Quinoline-based fluorescent probe for ratiometric detection of hydrogen peroxide in aqueous solution

Article abstract of DOI:10.1016/j.dyepig.2012.05.013

A novel quinoline-based fluorescent probe for detecting H₂O ₂ is described. In aqueous solution, the probe exhibits fluorescence emission at 542 nm originating from the monocationic species. The reaction between the probe and H₂O₂ causes quenching of the emission at 542 nm and simultaneously yields a significant hypsochromic shift of the emission maximum to 480 nm due to the H₂O₂-triggered boronate cleavage process. Thus, a single-excitation, dual-emission ratiometric measurement with a large blue shift in emission (Δλ = 62 nm) and remarkable changes in the ratio (F_{480 nm}/F_{542 nm}) of the emission intensity (R/R₀ up to 8.3-fold) can be established. Moreover, the probe can also afford high selectivity for detecting H ₂O₂ over other biological reactive oxygen species.

Full text of DOI:10.1016/j.dyepig.2012.05.013

Dyes and Pigments 95 (2012) 373e376
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Quinoline-based fluorescent probe for ratiometric detection of hydrogen peroxide
in aqueous solution
,
b
c
b
b,
**
Yuan-Yu Qian a , Lin Xue , De-Xin Hu , Guo-Ping Li , Hua Jiang
*
a Emergency Department of Chinese PLA General Hospital, Beijing 100853, PR China
b Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China
c Chemicals, Minerals & Metallic Materials Inspection Center, Tianjin Entry-Exit Inspection & Quarantine Bureau, Tianjin 300456, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel quinoline-based fluorescent probe for detecting H2O2 is described. In aqueous solution, the probe
exhibits fluorescence emission at 542 nm originating from the monocationic species. The reaction
between the probe and H2O2 causes quenching of the emission at 542 nm and simultaneously yields
a significant hypsochromic shift of the emission maximum to 480 nm due to the H2O2-triggered boronate
cleavage process. Thus, a single-excitation, dual-emission ratiometric measurement with a large blue
shift in emission (Dl ¼ 62 nm) and remarkable changes in the ratio (F480 nm/F542 nm) of the emission
intensity (R/R0 up to 8.3-fold) can be established. Moreover, the probe can also afford high selectivity for
detecting H2O2 over other biological reactive oxygen species.
Received 28 February 2012
Received in revised form
12 May 2012
Accepted 14 May 2012
Available online 23 May 2012
Keywords:
Fluorescent probe
Hydrogen peroxide
Quinoline
Ó 2012 Elsevier Ltd. All rights reserved.
Boronate cleavage
Protonation
Ratiometric
1. Introduction
measurements, because the intracellular fluorescence may suffer
from many sources of external interference. As an ideal solution,
The development of fluorescence imaging technology visual-
izing intracellular hydrogen peroxide (H2O2) has been attracting
more and more attention, because H2O2, a by-product of oxygen
metabolism, can accumulate in cells leading to oxidative stress and
subsequent serious health problems [1]. Recent evidence has
demonstrated that H2O2 is also a messenger in normal cellular
processes [2]. Therefore, design of highly sensitive and selective
H2O2 fluorescent probes will provide useful tools for intracellular
H2O2 detection and further H2O2-biological studies. Although
several fluorescent probes, such as 20,70-dichlorofluorescin [3], have
already been used for H2O2 imaging, the chemoselectivity from
other ROS/RNS (reactive oxygen or nitrogen species) was not
satisfactory. In the few past years, owing to the intelligent design
mechanisms and methods, including benzenesulfonyl ester [4],
benzil [5], or boronate ester chemistry [6], fluorescent probes with
improved sensitivity and selectivity have been constructed.
Nevertheless, most of them are fluorescence intensity-based
probes, and cannot provide sufficient accuracy for quantitative
ratiometric probes can provide self-calibration via two selected
emission wavelengths [7]. They are good candidates for accurate
and quantitative analysis. However, few ratiometric probes for
H2O2 have been achieved [8]; it is rather desirable to design
ratiometric H2O2-selective probes.
To this end, we now report the synthesis and photophysical
properties of a ratiometric probe (DQHP) for H2O2. As shown in
Scheme 1, the boronate-based benzyl cleavable group was conju-
gated to the 4-position of 6-dimethylamino-2-methyl quinolinone
(1). We anticipated that the probe can be protonated in neutral
aqueous solution and form a charge delocalized state due to the
resonance charge transfer from the oxygen atom to quinolinic
nitrogen atom (Scheme 2) [9]. If H2O2 triggers the boronate
cleavage to form compound 1 [6,8], the resonance will be shut
down and a distinct emission maxima shift will also be observed.
2. Experiment procedure
2.1. General
* Corresponding author.
All chemicals were purchased from Alfa Aesar or SigmaeAldrich
and used as received. All solvents were purified and dried by
** Corresponding author. Tel./fax: þ86 10 82612075.
E-mail addresses: qyy301@sina.com (Y.-Y. Qian), hjiang@iccas.ac.cn (H. Jiang).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2012.05.013

