A Ratiometric Fluorescent Probe Based on a Through-Bond Energy Transfer (TBET) System for Imaging HOCl in Living Cells

Article abstract of DOI:10.1002/chem.201503500

A simple ratiometric probe (Naph-Rh) has been designed and synthesized based on a through-bond energy transfer (TBET) system for sensing HOCl. In this probe, rhodamine thiohydrazide and naphthalene formyl were connected by simple synthesis methods to construct a structure of monothio-bishydrazide. Free probe Naph-Rh showed only the emission of naphthalene. When probe Naph-Rh reacted with HOCl, monothio-bishydrazide could be converted into 1,2,4-oxadiazole, which not only ensured that the donor and the acceptor were connected with electronically conjugated bonds, but also resulted in the spiro-ring opening and the emission of rhodamine. Therefore, a typical TBET process took place. The probe possessed high-energy transfer efficiency and large pseudo-Stokes shifts. As the first TBET probe for HOCl, Naph-Rh showed excellent selectivity and sensitivity toward HOCl over other reactive oxygen species (ROS)/reactive nitrogen species (RNS), and could respond fast to a low concentration of HOCl in the real sample. In addition, the probe was suitable for imaging HOCl in living cells due to its real-time response, excellent resolution, and reduced cytotoxicity.

Full text of DOI:10.1002/chem.201503500

DOI: 10.1002/chem.201503500
Full Paper
&
Fluorescent Probes
A Ratiometric Fluorescent Probe Based on a Through-Bond
Energy Transfer (TBET) System for Imaging HOCl in Living Cells
Yan-Ru Zhang,[a] Ning Meng,[b, c] Jun-Ying Miao,*[b] and Bao-Xiang Zhao*[a]
Abstract: A simple ratiometric probe (Naph-Rh) has been
designed and synthesized based on a through-bond energy
transfer (TBET) system for sensing HOCl. In this probe, rhoda-
mine thiohydrazide and naphthalene formyl were connected
by simple synthesis methods to construct a structure of
monothio-bishydrazide. Free probe Naph-Rh showed only
the emission of naphthalene. When probe Naph-Rh reacted
with HOCl, monothio-bishydrazide could be converted into
1,2,4-oxadiazole, which not only ensured that the donor and
the acceptor were connected with electronically conjugated
bonds, but also resulted in the spiro-ring opening and the
emission of rhodamine. Therefore, a typical TBET process
took place. The probe possessed high-energy transfer effi-
ciency and large pseudo-Stokes shifts. As the first TBET
probe for HOCl, Naph-Rh showed excellent selectivity and
sensitivity toward HOCl over other reactive oxygen species
(ROS)/reactive nitrogen species (RNS), and could respond
fast to a low concentration of HOCl in the real sample. In ad-
dition, the probe was suitable for imaging HOCl in living
cells due to its real-time response, excellent resolution, and
reduced cytotoxicity.
Introduction
In recent years, most turn-on fluorescent sensors based on
BODIPY, cyanine, and rhodamine were reported for HOCl/
ÀOCl.[19–25] On the one hand, these probes have high fluores-
cence quantum yield and emission bands at the red region of
the spectrum. On the other hand, they have high sensitivity
owing to the lack of background signal. However, their appli-
cations were limited because of small Stokes shifts and inter-
ference from the environment, probe concentration, and exci-
tation intensity.[26–34] Ratiometric probes based on fluorescence
resonance energy transfer (FRET) can give a solution to the tis-
sues, because they not only possess larger pseudo-Stokes
shifts, but also have a recorded ratio signal of two emission in-
tensities at different wavelength, which can afford a built-in
correction. Normally, the donor and the acceptor in FRET sen-
sors are usually linked with a nonconjugated spacer, and the
energy transfers from the donor to the acceptor through
space.[27,35–38] A substantial spectral overlap is necessary be-
tween the donor emission band and the acceptor absorption
band, which sometimes limits the choice and the design of
FRET sensors.[34] In contrast, for a TBET (through-bond energy
transfer) system to be effective, the donor and the acceptor
are linked with an electronically conjugated bond, through
which the energy can transfer from the donor to acceptor
without the need for a substantial spectral overlap between
the donor emission and acceptor absorbance.[39] These can
afford the desired flexibility in the choice of fluorophore and
the large emission shifts between the two emission peaks.
