Naked-eye visible and fluorometric dual-signaling chemodosimeter for hypochlorous acid based on water-soluble p-methoxyphenol derivative

Article abstract of DOI:10.1039/c1ob05550j

The oxidation of a simple p-methoxyphenol derivative by HClO induces an intramolecular charge transfer from the end phenyl units to the middle benzoquinone, which leads to colorimetric and fluorescent changes. This detection can be run in aqueous solution

Full text of DOI:10.1039/c1ob05550j

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PAPER
Naked-eye visible and fluorometric dual-signaling chemodosimeter for
hypochlorous acid based on water-soluble p-methoxyphenol derivative†
Wenjun Zhang, Chi Guo, Lihan Liu, Jingui Qin and Chuluo Yang*
Received 7th April 2011, Accepted 13th May 2011
DOI: 10.1039/c1ob05550j
The oxidation of a simple p-methoxyphenol derivative by HClO induces an intramolecular charge
transfer from the end phenyl units to the middle benzoquinone, which leads to colorimetric and
fluorescent changes. This detection can be run in aqueous solution with high selectivity over other
reactive oxygen species.
conjugated system. We anticipate that the oxidation of the p-
Introduction
methoxyphenol unit by HClO would induce an intramolecular
As a kind of reactive oxygen species (ROS), hypochlorous acid
charge transfer (ICT) from the end phenyl units to the middle
(HClO) plays a crucial role in our daily lives and in host defence
benzoquinone, which could in turn lead to colorimetric and
against invading pathogens.1–2 Under physiological conditions
fluorescent changes. In order to be applied in aqueous systems,
(with a pKa of 7.46 at 35 ◦C), approximately half of the HClO
the two end phenyl units are equipped with quaternary ammonium
dissociates to the hypochlorite anion (ClO-), one of the most
parts.
powerful natural oxidants.3–5 In living organisms, hypochlorous
acid is generated by the reaction of hydrogen peroxide with
chloride ions under the catalysis of the heme enzyme myeloper-
Results and discussion
oxidase (MPO), which is synthesized and secreted by activated
phagocytes.6–9 However, because of its high reactivity and non-
specificity,10 excessive hypochlorous acid can lead to damage of
host tissue that is implicated in a wide range of human diseases,
such as kidney disease,11–12 atherosclerosis,13–16 and arthritis.17–19
Therefore, sensitive and selective detection of hypochlorous acid
is of important significance.
PMOPP can be easily prepared in two steps. As shown in Scheme
1, the Suzuki coupling reaction of 2,5-dibromo-4-methoxyphenol
(1) and 2-(4-(6-bromohexyloxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (2) gave the precursor PMOPP-Br, which was then
followed by quaternization with trimethylamine to afford PMOPP.
Recently, a few probes for hypochlorous acid have been reported
based on the strong oxidation property of HClO.5,20–26 For example,
Nagano’s group20 and Libby’s group5 reported rhodamine- and
fluorescein-based fluorescent probes for HClO, respectively, but
the fluorescent probes involved complicated synthesis; Ma’s group
also developed two fluorescent probes for HClO,21,22 which work
in an organic co-solvent system. Moreover, to the best of our
knowledge, none of these probes can respond to HClO with colori-
metric and fluorescent changes simultaneously. A dual-signaling
chemodosimeter can combine the sensitivity of fluorescence with
the convenience and esthetic appeal of a colorimetric assay.27,28
It is known that p-methoxyphenol can be selectively oxidized to
benzoquinone by hypochlorous acid.20 Based on this reaction, we
designed a simple p-methoxyphenol derivative, namely PMOPP,
by putting the p-methoxyphenol as the middle unit of a terphenyl
Department of Chemistry, Hubei Key Lab on Organic and Polymeric
Optoelectronic Materials, Wuhan University, Wuhan 430072, P. R. China.
E-mail: clyang@whu.edu.cn
† Electronic supplementary information (ESI) available: ESI-MS spectra
of PMOPP and PQP, and NMR spectra of the key compounds. See DOI:
10.1039/c1ob05550j
Scheme 1 Synthesis of PMOPP.