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Y.-Y. Qian et al. / Dyes and Pigments 95 (2012) 373e376
recrystallized from methanol to give of the product as yellow
crystals (0.44 g, 73%). Mp: >300 ꢀC. TLC: Rf ¼ 0.30 (silica gel,
dichloromethane/0e2% methanol). FTIR (KBr, cmꢁ1): 3242, 3068,
2891, 2807, 2781, 1609, 1584, 1546, 1495, 1380, 1306, 1252, 1218,
1180, 1086, 964, 868, 823, 697, 617, 563, 498. 1H NMR (400 MHz,
DMSO-d6, ppm):
d
11.3 (s, 1H), 7.39 (d, J ¼ 9 Hz, 1H), 7.24e7.18 (m,
2H), 5.78 (s, 1H), 2.93 (s, 6H), 2.29 (s, 3H). 13C NMR (100 MHz,
MeOD, ppm):
d 179.7, 150.6, 149.4, 133.9, 126.4, 121.6, 119.9, 108.2,
105.2, 41.1, 19.7. (ESI): m/z Calcd for [M þ Hþ] C12H15N2O: m/z
203.12. Found: m/z 203.1.
Synthesis of DQHP. A mixture of compound 1 (202 mg, 1 mmol),
4-(bromomethyl)benzeneboronic acid pinacol ester (356 mg,
1.2 mmol), and Cs2CO3 (490 mg, 1.5 mmol) in N,N-
dimethylformamide (DMF; 15 mL) was heated at 80 ꢀC for 8 h.
After cooling, H2O (30 mL) was added to the mixture and extracted
with CH2Cl2 (10 mL ꢂ 3). The organic solutions were combined,
washed with water and brine, and dried with Na2SO4. The solvents
were evaporated to give the crude product, which was purified by
flash chromatography (silica gel, dichloromethane/0e2% meth-
anol) to give the desired products as a pale solid (0.24 g, 57%). TLC:
Rf ¼ 0.52 (silica, 25:2 CH2Cl2/MeOH). Mp: 149e150 ꢀC. FTIR (KBr,
cmꢁ1): 3423, 3031, 2979, 2925, 2803, 1620, 1594, 1564, 1511, 1451,
1397, 1357, 1324, 1271, 1246, 1219, 1191, 1143, 1093, 1014, 965, 886,
857, 826, 731, 707, 660, 604, 523, 438. 1H NMR (400 MHz, CDCl3,
Scheme 1. Synthesis and action of probe DQHP.
standard methods prior to use. Pure water (18.2
U) was used to
prepare all aqueous solutions. 1H NMR and 13C NMR spectra were
recorded on a Bruker AVANCE-400 400 MHz spectrometer. All
chemical shifts are reported in the standard notation of parts per
million using residual solvent protons as internal standard. IR data
were recorded on a Bruker Tensor-27 spectrometer. Mass spectra
(ESI) were obtained on LC-MS 2010 mass spectrometer. All titra-
tions were carried out in HEPES buffer (10 mM HEPES, 100 mM
NaCl, pH ¼ 7.0, 25 ꢀC). UVeVis absorption spectra were obtained
using Shimadzu UV-2550 spectrometer. Fluorescence spectra were
obtained using HITACHI F-4600 spectrometer. Various reactive
oxygen species were obtained according to the typical procedures
reported by Chang et al. [8,10].
ppm):
(d, J ¼ 2.84 Hz, 1H), 6.58 (s, 1H), 5.30 (s, 2H), 3.04 (s, 6H), 2.61 (s,
3H), 1.36 (s, 12H) 13C NMR (100 MHz, CDCl3, ppm):
160.0, 155.6,
d
7.87e7.83 (m, 3H), 7.52 (d, J ¼ 7.8 Hz, 2H), 7.34 (m, 1H), 7.23
d
148.0,142.8, 139.5, 135.2,128.9, 126.5, 120.9,119.4,101.8,100.2, 84.0,
69.9, 41.0, 25.7, 25.0. (ESI): m/z Calcd for [M þ Hþ] C25H32BN2O3: m/
z 419.25. Found: m/z 419.4.
2.2. Synthesis
3. Results and discussions
Synthesis of Compound 3. A solution of compound 2 (1.0 g,
