25
(t, J = 7.7 Hz, 2 H); 13C NMR (100MHz, DMSO-d6) δ (ppm) 138.61, 133.75, 122.91,
121.91, 121.11, 117.46.
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3. Results and Discussion
3.1 Synthesis and characteristic
The synthetic route to QC1 is illustrated in Scheme 2. Precursor 1 was
synthesized by alkylation reaction based on SN mechanism according to previously
published report [36-38]. Two boronic acid esters were introduced by palladium
catalysed
cross
coupling
reactions
with
bis(2,4-dimethylpentane-2,4-
glycolato)diboron as boron source. Pd(dppf)2Cl2 was chosen as the palladium catalyst.
Triphenylphosphine (TPP) group was attached on the end of the side chain through
the one-pot reaction of the toluene solution of 2 and 1.1 equiv. of Ph3P under the
reflux condition.
Fig. 1 shows the imperceptible changes on the structure of the probe and its
precursors (2 and TPP) by 1H and 13C NMR techniques. Firstly, we used 1H NMR to
study the migration of the methylene and phenyl groups. As shown in Fig. 1A, the
-CH2- and phenyls attached on the P+ migrated from 3.38 and 7.38 to 3.58 and 7.73,
13
respectively. The C NMR spectra can directly discover the slight variations in the
structure. The alkyl carbon and the phenyl carbon directly attached on the P+ moved
to 22 ppm and 120 ppm, respectively.
<Fig. 1>
3.2 Photophysical properties of QC1
3.2.1 Photophysical properties of QC1 in solid state and different solvents
QC1 showed distinct blue emission at solid state (Inset in Fig. 2A). Its
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absorption and emission spectra were shown in Fig. 2A. The absorption and emission
peaks of QC1 located at 403 and 485 nm, respectively. Then, the fluorescent property
was also explored in the solutions. When excited at 400 nm, the maximum emission
bands of QC1 varied from 430 nm to 482 nm in different solvents.
<Fig. 2>
3.2.2 Aggregated-induced Emission (AIE) property of QC1
AIE effect was first reported by Prof. Tang in 2001[39]. Here, we found that
QC1 can also work as the AIEgen[40, 41]. The emission property of QC1 was
investigated in DMSO-water mixture to prove its AIE characters (Fig. 3). In pure
DMSO solution, QC1 had very low emission intensity (Φf < 0.01). The emission
intensity of QC1 enhanced quickly with increasing fraction of water from 10% to
70%. Furthermore, the intensity was decreased for the stability of QC1 in the aqueous.
However, the about 2000-fold enhancement indicates the potential application of QC1
for ROS detection in water phase.
< Fig. 3>
3.3 Sensing of QC1 for ClOꢀ¯
3.3.1 Sensitivity of QC1
The sensing ability of QC1 for ClOꢀ was investigated by both UV−vis
absorption and emission spectroscopies. In the UV−vis absorption spectrum, the QC1
showed a weak visible band with the absorption wavelength at 313 nm and the strong
absorbance at 269 nm (Fig. 4a). Upon addition of ClOꢀ, the absorption peak around
313 nm was up, and the absorption peak around 269 nm was down. A linear fitting
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was obtained from 0 to 20 μM range implying that QC1 can work as the
colorimetric probe for detecting ClOꢀ in ratiometric method (Fig. 4b).
Compared with the uncolored UV-vis detection method, the bright blue emission
of QC1 indicated the practical application in fluorescent probe. As shown in Fig. 4c,
with gradual addition of ClOꢀthe emission peak at 381 nm turned on and the one at
486 nm was shutdown. Highly sensitive response was found in the range of 0 to 10
μM indicating that QC1 is more suitable in the fluorescent method than the
colorimetric method. The 3δ/k method was used to evaluate the detection limited (DL)
of QC1. As shown in Fig. S1, the DL value of QC1 reached 0.95 µM and 0.41 µM in
UV-vis and fluorescence approaches.
< Fig. 4>
< Fig. 5>
The quantum yields of QC1 was tested in aqueous and with the quinoline sulfate
(Φ=0.5) as a reference. The measured fluorescence quantum efficiency of QC1 is 0.21
in aggregated state and 0.48 in THF solution.
3.3.2 Mechanism of QC1 reacted with ClOꢁ
To verify the recognizing process of QC1 through the oxidant sulphur atom
pathway, we used 1H NMR titration experiments to spy the changes on the probe.
As shown in Fig. 5a, QC1 had broad bands were located at around 6.82 ppm and
7.6 ppm (marked with the blue areas), the same with phenothiazine (Fig. S2a).
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When 1 eq. of NaClO was added, both broad bands transformed to double bands
at the same locations (marked with the orange areas in Fig. 5b). Furthermore, we
o
added 100 eq. H2O2 to the QC1 sample and incubated it at 50 C for 10 min.
Then, the same spectrum was obtained in Fig. 5c. According to the previous
reports, the product of QC1 oxidized by NaClO coincided with the one oxidized
1
by H2O2. We also tested the H NMR spectrum of phenothiazine and NaClO. In
Fig. S2b, the broad bands of phenothiazine transformed to the double bands liked
QC1, which again supported the mechanism. On the contrary, this phenomenon
was not found in the titration process of (4-iodobutyl)triphenylphosphonium
iodide (Fig. S3). Additional, consideration of the strong signal at aryl region from
the bulk triphenylphosphine group, we chose the original phenothiazine dye for a
control experiment. As shown in Fig. S4, the oxide of phenothiazine showed the
characteristic peak at lower field around 7.0 ~ 8.0 ppm than the unoxidized dye.
3.3.3 Selectivity of QC1
Selectivity was another important parameter for a fluorescent probe. We applied
different ROS to evaluate the reaction activity of QC1. As shown in Fig. 6, the
maximum response of the emission intensity ratio at 381 nm and 482 nm reached 10.3
when 1 eq. of ClO－ was added in the system. The ratio value were lower than 5
when other ROS added, such as H2O2, t-BuOOH etc. The Fe2+ caused tiny variation
which was used to produce free radicals like the hydroxyl free radical.
< Fig. 6>
4. Conclusion
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In summary, we have developed a new phenothiazine based on AIE gen (QC1)
that can be used as a ratiometric fluorescent detector for probing ClOꢀ over other
common ROS species in HEPES buffer. Strong fluorescent signals obtained in solid
state (over 2000-fold than in DMSO) indicated this probe can be widely used in the
field of sensors. The high sensitivity of QC1 was recorded by titration experiment
(0~3 eq. of ClO－) and the calculation of detection limited value (3δ/k method, 0.41
µM). A new detecting process for ClO－ was performed based on the fast reaction
between ClO－ and phenothiazine. We believe that it can support a series of ClO－
-SENSORS with highly specific. Furthermore, we are working on modifying the
emission wavelength on the phenothiazine dyes.
Acknowledgements
This work was supported by NSFC/China (21404068), Talent Program of
Shanghai Municipal Education Commission (ZZGCD15029) and Shanghai University
of Engineering Science (A1-5300-14-020303, A1-5300-15-010228 and
E1-0501-15-0113). We thank Professor Zhihua Sun and his group for kindly helping
with organic synthesis site and recording of NMR spectra.
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Scheme and Figure Captions:
Scheme1. The descript of probe QC1 and its recognition mechanism towards NaClO.
Scheme 2. Synthesis Routine of probe QC1.
Fig. 1. 1H and 13C NMR spectra of probe QC1 and its precursors.
Fig. 2. (A) PL spectra of the QC1 in solid state. λex: 400 nm. (B) Normalized
emission spectra of QC1 in different solvents. λex: 400 nm. (Inset in Fig. 2A: solid
fluorescence of QC1)
Fig. 3. (A) Aggregated-induced emission spectra of QC1 performed in DMSO system
with different fraction of water (0~99%); (B) Emission enhancement of QC1 towards
different fraction of water.
Fig. 4. (A) Changes in the absorption spectra of QC1 (10 μM) upon ClOꢀ addition in
HEPES (pH = 7.4, contained 1% DMSO); (B) ratio of absorbance at 312 nm and
269nm with the addition of ClOꢀ; (C) Changes in the emission spectra of QC1 (10
μM) upon ClOꢀ addition in HEPES (pH = 7.4, contained 1% DMSO); (D) ratio of
fluorescent intensity at 381 nm and 482 nm with the addition of ClOꢀ.
1
Fig. 5. H NMR titration of QC1 with 0 eq. (A), 1 eq. NaClO (B) and 100 eq.
H2O2(C).
Fig. 6. Various ROS species selectivity profiles of QC1 (10 µM) in the presence of
various ROS species in HEPES (pH = 7.2, contained 1% DMSO).
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Scheme-1
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Scheme-2
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Figure-1
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Figure-2
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Figure-3
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Figure-4
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Figure-5
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Figure-6
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1. A phenothiazine probe (QC1) with double dioxaborolane moieties was designed
and synthesized.
2. A stability phenothiazine was chosen as the report unit to avoid oxidant bleaching
process.
3. The fluorescent sensor (QC1) was designed for detecting ClO－.
4. A new reaction and ratiometric signals for sensing HClO beyond other ROS.
5. QC1 showed lower detection limited in absorption and fluorescent titration
process.