A highly selective and sensitive fluorescence probe for the hypochlorite anion

Article abstract of DOI:10.1002/chem.200701677

A new rhodamine B-based fluorescent probe for the hypochlorite anion (OCl^-) has been designed, synthesized, and characterized. The probe comprises a spectroscopic unit of rhodamine B and an OCl^--specific reactive moiety of dibenzoylhydrazine. The probe itself is nearly nonfluorescent because of its spirolactam structure. Upon reaction with OCl^-, however, a largely enhanced fluorescence is produced due to the opening of the spirolactam ring by the oxidation of the exocyclic hydrazide and subsequently the formation of the hydrolytic product rhodamine B. Most notably, the fluorescence-on reaction shows high sensitivity and extremely high selectivity for OCl^- over other common ions and oxidants, which makes it possible for OCl^- to be detected directly in their presence. In addition, the reaction mechanism has been investigated and proposed. The OCl^- anion selectively oxidizes the hydrazo group in the probe, and forms the analogue of dibenzoyl diimide, which in turn hydrolyzes and releases the fluorophore. The reaction mechanism that is described here might be useful in developing excellent spectroscopic probes with cleavable active bonds for other species.

Full text of DOI:10.1002/chem.200701677

FULL PAPER
DOI: 10.1002/chem.200701677
A Highly Selective and Sensitive Fluorescence Probe for the Hypochlorite
Anion
Xinqi Chen, Xiaochun Wang, Shujuan Wang, Wen Shi, Ke Wang, and Huimin Ma*[a]
Abstract: A new rhodamine B-based
fluorescent probe for the hypochlorite
anion (OClÀ) has been designed, syn-
thesized, and characterized. The probe
comprises a spectroscopic unit of rho-
damine B and an OClÀ-specific reactive
moiety of dibenzoylhydrazine. The
probe itself is nearly nonfluorescent
because of its spirolactam structure.
Upon reaction with OClÀ, however, a
largely enhancedfluorescence is pro-
duced due to the opening of the spiro-
lactam ring by the oxidation of the
exocyclic hydrazide and subsequently
the formation of the hydrolytic product
rhodamine B. Most notably, the fluo-
rescence-on reaction shows high sensi-
tivity andextremely high selectivity for
OClÀ over other common ions andoxi-
dants, which makes it possible for OClÀ
to be detected directly in their pres-
ence. In addition, the reaction mecha-
nism has been investigatedandpro-
posed. The OClÀ anion selectively oxi-
dizes the hydrazo group in the probe,
andforms the analogue of idbenzoyl
diimide, which in turn hydrolyzes and
releases the fluorophore. The reaction
mechanism that is described here
might be useful in developing excellent
spectroscopic probes with cleavable
active bonds for other species.
Keywords: cleavage reactions
·
·
·
fluorescence
fluorescent
spectroscopy
probes
hypochlorite anion · rhodamine B
Introduction
able for OClÀ detection so far, and in particular for OClÀ-se-
lective detection.[6–8] For instance, 2,7-dichlorodihydrofluor-
escein, dihydrorhodamine 123 and 2-[6-(4’-amino)phenoxy-
3H-xanthen-3-on-9-yl]benzoic acidhave been suggestedas
probes for OClÀ detection, but their fluorescence responses
lack selectivity because of the cross-reactions with other
Hypochlorite anion (OClÀ), one of the biologically impor-
tant reactive oxygen species (ROS),[1] is produced in organ-
isms by the reaction of H2O2 with ClÀ ions under the cataly-
sis of a heme enzyme, myeloperoxidase.[2] Endogenous OClÀ
is essential to life andhas important antibacterial properties.
However, excessive or misplacedproduction of OCl
[7–9]
[6]
ROS andoxidizing agents.
Nagano andco-workers
re-
À
can
porteda fluorescence probe with goodselectivity for hypo-
lead to tissue damage and diseases, such as atherosclerosis,
chlorous acid, but its synthetic route was rather complicat-
ed.
It is notedthat OCl can selectively oxidize dibenzoylhy-
[2,3]
arthritis, andcancers.