6 mmol), ethyl acetoacetate (1.6 g, 12 mmol), and AcOH (0.5 mL) in
benzene (30 mL) was heated under reflux for 8 h, removing water
with a DeaneStark apparatus. This mixture was cooled to room
temperature and concentrated in vacuum. The residue was puri-
fied by flash chromatography (silica gel, petroleum ether/0e10%
ethyl acetate) to give the desired products as colourless oil
(63%). TLC: Rf ¼ 0.53 (silica, 5:1 petroleum ether/ethyl acetate).
Mp: 69e70 ꢀC. FTIR (KBr, cmꢁ1): 3271, 3032, 2991, 2915, 2803,
2305, 1652, 1613, 1521, 1488, 1437, 1387, 1351, 1263, 1159, 1058,
1020, 976, 947, 837, 784, 733, 688, 602, 555, 522, 492. 1H NMR
As shown in Scheme 3, compound 1 was synthesized according
to a typical procedure [9d]. The enamine 3 was obtained by
condensation of the 4-dimethylaminoaniline with ethyl acetoace-
tate. Compound 3 was cyclized by heating in diphenyl ether under
nitrogen to give quinolone 1 in 73% yield. O-alkylation using 4-
(bromomethyl)benzeneboronic acid pinacol ester under basic
conditions gave DQHP in moderate yield.
From the fluorimetric titration, the solubility of DQHP is about
15
mM in physiological buffer (10 mM HEPES, 100 mM NaCl,
pH ¼ 7.0), which is sufficient for spectral measurements or cellular
(400 MHz, CDCl3, ppm):
d
10.08 (s, 1H), 6.99 (d, J ¼ 8.44 Hz, 2H),
staining (Fig. S1).
6.68 (br, 2H), 4.61 (s, 1H), 4.17 (q, J ¼ 7.00 Hz, 2H), 2.94 (s, 6H), 1.87
(s, 3H), 1.30 (t, J ¼ 7.12 Hz, 3H). 13C NMR (100 MHz, CDCl3, ppm):
To evaluate whether our probe can be protonated under neutral
conditions, we measured the fluorescence spectra of DQHP at
various pH (pH below 5 or above 10 were not measured to avoid
potential hydrolysis of pinacol ester, Fig. 1). The pKa value of
7.47 ꢃ 0.05 clearly indicates that probe DQHP has the similar
protonation behaviour to the methylpropane-substituted analogues
(Scheme 2) [9d]. Under neutral conditions, DQHP exhibited a clear
d
170.7, 160.9, 148.8, 128.6, 126.9, 112.8, 84.0, 58.7, 40.9, 20.3, 14.8.
(ESI): m/z Calcd for [M þ Hþ] C14H21N2O2: m/z 249.16. Found: m/z
249.2. Calcd for [M þ Naþ] C14H20N2NaO2: m/z 271.44 Found:
271.2.
Synthesis of Compound 1. A mixture of compound 3 (0.74 g,
3 mmol) and diphenyl ether (10 mL) was heated with stirring at
250 ꢀC for 60 min under nitrogen. This mixture was cooled, and the
product began to precipitate in the reaction medium. After cooling,
the mixture was diluted with petroleum ether (100 mL) to
complete the precipitation. The solid was collected by filtration and
absorption band around 400 nm (
3
¼ 2.48 ꢂ 104 Mꢁ1 cmꢁ1
measured at 405 nm, Fig. 2). Upon addition of 1 mM H2O2 for 90 min,
this band gradually decreased with a concomitant increase around
346 nm. Meanwhile, the absorption spectrum of compound 1 also
Scheme 3. Synthetic procedure for compound 1. (a) Ethyl acetoacetate, AcOH,
benzene, reflux. (b) Diphenyl ether, 250 ꢀC.
Scheme 2. The protonation and resonance of the 4-isobutoxy substituted quinoline.

