A ratiometric fluorescent probe for hypochlorite based on a deoximation reaction

Article abstract of DOI:10.1002/chem.200802054

In this work, we have successfully provided a novel strategy for the rational design and synthesis of a ratiometric fluorescent probe for hypochlorite. The strategy is based on the deoximation reaction, which has not yet been used in the fluorescent hypoc

Full text of DOI:10.1002/chem.200802054

FULL PAPER
DOI: 10.1002/chem.200802054
A Ratiometric Fluorescent Probe for Hypochlorite Based on a Deoximation
Reaction
Weiying Lin,* Lingliang Long, Bingbing Chen, and Wen Tan[a]
Abstract: In this work, we have suc-
cessfully provided a novel strategy for
the rational design and synthesis of a
ratiometric fluorescent probe for hypo-
chlorite. The strategy is based on the
deoximation reaction, which has not
yet been used in the fluorescent hypo-
chlorite probe design. Interestingly, the
probe showed a ratiometric fluorescent
response to hypochlorite with the emis-
sion intensities ratio (I509/I439) increas-
ing from 0.28 to 2.74. Furthermore, the
probe displayed high selectivity for hy-
pochlorite over other species due to
the distinct deoximation conditions.
The probe developed herein represents
the first ratiometric fluorescent probe
for hypochlorite.
Keywords: charge transfer · fluo-
rescence spectroscopy · fluorescent
probes · hypochlorite · ratiometric
Introduction
ronment and probe distribution are problematic for quanti-
tative measurements.[10] By contrast, ratiometric fluorescent
probes allow the measurement of emission intensities at two
different wavelengths, which should provide a built-in cor-
rection for environmental effects and can also increase the
dynamic range of fluorescence measurement.[11]
To our best knowledge, no ratiometric fluorescent probes
for hypochlorite have been reported thus far. We therefore
wanted to develop a fluorescent probe which displayed a ra-
tiometric response to hypochlorite using intramolecular
charge transfer (ICT) as a signaling mechanism.[12] An ICT
system contains an electron donor and an electron acceptor.
As the ICT efficiency can be regulated by the variation in
electron donor/acceptor strength, we decided to employ this
key feature of ICT in our ratiometric fluorescent probe
design.
Recently, we have demonstrated that a specific reaction
promoted by an analyte could be employed to devise probes
with high selectivity for the analyte.[13] Therefore, for a
design of a highly selective probe for hypochlorite, we
turned our attention to select a specific reaction promoted
by hypochlorite. In organic synthesis, aldehyde groups can
be protected as oximes,[14] which are rapidly deprotected by
hypochlorite under mild conditions (i.e., at ambient temper-
ature) (Scheme 1).[15]
Hypochlorite (ClOÀ) is widely employed in our daily life,
such as household bleach, disinfection of drinking water,
cooling-water treatment, and cyanide treatment.[1] Typically,
it is used in the concentration range of 10À5–10À2 m.[1] How-
ever, concentrated hypochlorite solutions are a potential
health hazard to human and animals.[2] On the other hand,
hypochlorite is one of the important reactive oxygen species
(ROS) in living organisms,[3] and it plays a critical role in
the immune system. The endogenous hypochlorite is pro-
duced from peroxidation of chloride ions catalyzed by the
enzyme myeloperoxidase (MPO).[4] However, the abnormal
production of hypochlorite owing to variations in MPO
levels can lead to a variety of diseases including cardiovas-
cular diseases,[5] neuron degeneration,[6] arthritis,[7] and
cancer.[8] Thus, it is of great interest to detect hypochlorite
by sensitive and selective fluorescent probes. A limited
number of selective hypochlorite/hypochlorous acid fluores-
cent probes has been constructed only very recently,[9] all of
which respond to hypochlorite/hypochlorous acid with
changes only in fluorescent intensity. A major limitation of
intensity-based probes is that variations in the sample envi-
[a] Prof. W. Lin, L. Long, B. Chen, W. Tan
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University, Changsha, Hunan 410082 (PR China)
Fax : (+86)731-882-1464
E-mail: Weiyinglin@hnu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200802054.
Scheme 1. Protection of aldehydes as oximes and deprotection by ClOÀ.