Thus, TBET-based probes exhibit high energy transfer efficien-
cy, large pseudo-Stokes shifts,[40] and excellent resolution for
fluorescence imaging due to two well-resolved emission
peaks.[39] To the best of our knowledge, a fluorescent probe
based on TBET for HOCl has not been reported so far, although
Hypochlorous acid (HOCl)/hypochlorite (ÀOCl) is one of the
most important reactive oxygen species (ROS) generated from
hydrogen peroxide and chloride ions by the catalysis of myelo-
peroxidase (MPO) in living organisms.[1] Hypochlorite, as an en-
dogenous microbicidal agent, plays a vital role in defending
the invasion of pathogens.[2] On the other hand, uncontrolled
generation of hypochlorite is closely associated with some dis-
eases, such as arthritis, kidney disease, lung injury, atheroscle-
rosis, osteoarthritis, and cancer.[3–8] However, the roles of HOCl/
ÀOCl in these diseases are not quite clear owing to the limita-
tion of detection technology. Therefore, the development of
imaging techniques for HOCl/ÀOCl in vitro and in vivo is of sig-
nificant interest. The fluorescent probes for HOCl/ÀOCl have at-
tracted much attention due to their natural advantages includ-
ing excellent sensitivity, high selectivity, and rapid response
time.[9–18]
[a] Dr. Y.-R. Zhang, Prof. Dr. B.-X. Zhao
Institute of Organic Chemistry
School of Chemistry and Chemical Engineering
Shandong University, Jinan 250100 (P.R. China)
E-mail: bxzhao@sdu.edu.cn
[b] Dr. N. Meng, Prof. Dr. J.-Y. Miao
Institute of Developmental Biology, School of Life Science
Shandong University, Jinan 250100 (P.R. China)
E-mail: miaojy@sdu.edu.cn
[c] Dr. N. Meng
School of Biological Science and Technology
University of Jinan, Jinan 250022 (P.R. China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201503500.
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Scheme 1. Syntheses and proposed detection mechanism of Naph-Rh.
some TBET fluorescent probes were developed for the detec-
tion of other species.[39,41–43] Therefore, it is imperative to devel-
op ratiometric probes on TBET for imaging HOCl in living cells.
In our previous work,[44] we reported two FRET fluorescent
probes for HOCl/ÀOCl. Although the probes showed excellent
selectivity toward HOCl/ÀOCl, the sensitivity and energy trans-
fer efficiency are less than desirable. These limited their appli-
cation in imaging cells. In order to overcome the above short-
comings, in this work, we designed and synthesized a simple
TBET fluorescent probe (Naph-Rh, Scheme 1) for HOCl.
monothio-bishydrazide could be converted into electronically
conjugated bonds (1,2,4-oxadiazole confirmed by ESIMS, Fig-
ure S1, Supporting Information).[45] Thus, the spiro-ring of rhod-
amine was opened and the TBET system was setup simultane-
ously. The TBET system ensured that the energy could transfer
successfully from the donor to the acceptor. Moreover, to
avoid the spectral overlap between the donor emission and
the acceptor absorbance, and enlarge the pseudo-Stokes shifts
and emission shifts, we employed the naphthalene fluorophore
(excitation, 350 nm; emission, 440 nm) as a donor to match
with rhodamine fluorophore (absorbance, 570 nm; emission,
585 nm) as an acceptor. It can be seen from Figure 1 that the
spectral overlap is insignificant, yet the energy transfer efficien-
cy was calculated to be 83.4% (Figure S2, Supporting Informa-
tion).[32,42] To prove the TBET, we synthesized a model com-
pound Naph-A-Rh (Scheme 1) as a comparison. When Naph-A-
Rh reacted with HOCl, the spiro-ring of rhodamine was opened
Rhodamine thiohydrazide and naphthalene formyl were con-
nected by a simple synthesis method to construct a structure
of monothio-bishydrazide. In the free probe Naph-Rh, the
naphthalene fluorophore (donor) and rhodamine fluorophore
(acceptor) were linked by a nonconjugated bond (monothio-
bishydrazide), which does not seem to meet the requirement
of a TBET system. But when probe Naph-Rh reacted with HOCl,
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to give an 3-amino-1,2,4-oxadiazole derivative in which the
linker retained the nonconjugated bond (see Scheme S1 and
Figure S1, Supporting Information).[45] The energy-transfer effi-
ciency was calculated to be 45.3% (Figure S2, Supporting Infor-
mation). The results indicated that the energy transfer of
Naph-Rh should mainly be based on TBET. The pseudo-Stokes
shift is 235 nm and the emission shifts is 145 nm (Figure 1),
which are larger than those of reported TBET probes.[32,46] The
large emission shifts could improve imaging resolution in cells.