Y.-Y. Qian et al. / Dyes and Pigments 95 (2012) 373e376
375
Fig. 3. Fluorescence emission spectra (lex ¼ 405 nm) of DQHP (5
H2O2 (100 M) for 5, 15, 30, 60, 90, 120 min in HEPES buffer.
mM) upon addition of
Fig. 1. Fluorescence emission intensity (lem ¼ 540 nm) of DQHP (5
mM) over the pH
m
range 5e9, the solid lines represent the non-linear least-squares fits to the experi-
mental data.
480 and 542 nm were found to be 0.34 and 2.82 in the absence and
presence of H2O2, respectively, (R/R0 ¼ 8.3). These data imply DQHP
is a good candidate for ratiometric and turn-on detection of H2O2 in
aqueous solution because of the large emission shift (62 nm) and
ratio changes.
featured an aborption maximum at 346 nm, suggesting that the
H2O2-triggered boronate cleavage process occurred [6,8]
Next we evaluated fluorescence emission property of DQHP
(5
When excited at 405 nm, DQHP displayed a weak fluorescence
emission maximum at 542 nm (
¼ 0.07, Fig. 3), which is very
mM) in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH ¼ 7.0).
In addition, the chemoselectivity profile of DQHP for H2O2 was
investigated by fluorimetric titration of different ROS (Fig. 5). As
expected, only H2O2 significantly increases the emission ratio
(F480/F542 nm). But in the presence of other ROS, including tert-
butylhydroperoxide (TBHP), hypochlorite (ClOꢁ), hydroxyl radical
(ꢄOH), tert-butoxy radical (ꢄOtBu), nitric oxide (NO), and super-
oxide (Oꢁ2 ), no evident fluorescence emission ratio changes were
observed. Thus, DQHP also has excellent chemoselectivity for
H2O2 over other competing ROS. Furthermore, the emission ratio
of both DQHP and compound 1 displayed pH insensitivity over
biological window of 5.5e8 (Fig. S4). That implies subtle pH
changes do not interfere with the ratiometric detection of H2O2
under physiological conditions. In addition, it has been shown
F
similar to our previous observations with quinoline analogues [9d].
It clearly demonstrates the existence of the resonance charge
transfer in the monoprotonated form of DQHP. When treating
DQHP with 1 mM H2O2, the fluorescence showed remarkable
enhancements (17.5-fold, measured at 480 nm) and the emission
maximum gradually blue-shifted to 480 nm. Through measuring
reaction product of compound 1, we found 1 is in the neutral form
in HEPES buffer (Fig. S3). Its emission maximum and quantum yield
were characterized to be 480 nm and 0.58, respectively. Under
pseudo-first-order kinetics (1.25
mM DQHP, 1 mM H2O2), the
observed rate constant for
H2O2 deprotection is
kobs ¼ 1.40 ꢂ 10ꢁ3 sꢁ1 (Fig. 4). Therefore, it is clear that DQHP can
respond to H2O2 with significant ratiometric and turn-on fluores-
cence signal output, and the ratios of the fluorescence intensities at
Fig. 4. Time-course kinetic measurement of fluorescence response of DQHP to H2O2.
Data were collected under pseudo first-order conditions (1.25 mM DQHP, 1 mM H2O2)
in HEPES buffer. Excitation was provided at 405 nm, and the emission intensities at
480 nm were measured over various time points. Data collected from 3 to 90 min was
used to fit the rate constant (red line). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Fig. 2. UV/Vis absorption spectra of DQHP (10 mM), 1 (10 mM), and DQHP (10 mM) with
addition of H2O2 (1 mM) for 90 min in HEPES buffer (10 mM HEPES, 100 mM NaCl,
pH ¼ 7.0).

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Y.-Y. Qian et al. / Dyes and Pigments 95 (2012) 373e376
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4. Summary
In summary, we have synthesized and characterized a novel
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