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Although the mechanism of oxidative deoximation with
hypochlorite has not been described in the literature thus
far, we envisioned that this deprotection reaction could be
exploited as an interesting platform for ratiometric fluores-
cent ClOÀ probes. Notably, the deoximation reaction has
not been previously employed in fluorescent ClOÀ probe
design. Based on this platform, compound 1 was designed as
the first ratiometric fluorescent probe for hypochlorite
(Scheme 2). Compound 1 is composed of a phenanthroimi-
dazole dye[16] and an oxime protective group. We hypothe-
sized that the oxime protective group could be removed by
ClOÀ to give the aldehyde group. Thus, oxime 1 is trans-
formed into aldehyde 2. As phenanthroimidazole is an elec-
tron-rich fluorophore[16] and the electron-withdrawing ability
of the aldehyde group is stronger than that of the oxime
group, it was expected that the emission of compound 2
should have a redshift relative to that of compound 1 due to
stronger ICT. Therefore, “protected” 1 and “deprotected” 2
may be suitable for the development of ratiometric fluores-
cent ClOÀ probes based on regulation of the electron-with-
drawing ability of the electron acceptor in the ICT system.
Absorption and emission spectra properties: The optical
properties of probe 1 and compounds 2, 3 were studied in
0.1m potassium phosphate buffer, pH 9.0/DMF (v/v, 1:4) at
room temperature. The absorption spectra of compounds 1
and 2 exhibited a redshift relative to that of the reference 3
(Figure 1). This bathochromic shift is apparently attributed
to ICT between the phenanthroimidazole fluorophore and
&
*
Figure 1. Normalized absorption spectra of compounds 1 ( ), 2 ( ), and
~
3 ( ) in 0.1m potassium phosphate buffer, pH 9.0/DMF (v/v, 1:4).
the oxime or aldehyde group.
Additionally, the absorption of
compound 2 showed a longer
redshift than that of compound
1. This is consistent with the
fact that the aldehyde group
has stronger electron-withdraw-
ing ability than the oxime
group. In good agreement with
the bathochromic shift in the
absorption spectra, the fluores-
cence emission spectra of com-
pounds 1 (Ff =0.782, in DMF,
quinine sulfate as standard,[18]
see Supporting Information)
and 2 (Ff =0.567, in DMF) dis-
played a different extent of red-
shift when compared with that
of compound 3 (Ff =0.493, in
Scheme 2. Synthetic route to compound 1 and the structure of control compound 3. a) CH3COONH4,
CH3COOH, 1008C, 30 min; b) hydroxylamine hydrochloride, Et3N, ethanol, 608C, 3 h; c) NaOCl, potassium
phosphate buffer/DMF, ambient temperature.
DMF) (Figure 2). Compounds 1
and 2 have maximal emission at
439 and 509 nm, respectively.
The observation that the emis-
Results and Discussion
sion spectrum of compound 2 has a 70 nm redshift in com-
parison with that of compound 1 indicates that “protected”
oxime 1 is promising ratiometric fluorescent probe for ClOÀ
provided that 1 could be deprotected by ClOÀ to afford the
“deprotected” aldehyde 2.
Synthesis: Probe 1 was prepared by a standard procedure
(Scheme 2). Condensation of 9,10-phenanthrenequinone
with terephthalaldehyde and NH4OAc in AcOH by the
Steck and Day procedure[17] gave compound 2 in 72.6%
yield. The structure of the reference compound 3 is shown
in Scheme 2. Probe 1 was obtained in excellent yields by re-
acting compound 2 with hydroxylamine hydrochloride in
ethanol.
Sensing response to ClOÀ: To examine whether probe 1
could detect ClOÀ based on the deoximation reaction, probe
1 (10 mm) was treated with ClOÀ at ambient temperature,
and the progress of the reaction was monitored by fluores-
cence spectroscopy. In the absence of ClOÀ, compound 1 ex-
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Chem. Eur. J. 2009, 15, 2305 – 2309

A Ratiometric Fluorescent Probe for Hypochlorite
FULL PAPER
Figure 4. Fluorescence emission spectra of probe 1 (10 mm) in the pres-
ence of different concentrations of hypochlorite anion (0–30 equiv) in
0.1m potassium phosphate buffer, pH 9.0/DMF (v/v, 1:4). Excitation
wavelength was 394 nm.
&
Figure 2. Normalized fluorescent emission spectra of compounds 1 ( ), 2
~
*
(
), and 3 ( ) in 0.1m potassium phosphate buffer, pH 9.0/DMF (v/v,
1:4), excited at 394, 394, and 326 nm, respectively.
hibited no visible variations in the ratios of emission intensi-
ties at 509 and 439 nm (I509/I439 =0.28), suggesting that com-
pound 1 was stable in the assay conditions and not convert-
ed to deprotected 2 (Figure 3). However, upon addition of
ClOÀ at room temperature, a marked increase in the ratio
was observed within seconds, indicative of rapid deprotec-
tion of protected 1 to give deprotected 2, as anticipated.