&
Figure 1. Normalized emission spectra of the donor ( ), normalized absorp-
~
tion spectra of the acceptor after the addition of HOCl (5 mm; ), and nor-
Figure 2. a) Fluorescence spectrum changes of Naph-Rh toward HOCl (5 mm)
À
1
*
malized emission spectra of Naph-Rh after the addition of HOCl (5 mm; ).
Conditions: [Naph-Rh]=5 mm, [donor]=5 mm, [acceptor]=5 mm, Na2HPO4
(0.05m, pH 6)/EtOH (7:3, v/v), lex: 350 nm (slit widths: 10/5 nm).
C
C
and other ROS/RNS (50 mm, HO , ONOO , NO, H2O2, tBuOOH, tBuOO , O2,
ÀO2). b) The relative ratio (I585/440ÀINaph-Rh585/440)/INaph-Rh585/440) of fluorescence in-
tensity ratio changes, I585/440 is the ratio (I585/I440) of Naph-Rh with addition of
ROS/RNS, INaph-Rh
the ratio (I585/I440) of Naph-Rh. Conditions: [Naph-
585/440
Rh]=5 mm, Na2HPO4 (0.05m, pH 6)/EtOH (7:3, v/v), lex: 350 nm (slit widths:
10/5 nm).
Results and Discussion
To evaluate the selectivity of Naph-Rh toward HOCl among
ROS/reactive nitrogen species (RNS), the absorbance and emis-
sion spectra of Naph-Rh were measured in the presence of
ROS/RNS at pH 6. When 5 mm HOCl was added to the solution
of Naph-Rh (5 mm) in a mixture of Na2HPO4 (0.05m, pH 6)/EOH
(7:3, v/v), a characteristic absorption peak of rhodamine at
570 nm appeared and a typical absorption peak of naphtha-
lene at 350 nm changed little (Figure S3, Supporting Informa-
tion). Simultaneously, a typical emission peak of naphthalene
(440 nm) decreased dramatically and a characteristic emission
peak of rhodamine (585 nm) appeared with the addition of
HOCl (Figure 2a). The results should be attributed to the TBET
system between naphthalene and rhodamine. The free probe
Naph-Rh was in a spiro ring-closing state and the TBET was off
in the absence of HOCl, so only emission of naphthalene was
observed in its fluorescent spectra. When the probe reacted
with HOCl, the oxadiazole was generated resulting in the spiro
ring-opening and the TBET. Therefore, the emission spectrum
of rhodamine appeared and the fluorescent intensity of naph-
thalene weakened with excitation at 350 nm. Other ROS/RNS
affected little the absorption and fluorescence spectra of
Naph-Rh, although the concentration of other ROS/RNS
The titration experiments were implemented in a mixed so-
lution of Na2HPO4 (0.05m, pH 6)/EtOH (7:3, v/v). The fluores-
cence intensity at 440 nm diminished significantly with the ad-
dition of HOCl from 0 to 5 mm and a new emission peak at
585 nm enhanced gradually (Figure 3a). Meanwhile, the corre-
sponding ratio (I585/I440) increased gradually and an excellent
linearity was established between the ratio and the concentra-
tion of HOCl from 2.5 to 5 mm (Figure 3b) with the detection
limit 0.1 mm (S/N=3). Furthermore, it could also be seen from
Figure 3a that the fluorescence intensity changed obviously at
a low concentration of HOCl (0.5 mm). These results indicated
that probe Naph-Rh showed excellent sensitivity toward HOCl
and could detect a low concentration of HOCl in the real
sample. The absorption spectra (Figure S4, Supporting Informa-
tion) showed that the characteristic peak (570 nm) of rhoda-
mine enhanced gradually with the addition of HOCl, which
also illustrated that the rhodamine moiety of probe Naph-Rh
could react with HOCl to induce spiro-ring opening.
We investigated the pH effect on the ratiometric response of
Naph-Rh toward HOCl (Figure S5, Supporting Information). The
results showed that probe Naph-Rh could effectively detect
HOCl at pH 3–7. Thus, we deduced that Naph-Rh is suitable for
imaging in vivo. Notably, the changes of time-dependent fluo-
rescence intensity ratio (I585/I440) indicated that probe Naph-Rh
could rapidly respond to HOCl and reached a plateau within
50 s (operation time), which was very important for real-time
imaging of HOCl in situ (Figure S6, Supporting Information).