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The final product shows good water-solublity owing to the charged
trimethylammonium groups.
The value is comparable to the reported probes for hypochlorous
acid.21,22
In order to mimic the environment in living organisms, the
optical responses of PMOPP toward HClO were examined in
10 mM phosphate buffered saline (PBS) solution (pH 7.4). The
absorption spectra of PMOPP upon addition of hypochlorite are
shown in Fig. 1. After addition of hypochlorite, the absorption
at 314 nm decreases; synchronously, a new band at 393 nm
appears. A clear isosbestic point at 341 nm indicates that the
reaction of hypochlorite with PMOPP is a clean process. The
broad absorption band centred at ca. 393 nm could be attributed
to an ICT transition from the end phenyl units to the middle
benzoquinone. It is noteworthy that the process is accompanied
by a color change from colorless to yellow (the inset of Fig.
1), which makes PMOPP capable for the naked-eye detection of
HClO. Compared to fluorometric sensors, naked-eye detect in a
straightforward and inexpensive manner, offers qualitative and
quantitative information without using expensive equipment.29
We presumed that the color change and fluorescence quenching
could be attributed to the oxidation of PMOPP to its benzo-
quinone derivative PQP (Scheme 2). To further confirm the pro-
cess, we carried out 1H NMR titrations. Superimposed 1H NMR
spectra of PMOPP after addition of 0–3 equiv. of hypochlorite are
shown in Fig. 3. Upon the addition of hypochlorite, the signals
assigned to OH at d ª 6.7 ppm and OCH3 at d ª 3.1 ppm are
gradually weakened, instead a new resonance at d ª 3.2 ppm
appears, which may correspond to ClCH3. Accordingly, the signals
assigned to the phenyl group also show changes in chemical shift.
The 1H NMR titrations confirm that the p-methoxyphenol is
actually oxidized to benzoquinone by hypochlorite. More direct
evidence comes from the ESI-MS result, in which the oxidation
product PQP is observed (see the ESI†).
Fig. 1 UV/vis spectra of PMOPP (final concentration: 50 mM) in 0.01
M PBS solution (pH 7.4) after the addition of ClO- (ranging from 0 to
100 mM). Inset: the color change of the solution.
Scheme 2 Reaction of PMOPP with HClO.
The fluorescent response of PMOPP to hypochlorite was sub-
sequently investigated. As indicated in Fig. 2, the fluorescence of
PMOPP decreases dramatically upon increasing the hypochlorite
concentration. After treatment with 24 mM hypochlorite, the fluo-
rescence of the solution is quenched completely. Correspondingly,
the fluorescent quantum yield decreased from 72.6% to 0.8%.
In the range of 1–10 mM (the inset of Fig. 2), the fluorescence
intensity is directly proportional to the ClO- concentration. The
detectionlimit for HClOis determinedtobe0.8 mM (S/N = 3) from
the fluorescence titration profile of PMOPP (5 mM) with HClO.
Fig. 3 1H NMR spectra of PMOPP (12 mM) in D2O after addition of
0–3 equiv. of ClO-.
Finally, we investigated the reactivity of PMOPP toward various
∑
∑
ROS, including HClO, H2O2, O2 -, NO, OH, ONOO-. The
photograph in Fig. 4 shows the solution color after addition
of various ROS in PBS solution. It is clear that the solution
remains colorless upon addition of various ROS, except HClO.
Similarly, PMOPP exhibits a selective fluorometric detection for
hypochlorite (Fig. 5). It is obvious that fluorescence quenching
occurs only upon addition of HClO.
Fig. 2 Fluorescence spectra of PMOPP (final concentration: 5 mM) in
0.01 M PBS solution (pH 7.4) after the addition of ClO- (ranging from 0
to 24 mM). The excitation wavelength is 320 nm.
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mass spectrophotometer. LC-mass spectra were measured on a
Waters ZQ-Mass ESI. UV-vis absorption measurements were
performed on a Shimadzu 2550 spectrophotometer. Fluorescence
measurements were performed on a Hitachi F-4500 fluorescence
spectrophotometer.