Furthermore, the rapid conversion of 1 into 2 by ClOÀ sug-
gests that probe 1 can be used to monitor ClOÀ in real time.
to the standard characterization. The 1H NMR (Figure 5),
Mass, absorption, excitation, and emission spectra of the iso-
lated product are identical with those of the standard com-
pound 2, demonstrating that, indeed, probe 1 was deprotect-
ed by ClOÀ ions to give aldehyde 2.
Figure 5. Partial 1H NMR (400 MHz) spectra of a) probe 1, b) the sepa-
rated product of probe 1+ClOÀ, and c) the standard compound 2.
~
Figure 3. Reaction–time profile of probe 1 (10 mm) in the absence (
)
À
&
and presence ( ) of 30 equiv of ClO in 0.1m potassium phosphate
buffer, pH 9.0/DMF (v/v, 1:4). Kinetic studies were performed at room
temperature. The emission ratio changes (I509/I439) were continuously
monitored at time intervals.
Effect of pH: The ratiometric responses of the probe toward
ClOÀ at different pH conditions were investigated. In the
absence of ClOÀ, the probe was stable over a wide range of
pH values from 2.5 to 10.5 (Figure 6). However, the ratio-
metric responses of the probe toward ClOÀ were pH-depen-
dent. Notably, the fluorescent ratio was increased dramati-
cally above pH 7.5. As the pKa of HOCl is 7.6, we reasoned
that probe 1 senses ClOÀ instead of HClO.[9a–b] We decided
to study the photophysical and sensing properties of probe 1
at pH 9.0, as the probe was much more sensitive. However,
the probe could still show a marked ratiometric response to
ClOÀ at pH as low as 7.8 (Figure S1), indicating that probe
1 may also be used in the near physiological pH range.
Unlike the HClO probes which function well under acidic
conditions,[9b–c] the ClOÀ probes work better at basic condi-
tions due to the fact that the pKa of HOCl is 7.6. For exam-
ple, an intensity-based ClOÀ probe was reported[9a] to be
used at pH 12.
When increasing concentrations of ClOÀ ions were intro-
duced, the fluorescent spectra of probe 1 exhibited signifi-
cant changes. The intensity of the emission maximum at
439 nm was gradually decreased with the simultaneous ap-
pearance of a new redshifted emission band centered at
509 nm (Figure 4). Importantly, the ratios of emission inten-
sities at 509 and 439 nm (I509/I439) displayed a large increase
from 0.28 to 2.74. Probe 1 is able to detect low micromolar
concentrations of hypochlorite. As the pathophysiologically
relevant concentrations of HOCl are in the low to medium
micromolar concentrations,[5b] our probe is potentially suita-
ble for medical and biological use. Furthermore, a definite
isoACHTUNGTRENNUNGemission point was observed at 470 nm which may sug-
gest that only one new species was formed during the titra-
tion process. The reaction product was isolated and subject
Chem. Eur. J. 2009, 15, 2305 – 2309
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W. Lin et al.
iently for hypochlorite detection by simple visual detection.
We further examined the ratiometric response of the probe
toward ClOÀ in the presence of other potentially competing
species. Most of other species only displayed minimum inter-
ference (Figure 9). This clearly indicates that probe 1 is
useful to sense ClOÀ in the presence of other related
AHCTUNGTRENNsGUN pecies.
Figure 6. Ratios of the fluorescent emission intensities at 509 and 439 nm
À
~
*
(I509/I439) of probe 1 (10 mm) in the absence ( ) or presence ( ) of ClO
(30 equiv) at various pH values. Excitation wavelength was 394 nm.
Selectivity studies: Probe 1 was treated with a wide variety
of cations, anions, and oxidants to examine the selectivity.
As shown in Figure 7, the addition of ClOÀ induced a signif-
Figure 9. Fluorescence ratiometric response of probe
1 (10 mm) to
30 equiv of NaClO in the presence of 30 equiv of different species in
0.1m potassium phosphate buffer, pH 9.0/DMF (v/v, 1:4): 1) the organic
aqueous buffer; 2) ZnCl2; 3) AlCl3; 4) NaNO2; 5) CuCl2; 6) LiClO4; 7)
NaOAc; 8) KClO3; 9) Na2SO4; 10) NaCO3; 11) MgCl2; 12) H2O2. Excita-
tion at 394 nm.
Conclusion
In summary, we have rationally designed compound 1 as a
ratiometric fluorescent probe for hypochlorite via the depro-
tection of the oxime into the aldehyde. Notably, this deoxi-
mation reaction has not been previously used in fluorescent
hypochlorite probe development. Probe 1 was readily syn-
thesized in two steps. Importantly, the probe exhibited a ra-
tiometric fluorescent response to hypochlorite with the
emission intensities ratio (I509/I439) increasing from 0.28 to
2.74. Additionally, the probe showed high selectivity for hy-
pochlorite over other species due to distinct deoximation
conditions. To our best knowledge, the probe developed
herein represents the first ratiometric fluorescent probe for
hypochlorite. As the probe features a ratiometric fluorescent
signal and a low micromolar detection limit, it may be favor-
able for interesting applications in many environmental and
biological settings.
Figure 7. Fluorescence spectra of probe 1 (10 mm) in the absence and
presence of 30 equiv of various species in 0.1m potassium phosphate
buffer, pH 9.0/DMF (v/v, 1:4). Excitation wavelength was 394 nm.
icant redshift of the fluorescence emission spectra. However,
representative species such as Li+, Na+, K+, Mg2+, Zn2+
,
À
À
Cu2+, Al3+, ClÀ, CH3COOÀ, SO42À, CO32À, NO2 , ClO3 ,
À
ClO4 , and H2O2 elicited almost no changes in the fluores-
cence spectra. Although Cu2+, H2O2, or NO2 are known to
À
be able to deprotect oximes into aldehydes, the deprotection
by these species have to be performed under microwave ir-
radiation,[19] in the presence of other catalysts,[20] or at ele-
vated temperature.[21] Thus, distinct reaction conditions re-
quired for deoximation could account for the high selectivity
of the probe for ClOÀ over other species. Furthermore, the
visual fluorescence response of probe 1 to various species
(Figure 8) demonstrates that the probe can be used conven-
Experimental Section
General information: Unless otherwise stated, all reagents were pur-
chased from commercial suppliers and used without further purification.
Solvents used were purified and dried by standard methods prior to use.
Twice-distilled water was used throughout all experiments. Electronic ab-
sorption spectra were obtained on a Shimadzu UV-2450 spectrometer.
Photoluminescent spectra were recorded with a Hitachi F4500 fluores-
cence spectrophotometer with the excitation and emission slit widths at
2.5 nm. Melting point of compounds were measured on a Beijing Taike
XT-4 microscopy melting point apparatus, all melting points were uncor-
rected. Mass spectrometry analyses were performed using an Agilent
1100 HPLC/MSD spectrometer. 1H NMR spectra were recorded on an
INOVA-400 spectrometer, using TMS as an internal standard. Elementa-
Figure 8. Visual fluorescence changes of 1 (10 mm) in the absence and
presence of different species (30 equiv) in 0.1m potassium phosphate
buffer, pH 9.0/DMF (v/v, 1:4). The photos were taken under a handheld
UV (365 nm) lamp.
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A Ratiometric Fluorescent Probe for Hypochlorite
FULL PAPER
ry analyses were obtained on a Vario El III Elemental Analyzer. TLC
analyses were performed on silica gel plates and column chromatography
was conducted over silica gel (mesh 200–300), both of which were ob-
tained from the Qingdao Ocean Chemicals. Stock solution of probe 1
was prepared at 5ꢁ10À4 m in DMF. The solutions of various testing spe-
cies were prepared from ZnCl2, CuCl2·2H2O, MgCl2·6H2O, NaNO2, Li-
ClO4·3H2O, KMnO4, Na2CO3, CH3COOK, AlCl3·6H2O, Na2SO4, KClO3,
10% NaClO, 30% H2O2, respectively. K2HPO4 and KH2PO4 were uti-
lized to prepare potassium phosphate buffer. Test solutions in 0.1m potas-
sium phosphate buffer, pH 9.0/DMF (v/v, 1:4) were prepared by placing
0.1 mL of probe 1 stock solution, 1 mL potassium phosphate buffer solu-
tion, and an appropriate aliquot of each species stock into a 5 mL volu-
metric flask, and then diluting the solution to 5 mL with DMF. The re-
sulting solution was shaken well before recording the absorption and
emission spectra.
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4-(1H-Phenanthro
ACHTUNGTRENNUNG[9,10-d]imidazol-2-yl)benzaldehyde oxime (1): Com-
pound (50 mg, 0.155 mmol), hydroxylamine hydrochloride (16 mg,
2
0.233 mmol), and Et3N (23.6 mg, 0.233 mmol) in ethanol were heated to
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(m,
4H);
UV/Vis
(DMF):
lmax
(e)=370 nm
(10500 molÀ1 dm3 cmÀ1); MS (ESI): m/z: 338.1 [M+H]+; elemental analy-
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Acknowledgements
Funding was partially provided by the Key Project of Chinese Ministry
of Education (No:108167), the National Science Foundation of China
(20872032), the Scientific Research Foundation for the Returned Over-
seas Chinese Scholars, State Education Ministry (2007-24), and the
Hunan University research funds.
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Received: October 6, 2008
Published online: January 20, 2009
Chem. Eur. J. 2009, 15, 2305 – 2309
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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