Murine RAW264.7 can produce HOCl in the immune system
by stimulation with lipopolysaccharide (LPS),[19,47] So we em-
reached up to 50 mm. Figure 2b showed that the ratio (I585/I440
)
of probe Nph-Rh in the presence of HOCl increased by about
10-fold compared to the ratio (I585/I440) of free probe Nph-Rh.
The ratio (I585/I440) changed little with addition of other ROS/
RNS into probe Nph-Rh. These results indicate that probe
Naph-Rh has excellent selectivity toward HOCl over other ROS/
RNS.
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Figure 3. a) Fluorescence spectra and b) fluorescence intensity ratio (I585/I440
)
changes of Naph-Rh with the addition of HOCl (0–5 mm). Conditions: [Naph-
Rh]=5 mm, Na2HPO4 (0.05m, pH 6)/EtOH (7:3, v/v), lex: 350 nm (slit widths:
10/5 nm).
ployed RAW264.7 cells to investigate probe Naph-Rh for imag-
ing endogenous HOCl in living cells. In the control group, after
RAW264.7 cells had been incubated with Naph-Rh (10 mm) for
4 h, strong blue fluorescence (Figure 4a) and very faint red
fluorescence (Figure 4b) were observed. When RAW264.7 had
been incubated with probe Naph-Rh for 4 h after stimulating
with LPSs for 12 h, the strong blue fluorescence darkened (Fig-
ure 4c) and the faint red fluorescence brightened (Figure 4d).
The corresponding ratio (red to blue) of fluorescence intensity
increased obviously (Figure 4e). The results indicated that
probe Naph-Rh was suitable for imaging endogenous HOCl in
the living cells. In addition, the cytotoxicity of Naph-Rh was
evaluated in RAW264.74 cells. Little toxic effect on cell viability
was observed after incubation with Naph-Rh (10 mm) for 4 h
(Figure 4 f), which demonstrated that Naph-Rh could be used
for clinical applications in living organisms.
Figure 4. Fluorescence images of RAW264.7 cells incubated with a 10 mm
probe for 4 h from a) blue channel and b) red channel. Fluorescence images
of RAW264.7 cells pretreated with 1 mgmLÀ1 lipopolysaccharide (LPS) for
12 h, then incubated with 10 mm probe for 4 h from c) blue channel and
d) red channel. e) The ratio (red to blue) of fluorescence intensity, *p<0.05.
The results are presented as means ÆSE with replicates n=5. f) Viability of
RAW264.7 cells after treatment with the probe for 4 h at a concentration of
10 mm. Excitation: 405 nm; blue channel: 405–550 nm and red channel:
550–650 nm.
Experimental Section
Preparation of the test solutions
Naoh-Rh was dissolved in EtOH for a stock solution (1 mm). Test
solutions were prepared by displacing 50 mL of the stock solution
into a 10 mL volumetric flask. The solution was diluted to 10 mL in
a mixture of phosphate buffer (0.05m, pH 6) and EtOH (7:3, v/v).
Small aliquots of each testing species solution were added. The re-
sulting solutions were shaken well and incubated for 1 h at room
temperature before recording spectra.
Conclusions
In summary, a TBET strategy is employed to design a ratiometric
probe Naph-Rh for imaging endogenous HOCl in living cells.
As we know, this probe is the first TBET-based fluorescent
probe for HOCl. It shows two well-resolved emission peaks
with high energy transfer efficiency and large pseudo-Stokes
shifts. This probe exhibits high selectivity toward HOCl over
other ROS/RNS and can respond fast to a low concentration of
HOCl in the real sample. Little cytotoxicity is crucial for Naph-
Rh to be used for clinical applications in living organisms. We
hope this strategy might promote the development of TBET-
based probes for HOCl.
Cell culture and imaging
RAW264.7 cells were obtained from the Cell Bank of the Chinese
Academy of Sciences (Shanghai, China) and were cultured as rou-
tine in DMEM medium containing 10% fetal bovine serum. All cells
were maintained at 378C under humidified conditions and 5%
CO2. RAW264.7 cells were passed on small glasses and incubated
for 24 h, and then incubated with LPS 1 mgmLÀ1 for 12 h. Before
the staining experiment, cells were washed 3 times with PBS, incu-
bated with 10 mm probe for 4 h, and then washed 3 times with
PBS and underwent imaging measurements with a confocal micro-
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scope (Zeiss LSM780, Carl Zeiss Canada) at 405 nm excitation. The
emission of the blue channel was 405–550 nm and the red channel
550–650 nm.