Therefore, much effort has recently
À
been focused on the studies of the biological functions and/
or deleterious effects of OClÀ,[3b,4] anda number of sensitive
andselective analytical methosd have been proposedfor
conducting such research, among which fluorescent probes
drazine into dibenzoyl diimide, which can further undergo a
decomposition in some nucleophilic solvents (Scheme 1).[10]
play an important role in this respect due to their high-time
[5,6]
andspatial resolution capability.
Unfortunately, only a
limitednumber of fluorescent probes have been made avail-
[a] Dr. X. Chen, X. Wang, S. Wang, W. Shi, K. Wang, Prof. Dr. H. Ma
Beijing National Laboratory for Molecular Sciences
Institute of Chemistry, Chinese Academy of Sciences
Beijing 100080 (China)
Fax : (+86)10-6255-9373
E-mail: mahm@iccas.ac.cn
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Scheme 1. The OClÀ anion oxidizes dibenzoylhydrazine into dibenzoyl
diimide.
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However, such a reaction has never been usedto develop a
selective fluorescent probe for OClÀ. In the present work,
we attemptedto accomplish this. Specifically, herein we de-
scribe the design of N-benzoyl rhodamine B–hydrazide 1
(Scheme 2) as a novel OClÀ fluorescent probe by engineer-
ing a dibenzoylhydrazine moiety into rhodamine B, which is
an excellent fluorophore.[11] The probe, 1, can be synthesized
by a simple synthetic procedure, and as expected, the probe
exhibits a highly selective fluorescence response to OClÀ
only, insteadof other common ions andoxidants.
Figure 1. X-ray crystal structure of 1.
fluorescent.[13] This is very desirable because it affords a low
backgroundsignal in detection.
Absorption spectral properties of 1: The absorption spectra
of 1 andits reaction solution with various concentrations of
OClÀ are shown in Figure 2. As expected, 1 has nearly no
absorption in the visible region (curve 1) because of its
[13]
closedspirolactam form.
Upon reaction with OClÀ, how-
ever, the pink color that is indicative of rhodamine B is
À
largely restoredwith the increase in OCl
concentration
(curves 2–20). This can be ascribedto the selective oxidation
of dibenzoylhydrazine by OClÀ, which promotes the opening
of the closed spirolactam ring. In addition, the probe is
stable because no obvious change in the absorption spec-
trum was observedafter the probeꢁs solution was storedat
room temperature for a week.
Scheme 2. Synthesis of 1.
Results and Discussion
Design and synthesis of 1: Spectroscopic probes usually con-
sist of two units:[12] 1) a spectroscopic or signaling unit, the
properties of which shouldbe changedupon reaction with
the analyte of interest, and2) a labeling or recognition unit
that is responsible for the selective reaction with the analyte.
To design a selective OClÀ fluorescent probe, the fluoro-
phore rhodamine B was chosen as the signaling unit. How-
ever, there still exists a challenge in choosing a suitable rec-
ognition unit because other oxidizing agents not only coex-
ist, but also often have similar reactivity. In this respect, we
take advantage of the selective oxidation of dibenzoylhydra-
zine by OClÀ. As a result, the probe 1 was readily prepared
from rhodamine B by a two-step reaction (Scheme 2). The
reaction of rhodamine B with hydrazine gave rhodamine B–
hydrazide (yield: 79%), which was then converted into the
designed product 1 (yield: 65%) by further reaction with
benzoyl chloride. The structure of 1 was confirmedby
1H NMR spectroscopy, mass spectrometry, andelemental
analysis. A single crystal of 1 was grown at room tempera-
ture from the mixedsolvents of petroleum ether/ethyl ace-
tate (3:1, v/v) andcharacterizedby X-ray crystallography.
As shown in Figure 1, the probe has a five-memberedspiro-
lactam structure, which causes it to be colorless andnon-
Figure 2. Absorption spectra of probe 1 (10 mM, curve 1) andits reaction
solution with OClÀ at various concentrations (1-100 mm, curves 2–20)
against the corresponding reagent blank. The reaction was carried out in
0.03m Na2B4O7/NaOH buffer (pH 12) that contained30% (v/v) THF.
The inset depicts the absorbance at the absorption maximum of 554 nm
in the curves 2–20 as a function of OClÀ concentration.