All chemicals were purchased from commercial sources and
were used without further purification unless otherwise noted. 2,5-
Dibromobenzene-1,4-diol and 2,5-dibromo-4-methoxyphenol (1)
were synthesized following literature procedures.30
Fig. 4 Color changes of PBS solution (pH = 7.4, 0.01 M) of PMOPP
(50 mM) in the presence of the following ROS: from left to right: none,
Synthesis of 2-(4-(6-bromohexyloxy)phenyl)-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (2)
∑
HClO, H2O2, O2 -, NO, ∑OH and ONOO-. HClO: 50 mM Ca(ClO)2. H2O2:
∑
20 mM H2O2. O2 -: 20 mM KO2 was added and the mixture was stirred
at 37 ◦C for 30 min. NO: 20 mM sodium nitroferricyanide(III) was added
A
flask charged with 1-bromo-4-(6-bromohexyloxy)benzene
∑
and the mixture was stirred at 25 ◦C for 30 min. OH: 10 mM ferrous
(1.01 g, 3.0 mmol), bis(pinacolato)diborane (1.26 g, 3.3 mmol),
potassium acetate (0.88 g, 9.0 mmol), Pd(dppf)Cl2 (0.73 g,
0.1 mmol), and 20 mL of anhydrous dioxane was degassed for
15 min. The mixture was stirred at 80 ◦C for 12 h. After being
cooled to room temperature, the mixture was poured into ice water,
and then extracted with ethyl acetate. The combined organic layers
were dried over anhydrous Na2SO4. After the solvent was removed
by rotary evaporation, the residue was purified by silica gel column
chromatography (15 : 1 petroleum ether/ethyl acetate) to afford a
perchlorate and 20 mM H2O2. ONOO-: 20 mM sodium peroxynitrite.
1
white solid. Yield: 0.90 g, 78%. H NMR (300 MHz, CDCl3): d
7.72 (d, 2H, J = 8.4 Hz), 6.87 (d, 2H, J = 8.4 Hz), 3.97 (t, 2H, J =
6.3 Hz), 3.41 (t, 2H, J = 6.6 Hz), 1.88 (m, 2H), 1.78 (m, 2H), 1.49
(m, 4H), 1.32 (s, 12H). 13C NMR (75 MHz, CDCl3): d 161.8, 136.7,
114.0, 83.7, 67.7, 34.0, 32.9, 29.2, 28.1, 25.5, 25.1, 25.0. EI-MS:
m/z 382 (M+). Anal. Calcd for C18H28BBrO3: C, 56.43; H, 7.37.
Found: C, 56.36; H, 7.63.
Fig. 5 Fluorescence intensity changes of PMOPP (final concentration:
5 mM) in PBS solution (pH = 7.4, 0.01 M) with various ROS generating
systems. The fluorescence intensity (388 nm) was determined with exci-
∑
-
tation at 320 nm. HClO: 10 mM Ca(ClO)2. H2O2: 100 mM H2O2. O2
:
100 mM KO2 was added and the mixture was stirred at 37 ◦C for 30 min.
NO: 100 mM sodium nitroferricyanide (III) was added and the mixture was
stirred at 25 ◦C for 30 min. ∑OH: 50 mM ferrous perchlorate and 100 mM
H2O2. ONOO-: 100 mM sodium peroxynitrite.
4,4¢¢-Bis(6-bromohexyl)oxy-5¢-methoxy-1,1¢:4¢,1¢¢-terphenyl-2¢-ol
(PMOPP-Br)
A flask charged with 1 (0.14 g, 0.5 mmol), 2 (0.45 g, 1.2 mmol),
Pd(PPh3)4 (0.02 g, 0.02 mmol), and sodium carbonate (1.06 g,
10.0 mmol), in 20 mL THF/H2O (3 : 1) was degassed for 15 min.
The mixture was refluxed for 24 h. After being cooled down to
room temperature, the mixture was neutralized by acetic acid,
and then extracted with CHCl3. The organic layer was washed
with water and brine and dried with anhydrous Na2SO4. After
the solvent was removed by rotary evaporation, the residue was
purified by silica gel column chromatography (5 : 1 petroleum
ether/ethyl acetate) to give a pale yellow solid. Yield: 0.25 g, 79%.
1H NMR (300 MHz, CDCl3): d 7.51 (d, 2H, J = 8.1 Hz), 7.45
(d, 2H, J = 8.7 Hz), 7.03 (d, 2H, J = 8.1 Hz), 6.96 (s, 1H), 6.94
(d, 2H, J = 7.5 Hz), 6.83 (s, 1H), 4.98 (s, 1H), 4.05–3.99 (m, 4H),
3.77 (s, 3H), 3.47–3.42 (m, 4H), 1.91–1.83 (m, 8H), 1.53 (br, 8H).
13C NMR (75 MHz, CDCl3): d 158.8, 158.3, 150.6, 146.4, 130.7,
130.5, 130.2, 129.2, 126.6, 117.8, 115.2, 114.1, 113.6, 67.9, 67.7,
56.4, 33.9, 32.7, 29.2, 29.1, 28.0, 25.3. EI-MS: m/z 632 (M+) Anal.
Calcd for C31H38Br2O4: C, 58.69; H, 6.04. Found: C, 58.75; H,
6.20.
Conclusions
In conclusion, we have designed and synthesized a simple and
water-soluble p-methoxyphenol derivative that shows colorimetric
and fluorometric dual-signaling responses for hypochlorite acid
in the aqueous system. The detection process gives rise to
a color change that is clearly visible to the naked eye. This
makes the colorimetric chemodosimeters helpful for “in-the-
field” measurement. Furthermore, both the colorimetric and
fluorometric detection exhibit high selectivity over the usual
reactive oxygen species. The advantages of dual-signaling, color-
change and running environment in physiological conditions make
the new probe promising for the detection of hypochloric acid,
especially in biological system, and this work is under way.
Experimental
General information
6,6¢-[(2¢-Hydroxy-5¢-methoxy-1,1¢:4¢,1¢¢-terphenyl-4,4¢¢-diyl)bis-
(oxy)]bis(N,N,N-trimethylhexan-1-aminium) dibromide
(PMOPP)
1H NMR and 13C NMR spectra were obtained in the indi-
cated solvent with tetramethylsilane as an internal standard on
MECUYRVX300 spectrometers. Elemental analysis of carbon,
hydrogen, and nitrogen were performed on a Carlorerba-1106
microanalyzer. Mass spectra were measured on a ZAB 3F-HF
To a solution of PMOPP-Br (0.15 g, 0.2 mmol) in 10 mL THF
was added N(CH3)3 aqueous solution (~2.4 mL). After stirring for
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0.5 h, 1 mL H2O was added to the mixture. Then excess N(CH3)3
aqueous (~2.4 mL) was added and the mixture was stirred for 48
h. After the solvent was removed by rotary evaporation, the crude
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1
product was recrystallized from ethanol. Yield: 0.10 g, 58%. H
NMR (300 MHz, D2O): 7.01 (br, 4H), 6.73 (s, 1H), 6.49–6.41 (br,
6H), 3.42–3.38 (br, 4H), 3.05 (s, 3H), 2.82 (br, 4H), 2.72 (s, 9H),
2.70 (s, 9H), 1.26 (br, 8H), 0.90 (br, 8H). 13C NMR (75 MHz,
D2O): d 157.8, 150.1, 147.5, 130.7, 129.4, 127.0, 118.0, 114.5, 68.3,
66.6, 56.0, 53.0, 28.8, 25.7, 25.1, 22.6. ESI-MS: m/z 671 (M+ -
Br). Anal. Calcd for C37H56Br2N2O4: C, 59.04; H, 7.50. Found: C,
59.26; H, 7.51.
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A 5 mM stock solution of PMOPP was prepared in PBS buffer.
Testing solutions were prepared by adding 2 or 20 mL of the probe
stock solution to 2 mL PBS buffer in a test tube and then adding
an appropriate aliquot of each ROS stock solution. All of the
absorption and fluorescence sensing of ROS was run immediately
after the ROS were added.
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We are grateful for financial support from the National Natural
Science Foundation of China (No.20874077), the Fundamental
Research Funds for the Central Universities and the program for
Changjiang Scholars and Innovative Research Team in University
of the Ministry of Education.
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