[M+H]+; found: 670.3261; m/z [M+2H]2+/2 calcd for C41H45N5O2S:
335.6647; found: 335.6663.
Synthesis of acceptor: To the solution of compound 3 (94 mg,
0.20 mmol) in dry pyridine (4 mL), benzoyl chloride (0.3 mL) was
added at RT under a N2 atmosphere. The reaction solution was
stirred at RT for 2 h, and was then heated and kept at reflux for
6 h. The solvent was removed under reduced pressure and the
crude product was purified by column chromatography (petroleum
ether/ethyl acetate=4:1) to give a pale-red solid (31.7 mg). Yield:
Synthesis of compound 1: Dissolve 6-bromo-N,N-dimethylnaph-
thalen-2-amine (5.0 g, 20 mmol) in dry THF (100 mL) and transfer
the solution into a pressure-equalizing dropping funnel. First,
about 5 mL of the above solution was dropped into the reaction
flask containing 480 mg magnesium under a N2 atmosphere, and
a little iodine was added to initiate the reaction. Then, the remain-
der of 6-bromo-N,N-dimethylnaphthalen-2-amine solution was
slowly dropped into the reaction flask under a boiling state; the
mixture was kept refluxing for 2 h after addition.
1
28.2%; H NMR (300 MHz, [D6]DMSO): d=10.69 (s, 1H), 8.02 (d, J=
6.8 Hz, 1H), 7.95 (d, J=7.4 Hz, 2H), 7.61 (d, J=7.5 Hz, 3H), 7.50 (t,
J=7.4 Hz, 3H), 7.40 (d, J=7.3 Hz, 1H), 7.15 (d, J=6.6 Hz, 1H), 6.45
(d, J=8.3 Hz, 1H), 6.35 (d, J=8.3 Hz, 3H), 3.31 (d, J=6.8 Hz, 8H),
1.07 ppm (t, J=6.7 Hz, 12H); 13C NMR (75 MHz, DMSO): d=167.23,
164.32, 153.20, 148.57, 135.97, 132.93, 132.78, 131.52, 130.71,
129.60, 129.19, 128.78, 128.49, 128.03, 127.70, 124.31, 107.63,
97.01, 43.60, 39.96, 12.36 ppm; HRMS: m/z: calcd for C35H37N4O2S:
577.2637 [M+H]+; found: 557.2728; m/z: calcd for C35H38N4O2S:
289.1358 [M+2H]2+/2; found: 289.1394.
The above reaction solution was cooled to room temperature. Sub-
sequently, CO2 was bubbled into the reaction solution for 10 h,
and then the mixture was poured into ice-water, extracted with di-
chloromethane, and purified by column chromatography to give
a
pale-yellow solid (2.3 g). Yield: 53.5%; 1H NMR (300 MHz,
[D6]DMSO): d=12.60 (s, 1H), 8.38 (s, 1H), 7.89 (d, J=9.2 Hz, 1H),
7.80 (dd, J=8.6, 1.7 Hz, 1H), 7.68 (d, J=8.7 Hz, 1H), 7.27 (dd, J=
9.1, 2.5 Hz, 1H), 6.96 (d, J=2.2 Hz, 1H), 3.06 ppm (s, 6H); 13C NMR
(75 MHz, DMSO): d=167.67, 149.90, 137.00, 130.38, 130.13, 125.73,
125.47, 124.66, 123.18, 116.46, 104.78, 39.95 ppm; HRMS (ESI): m/z:
calcd for C13H14NO2: 216.1025 [M+H]+; found: 216.1022.
Synthesis of Naph-A-Rh: Naph-A-Rh was synthesized according to
the reported method.[48] Briefly, to the solution of potassium thio-
cyanate (97.2 mg, 1 mmol) in acetonitrile (20 mL), compound 2
(233 mg, 1 mmol) was added slowly at RT. After addition, the reac-
tion solution was heated and kept at reflux for 45 min, and then fil-
tered to remove KCl. Rhodamine B hydrazide (456.6 mg, 1 mmol)
was added into the above filtrate, and kept at reflux for 2 h. Aceto-
nitrile was removed under reduced pressure and the crude com-
pound was purified by column chromatography (petroleum ether/
ethyl acetate/triethylamine=10:10:1) pale-yellow solid (357 mg).