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Fluorescence Probe for Hypochlorite
FULL PAPER
Fluorescence properties and selectivity studies: Figure 3
shows the fluorescence spectra of 1 andits reaction solution
with various species under the same conditions. Similar to
its absorption properties, 1 itself is essentially nonfluores-
cent due to its five-membered spirolactam structure.[13]
Upon the introduction of OClÀ into the solution of 1, how-
ever, fluorescence (lem =578 nm) that is characteristic of
rhodamine B appeared.
Figure 4. Effect of pH on the reaction of 1 (10 mm) with OClÀ (40 mm);
1+OClÀ
(
), 1 ( ). Fluorescence intensity was measuredat lex/em =520/
&
*
578 nm.
present system; milder pH conditions, such as about pH 10,
can also be used, but the sensitivity is relatively low. Time-
course studies revealed that the fluorescence intensity in-
creasedwith the increase of reaction time at room tempera-
ture (about 258C). However, 30 min later a plateau of fluo-
rescence enhancement was achieved. As a result, a reaction
medium of 0.03m Na2B4O7/NaOH buffer (pH 12) that con-
tained30% (v/v) THF, anda reaction time of 30 min at
room temperature were chosen for the present reaction, be-
cause under these conditions a plateau of fluorescence en-
hancement couldbe produced(see Figure 4 andFigures S1
andS2 in the Supporting Information). Obviously, under the
above-determined conditions, the detection of OClÀ should
be performedin vitro insteadof in vivo for biological stud-
ies.
Figure 3. Fluorescence spectra (lex =520 nm) of 1 (10 mM) at pH 12 in the
presence of 0.5–500 equivalents of various species (500 equiv of ClÀ or
K+; 100 equiv of H2O2, NO3À, PO43À, SiO32À, or SO42À; 30 equiv of Mg2+
15 equiv of Ca2+; 10 equiv of ClO3À, Ni2+, or Zn2+; 2 equiv of Mn2+
;
;
1 equiv of Cu2+, Fe2+, MnO4À, or OClÀ; 0.5 equiv of Fe3+, Hg2+, or Pb2+
were used).
To test the selectivity of the reaction, a solution of probe
1 (10 mm) was preparedin 0.03 m Na2B4O7/NaOH buffer
(pH 12) that contained30% (v/v) THF, and0.5–500 equiva-
lents of various species were then added. The reactions were
conducted at room temperature (about 258C) for 30 min,
andthen the fluorescence spectrum was monitored. The spe-
cies that were testedwere Ca 2+, Cu2+, Fe3+, Fe2+, Hg2+, K+,
Linearity: Under the optimized conditions that were deter-
minedabove, the enhancement value ( DF) of fluorescence
intensity in contrast to the blank solution without OClÀ
couldremain stable for at least 5 h, andit was directly pro-
portional to the OClÀ concentration (C) in a range of 1–
10 mm (Figure 5). The linear regression equation was deter-
minedto be DF=52.69”CÀ29.83 (n=10, g=0.9992) with a
detection limit of 27 nm (S/N=3), which also shows a highly
sensitive feature. Reproducibility tests (n=6) showedthat
the relative standard deviation of fluorescence was 4.0% for
10 mm OClÀ.
À
Mg2+, Mn2+, Ni2+, Pb2+, Zn2+, MnO4 , H2O2, ClÀ, OClÀ,
À
À
ClO3 , SO42À, NO3 , PO43À, andSiO 2À. As shown in
3
Figure 3, probe 1 displays a highly selective fluorescence-on
response to OClÀ only. This remarkable property makes 1 of
great potential for detecting OClÀ directly in the presence
of other species.
Optimization of experimental conditions: Various experi-
mental conditions, such as the concentration of probe 1, re-
action media, pH, temperature and time, were examined to
optimize the reaction conditions. The results show that both
the backgroundandresponse signals increase with increas-
ing concentration of 1. In this study a concentration of
10 mm of 1 was usedbecause sufficient fluorescence signal
couldbe obtainedat this concentration. The water solubility
of 1 is limited, and the use of 30% (v/v) THF as a co-solvent
is requiredin this system. Figure 4 depicts the effect of pH
on the reaction. As can be seen, pH 12 can be usedfor the
Interference studies: The effects of various ions on the de-
termination of OClÀ were investigatedby analyzing synthet-
ic sample solutions that contained10 mm of OClÀ. The toler-
able concentration was estimatedby the criterion at which a
species gave a relative error of no more than 10% as deter-
minedby the recovery of OCl À. The results are listedin
Table 1, from which it can be seen that the probe 1 exhibits
a high selectivity for OClÀ over a wide range of common
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H. Ma et al.
Figure 5. Fluorescent spectra of probe 1 (10 mM, curve 1) andits reaction
solutions with various concentrations of OClÀ (1–100 mM, curves 2–20)
according to the general procedure. The inset shows the plot of fluores-
cence enhancement value DF against OClÀ concentration.
Figure 6. HPLC chromatograms of different reaction products in 0.03m
Na2B4O7/NaOH buffer (pH 12) that contained30% THF. A) 100 mm
probe 1, B) 100 mm rhodamine B, C) 100 mm 1 plus 100 mm OClÀ, and
(blank) 100 mm OClÀ. HPLC analyses were performedon a HiQ sil C18W
(4.6”200 mm) column by using a Jasco HPLC system that consistedof a
PU-2086-plus pump anda UV-2075-plus detector at 300 nm with metha-
nol/water (6:1, v/v) as eluent (flow rate: 0.8 mLminÀ1). The assignment
of the peaks: 1) 4.71 min, rhodamine B, 2) 5.75–6.81 min, unidentified
products, 3) 12.57 min, and 4) 13.69 min, two tautomers of probe 1.
Table 1. Recovery of 10 mm OClÀ in the presence of various coexisting
species.
Species
Concn [mm]
Molar ratio of the
Recovery [%]
added species to OClÀ
K+
5000
150
300
10
10
5
500
15
30
1
96.2
98.7
100.4
93.9
103.1
101.3
107.0
101.0
101.8
106.4
98.5
Ca2+[a]
Mg2+[a]
Cu2+[a]
Fe2+[a]
Fe3+[a]
Hg2+[a]
Mn2+[a]
Ni2+
1
0.5
0.5
2
10
0.5
10
500
10
1
100
100
100
100
5
20
100
5
100
5000
100
10
1000
1000
1000
1000
Scheme 3. Two possible tautomers of probe 1.
Pb2+[a]
Zn2+
ClÀ
96.2
compounds such as luminol[14]). After reaction with OClÀ,
the two chromatographic peaks of 1 decrease markedly, and
several new peaks emerge (curve C), among which the
major peak at 4.71 min indicates the formation of rhodami-
ne B.
À
ClO3
MnO4
NO3
PO4
SiO3
SO4
104.5
106.0
103.4
107.6
102.9
106.9
À
À
3À
2À
2À
To further confirm the generation of rhodamine B, the re-
action solution also underwent MALDI-TOF MS analysis.
The experimental results (see Figure S3 in the Supporting
Information) showedthat, besides the peak of probe 1 (m/z:
561 [M+H]+), there are two more peaks that appear at m/z:
443 and427, respectively. Among them, the peak at m/z:
443 represents the formation of rhodamine B (m/z: 443
[M+H]+). Basedon our data andprevious observations that
OClÀ can selectively oxidize dibenzoylhydrazine into diben-
zoyl diimide, which can further undergo a decomposition in
some nucleophilic solvents (Scheme 1),[10] we propose that
the fluorescence-on reaction in this system proceeds mainly
through the route that is depicted in Scheme 4 to release the
fluorophore of rhodamine B.
[a] A higher concentration of these ions ledto the precipitation of metal
hydroxides at pH 12.
À
cations, anions, andeven some oxidants, such as ClO
MnO4 .
and
3
À
Reaction mechanism: The reaction mechanism in the pres-
ent system was studied. The generation of rhodamine B as a
product might be responsible for the recovery of both ab-
sorption andfluorescence. To prove this, the reaction prod-
ucts of probe 1 with OClÀ were subjectedto HPLC analysis.
As shown in Figure 6, the chromatographic peak of rhoda-
mine B appears at 4.71 min (peak 1 in curve B), whereas the
probe 1 has two chromatographic peaks (curve A) that
appear at 12.57 and13.69 min, respectively, which may be
ascribedto the two tautomers of the probe (see Scheme 3;
this enolization phenomenon has been reportedfor similar
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Fluorescence Probe for Hypochlorite
FULL PAPER
Scheme 4. The possible ring-opening reaction of 1 that is induced by the oxidative action of OClÀ.
to 10 mL with distilled, deionized water. After 30 min at room tempera-
ture, a reaction aliquot (3 mL) was transferredto a 1 cm quartz cell to
Conclusion
measure an absorbance or fluorescence intensity/spectrum with lex/em
=
520/578 nm andboth excitation andemission slit widths of 10 nm. In the
meantime, a blank solution that containedno OCl was preparedand
measured under the same conditions for comparison.
N-benzoyl rhodamine B–hydrazide has been characterized
À
as a new fluorescent probe for OClÀ. The probe displays
À
high sensitivity andextremely high selectivity for OCl
,
which makes it of great potential for the selective detection
of OClÀ in the presence of other common ions andoxidants.
In addition, the reaction mechanism that is revealed here
might be useful in developing excellent spectroscopic probes
with cleavable active bonds for other species.
Acknowledgement
We are grateful to the NSF of China (Nos. 20525517 and20435030), the
Ministry of Science andTechnology of China, andthe Chinese Academy
of Sciences for financial support.
Experimental Section
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N-Benzoyl rhodamine B–hydrazide 1: Probe 1 can be readily prepared
from rhodamine B by a two-step reaction (Scheme 2). First, the rhodami-
ne B–hydrazide was prepared by a similar procedure to that reported
previously.[15] In brief, 85% hydrazine hydrate (4 mL) was added to a so-
lution of rhodamine B (1 g, 2.09 mmol) in methanol (40 mL). The solu-
tion was refluxedfor 6 h with stirring. Then, the reaction mixture was
evaporated under reduced pressure to give an orange oil, which was then
recrystallized from methanol/water to afford rhodamine B–hydrazide as a
light-orange crystal (750 mg, 79%). ESI-MS: m/z: 457 [M+H]+.
Benzoyl chloride (0.3 mL, 2.6 mmol) in THF (5 mL) was added dropwise
to a mixed solution of rhodamine B–hydrazide (200 mg, 0.44 mmol) and
triethylamine (0.4 mL, 2.9 mmol) in THF (5 mL)/water (1 mL) at 0–58C
with stirring. Then, the mixture was stirredat room temperature over-
night. The solvent was removed under reduced pressure to give an
orange oil. The oil was dissolved in CH2Cl2 (20 mL), andthe solution was
successively washedwith dilute NaOH solution andbrine. The organic
phase was dried over anhydrous Na2SO4, concentratedandpurifiedby
silica-gel column chromatography with petroleum ether (60–908C)/
EtOAc (3:1, v/v) as the eluent to afford 1 as a colorless crystal (160 mg,
65%). 1H NMR (300 MHz, [D6]DMSO, 373 K): d=9.54 (s, 1H), 7.86 (d,
J=7.2 Hz, 1H), 7.60–7.54 (m, 4H), 7.45 (t, J=6.8 Hz, 1H), 7.34 (t, J=
7.0 Hz, 2H), 7.09 (d, J=7.0 Hz, 1H), 6.57 (d, J=8.5 Hz, 2H), 6.37–6.33
(m, 4H), 3.33 (q, J=6.9 Hz, 8H), 1.12 ppm (t, J=6.9 Hz, 12H); ESI-MS:
m/z: 561 [M+H]+; elemental analysis calcd(%) for C 35H36N4O3: C 74.98,
H 6.47, N 9.99; found: C 74.96, H 6.59, N 10.11; crystal data for 1: triclin-
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¯
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1cald =1.415 gcmÀ3; m(MoKa)=0.71073 mmÀ1; 6738 reflections measured,
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General procedure for OClÀ determination: Unless otherwise noted, all
measurements were made in 0.03m Na2B4O7/NaOH buffer (pH 12) that
contained30% (v/v) THF according to the following procedure. In a
10 mL tube, the 1 mm stock solution of 1 (0.1 mL) in THF was diluted
with THF (2.9 mL), followed by the addition of distilled, deionized water
(2 mL) and0.1 m Na2B4O7-NaOH buffer (3 mL, pH 12). Then an appro-
priate volume of the OClÀ sample solution (final concentration of OClÀ
was not more than 10 mm) was added, and the final volume was adjusted
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Received: October 24, 2007
Publishedonline: April 2, 2008
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