Synthesis of compound 2: Compound 1 was added into thionyl
chloride (3 mL) at 08C and kept stirring for 2 h. After the reaction
completed, thionyl chloride was removed under reduced pressure
to give a gray/white solid.
Synthesis of compound 3: Rhodamine B hydrazide (1.0 g,
2.2 mmol) and Lawesson reagent (890 mg, 2.2 mmol) were dis-
solved in dry toluene (40 mL). The reaction solution was heated
and kept at reflux for 8 h under a N2 atmosphere. Toluene was re-
moved under reduced pressure and the crude compound was pu-
rified by column chromatography (petroleum ether/ethyl acetate=
1
Yield: 50.1%; H NMR (300 MHz, [D6]DMSO): d=11.48 (s, 1H), 8.40
(s, 1H), 7.91 (d, J=6.3 Hz, 1H), 7.82 (d, J=9.2 Hz, 1H), 7.76 (dd, J=
5.8, 2.6 Hz, 2H), 7.66–7.57 (m, 3H), 7.27 (dd, J=9.2, 2.4 Hz, 1H),
7.11 (d, J=6.5 Hz, 1H), 6.93 (d, J=2.1 Hz, 1H), 6.40 (d, J=14.7 Hz,
6H), 3.34 (d, J=4.1 Hz, 8H), 3.05 (s, 6H), 1.10 ppm (s, 12H);
13C NMR (75 MHz, DMSO): d=167.95, 165.16, 153.02, 152.94,
151.77, 150.20, 148.58, 148.03, 137.13, 132.27, 129.53, 128.01,
127.59, 123.40, 122.04, 116.49, 107.71, 105.38, 103.21, 97.34, 64.66,
59.64, 43.58, 12.40 ppm; HRMS (ESI): m/z: calcd for C42H45N6O3S:
713.3274 [M+H]+; found: 713.3264.
1
4:1) to give a white solid (263 mg). Yield: 25.3%; H NMR (300 MHz,
[D6]DMSO): d=7.94–7.83 (m, 1H), 7.62–7.46 (m, 2H), 7.05 (dd, J=
5.6, 2.7 Hz, 1H), 6.40 (d, J=2.4 Hz, 2H), 6.37 (d, J=2.5 Hz, 1H), 6.34
(d, J=2.5 Hz, 1H), 6.17 (d, J=8.8 Hz, 2H), 5.36 (s, 2H), 3.38–3.27
(m, 8H), 1.09 ppm (t, J=6.9 Hz, 12H); 13C NMR (100 MHz, DMSO):
d=181.02, 153.33, 149.71, 149.13, 136.35, 132.43, 129.12, 128.20,
124.11, 123.40, 108.49, 103.76, 97.88, 73.13, 44.17, 12.89 ppm;
HRMS (ESI): m/z calcd for C28H33N4OS: 473.2375 [M+H]+; found:
473.2402; m/z: calcd for C28H34N4OS: 237.1227 [M+2H]2+/2; found:
237.1237.
Acknowledgements
This study was supported by the Natural Science Foundation
of Shandong Province (ZR2014M004) and the National Natural
Science Foundation of China (91313303).
Synthesis of Naph-Rh: To the solution of compound 3 (118 mg,
0.25 mmol) in dry pyridine (5 mL), compound
2 (58.3 mg,
0.25 mmol) was added at RT under a N2 atmosphere. The reaction
solution was stirred at RT for 2 h, and was then heated and kept at
reflux for 6 h. The solvent was removed under reduced pressure
and the crude product was purified by column chromatography
(petroleum ether/ethyl acetate=3:1) to give a pale-yellow solid
Keywords: fluorescent probes
naphthalene · rhodamine · through-bond energy transfer
·
hypochlorous acid
·
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7.68–7.48 (m, 4H), 7.24 (dd, J=9.1, 2.3 Hz, 1H), 7.13 (d, J=6.5 Hz,
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132.92, 129.73, 129.65, 128.74, 128.20, 125.34, 125.11, 124.74,
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Published online on November 16, 2015
Chem. Eur. J. 2015, 21, 19058 – 19063
www.chemeurj.org
19063
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim