Fluorescent detection of hypochlorous acid from turn-on to FRET-based ratiometry by a HOCl-mediated cyclization reaction

Article abstract of DOI:10.1002/chem.201101918

Hypochlorous acid (HOCl), a reactive oxygen species (ROS), plays a significant biological role in living systems. However, abnormal levels of HOCl are implicated in many inflammation-associated diseases. Therefore, the detection of HOCl is of great import

Full text of DOI:10.1002/chem.201101918

DOI: 10.1002/chem.201101918
Fluorescent Detection of Hypochlorous Acid from Turn-On to FRET-Based
Ratiometry by a HOCl-Mediated Cyclization Reaction
Lin Yuan, Weiying Lin,* Yinan Xie, Bin Chen, and Jizeng Song[a]
Abstract: Hypochlorous acid (HOCl),
a reactive oxygen species (ROS), plays
a significant biological role in living
systems. However, abnormal levels of
HOCl are implicated in many inflam-
mation-associated diseases. Therefore,
the detection of HOCl is of great im-
portance. In this work, we describe the
HOCl-promoted cyclization of rho-
transfer (FRET) signaling mechanism,
2 was further converted into 1a and
1b, which represent the first paradigm
of FRET-based ratiometric fluorescent
HOCl probes. The outstanding features
of 1a and 1b include well-resolved
emission peaks, high sensitivity, high
selectivity, good functionality at physio-
logical pH, rapid response, low cytotox-
icity, and good cell-membrane permea-
bility. Furthermore, these excellent at-
tributes enable us to demonstrate, for
the first time, the ratiometric imaging
of endogenously produced HOCl in
living cells by using these novel ratio-
metric probes. We expect that 1a and
1b will be useful molecular tools for
studies of HOCl biology. In addition,
the HOCl-promoted cyclization reac-
tion of rhodamine-thiosemicarbazides
to rhodamine-oxadiazoles should be
widely applicable for the development
of different types of fluorescent HOCl
probes.
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ploited as a novel design strategy for
the development of a new fluorescence
turn-on HOCl probe 2. On the basis of
the fluorescence resonance energy
Keywords: fluorescent probes
·
FRET · oxadiazole · ratiometry ·
sensors
Introduction
the known fluorescent probes respond to HOCl with
changes only in fluorescent intensity. A major limitation of
intensity-based probes is that fluorescence measurement is
affected by variations in the sample environment and probe
distribution. By contrast, ratiometric fluorescent probes
allow the measurement of emission intensities at two differ-
ent wavelengths; this should provide a built-in correction
for environmental effects and can also increase the dynamic
range of fluorescence measurement.[15] Our group previously
reported a ratiometric fluorescent OClÀ probe operating
through an intramolecular charge transfer (ICT) signaling
mechanism.[16] The ratiometric fluorescent OClÀ probe was
designed on the basis of the specific hypochlorite-mediated
transformation of oximes to aldehydes. Unfortunately, this
oxime-based ratiometric OClÀ probe only functions at a
high pH value (pH 9.0), so it is not suitable for applications
in living cells. To our best knowledge, ratiometric fluores-
cent probes suitable for ratiometric imaging of HOCl in
living cells have not yet been reported. Therefore, there is a
need to develop ratiometric fluorescent probes for HOCl
with favorable characteristics for imaging applications by a
new design strategy, which should be compatible with bio-
logical conditions. Since the pioneering work of Czarnikꢀs
group regarding a rhodamine-based Cu2+ fluorescent che-
modosimeter,[17] rhodamine derivatives have been widely
employed as an effective platform for the construction of
small-molecule fluorescence “turn-on” probes for diverse
species such as Cu2+, Hg2+, Fe3+, Cr3+, Pb2+, Zn2+, Ag+,
Au3+, Pd2+, Pt2+, HOCl, and NO, through exploitation of
the unique spirolactam ring-opening process of rho-
Reactive oxygen species (ROS) mediate a wide variety of
biological processes.[1] Hypochlorous acid (HOCl), a biologi-
cally significant ROS, is produced by the peroxidation of
chloride ions catalyzed by the enzyme myeloperoxidase
(MPO) in activated leukocytes.[2] HOCl is also a critical mi-
crobicidal agent in natural defense.[3] However, abnormal
levels of HOCl are implicated in many inflammation-related
diseases, including cardiovascular diseases,[4] damage of
human red blood cells,[5] lung injury,[6] rheumatoid arthritis,[7]
and cancer,[8] so the detection of HOCl is of great impor-
tance. A number of methods such as electroanalysis,[9] po-
tentiometry,[10] spectrophotometry,[11] and chemilumines-
cence[12] for the detection of HOCl have been developed.
However, fluorescence sensing is advantageous because of
its high sensitivity and selectivity.[13] In addition, in conjunc-
tion with microscopy, it is very useful for bio-imaging appli-
cations in living cells. Therefore, the construction of small-
molecule fluorescent probes for the specific detection of
HOCl has received intense attention.[14] However, most of
[a] L. Yuan, Prof. W. Lin, Y. Xie, B. Chen, J. Song
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University, Changsha, Hunan 410082 (P. R. China)
Fax : (+86)88821464
E-mail: weiyinglin@hnu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101918.
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FULL PAPER
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
cal signal change only in their fluorescence intensity. Fluo-
rescence resonance energy transfer (FRET) is a common
signaling mechanism used in the design of dual-emission ra-
tiometric fluorescent probes,[19] in which the excitation
energy of the energy donor is transferred to the energy ac-
ceptor. The aim of this work was to develop FRET-based ra-
tiometric fluorescent probes for the ratiometric imaging of
HOCl in living cells. In the preliminary work, we found that
the reaction of rhodamine-thiosemicarbazide 2 with NaOCl
at room temperature could result in the formation of rho-
Scheme 2. Structures of the new ratiometric fluorescent HOCl probes 1a
and 1b and the control compound 8.
fluorescence imaging studies for these ratiometric fluores-
cent HOCl probes.
Results and Discussion
Scheme 1. Reaction of 2 with NaOCl.
Synthesis: The synthesis of the ratiometric probes 1a and
1b started with the preparation of the intermediate 3a/b
(Scheme 3). Condensation of 3-(diethylamino)phenol 1 with
commercially available 1, 2, 4-benzenetricarboxylic anhy-
dride 2 led to the formation of the mixture of 4’- and 5’-car-
ACHTUNGTRENNUNGdamine-1,3,4-oxadiazole 7 (Scheme 1). The observation that
the conversion of thiosemicarbazides into oxadiazoles can
be mediated by NaOCl under very mild conditions suggests
that this reaction is promising
as a new platform for the devel-
opment of fluorescent HOCl
probes. In this contribution, we
initially constructed a new fluo-
rescence turn-on HOCl probe 2
(Scheme 1) by a novel design
approach, that is, the HOCl-
mediated cyclization of rho-
ACHTUNGTRENNUNGdamine-thiosemicarbazides to
rhodamine-oxadiazoles. To ful-
fill the aim of this work, we de-
cided to further translate the
fluorescence turn-on probe 2
into novel ratiometric fluores-
cent HOCl probes based on a
FRET signaling mechanism.
The coumarin dye was selected
as the energy donor to render a
large emission shift between
the coumarin donor and the
rhodamine acceptor (Figure S1
in the Supporting Information).
Thus, the 4- and 5-position re-
gioisomers of compounds 1a
and 1b (Scheme 2) were de-
signed as new candidates for
FRET-based ratiometric fluo-
rescent HOCl probes. Herein,
we describe the design, synthe-
Scheme 3. Synthesis of 4- and 5-position regioisomers of ratiometric probes 1a and 1b. Reagents and Condi-
tions: a) 85% H3PO4, 170–1808C; b) DCC, DMAP, CH2Cl2; c) NH2NH2·H2O, DCC, DMAP, CH2Cl2; d) sepa-
sis, spectral properties, and ration by column chromatography (C2H5OH:CH2Cl2 =1:200 to 1:20); e) phenylisothiocyanate, DMF.
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W. Lin et al.
boxyrhodamines 3a/b. Condensation of 3a/b with coumarin
amine 6 by standard coupling chemistry gave the FRET
dyads 4a/b. Notably, the coupling occurred almost exclusive-
ly at the 4- or 5-position carboxylic acid. The high regiose-
lectivity can be attributed to the lower steric hindrance of
the 4- or 5-position carboxylic acid compared to the 2-posi-
tion one. Reaction of 4a/b with hydrazine hydrate gave
compounds 5a/b. Separation of the mixture of compounds
5a/b could be readily achieved by standard column chroma-
tography to give the individual compounds 5a and 5b in
pure isomeric form. Alternatively, the pure isomeric forms
4a and 4b can be obtained by preparative thin-layer chro-
matographic separation (CH2Cl2:CH3OH=9:1) from the
mixture of 4a/b (Scheme S1 in the Supporting Information)
or from the pure isomeric forms 3a and 3b (Scheme S2 in
the Supporting Information), respectively. The pure isomeric
forms 5a and 5b can also be obtained from the pure isomer-
ic forms 4a and 4b (Scheme S2 in the Supporting Informa-
tion). With the pure intermediates 5a and 5b in hand, pro-
bes 1a and 1b were readily synthesized by reaction of 5a
and 5b, respectively, with phenylisothiocyanate (Scheme 3).
The control compound 8 (Scheme 2) was previously synthe-
sized.[18i] The synthesis of the intermediate 6 and energy
donor 10 are shown in Scheme S3 of the Supporting Infor-
mation. The energy acceptors 9a and b were obtained ac-
cording to Scheme S4 of the Supporting Information. Com-
pound 2 was prepared in one step by the reaction of rho-
A
hydrazide
13
with
phenylisothiocyanate
(Scheme S5, Supporting Information). The compounds 2,
1a, and 1b were fully characterized by 1H NMR and
13C NMR spectroscopy and HRMS.
Figure 1. Absorption (a) and fluorescence (b) titration spectra of 2 (5 mm)
in PBS/DMF (pH 7.4, 1:1) upon addition of increasing concentrations of
NaOCl (0–30 equiv). c) Fluorescence spectra of 2 (5 mm) in the presence
of various biologically relevant analytes (7 equiv) including hydrogen per-
oxide, hydroxyl radicals, superoxide, HOCl, CH3COOOH, Fe3+, Cu2+
,
Photophysical properties and sensing responses of probe 2:
As shown in Figures 1A and B, the free compound 2 dis-
played almost no considerable absorption or emission bands
above 500 nm (F<0.001), indicating that it exists as the
ring-closed form. However, significantly, treatment of
NaOCl with compound 2 induced the formation of a strong
visible absorption band centered at 569 nm and an intense
emission band (Ff =0.29) at around 590 nm (Figure 1A, B),
suggesting that HOCl promotes the transformation of com-
pound 2 from the ring-closed form to the ring-opened form.
Furthermore, 2 displayed a high selectivity for HOCl over
other biologically relevant species represented by hydrogen
Co2+, Zn2+, NO2À, NO3À, HCO3À, Val, cytosine, thymine, adenine, glu-
cose, GSH, vitamin C.
C2H5OH=10:1) to afford a dark purple solid, which was un-
ambiguously identified to be rhodamine-1,3,4-oxadiazole 7
1
(Scheme 1) by studies of the H NMR, 13C NMR, ESI-MS,
HRMS, and absorption and emission spectra (Figures S2–S4
in the Supporting Information). To gain some insight into
the reaction mechanism, control compound 8, which con-
tains a semicarbazide instead of the thiosemicarbazide
group, was treated with NaOCl at room temperature. How-
ever, no reaction was observed (Scheme S6, Supporting In-
formation). Consistently, control compound 8 exhibited
almost no fluorescence response toward NaOCl (Figure S5,
Supporting Information). These findings indicate that the
sulfur atom plays a significant role in the reaction of probe
2 with NaOCl. On the other hand, although it is known that
iodine (or other oxidants) can transfer thiosemicarbazides to
1,3,4-oxadiazoles through iodine- (or other oxidant)-mediat-
ed oxidation in the presence of a base (under basic condi-
tions) and the reaction is widely used in organic synthesis,[20]
it is noteworthy that the reaction mechanism is still unclear.
In addition, it has been reported that thiones can be oxi-
peroxide, hydroxyl radicals, superoxide, CH3COOOH, Fe3+
,
Cu2+, Co2+, Zn2+, NO2 , NO3 , HCO3 , Val, cytosine, thy-
mine, adenine, glucose, GSH, and vitamin C (Figure 1C).
Taken together, these results indicate that compound 2 is a
new fluorescence turn-on probe for HOCl. As the aim of
this work was to construct new fluorescent HOCl probes, we
decided to investigate the signaling mechanism of probe 2
for sensing HOCl. Obviously, the identity of the fluorescent
product arising from treatment of probe 2 with HOCl is
highly valuable for the fluorescence sensing mechanism.
À
À
À
Thus, rhodamine-thiosemicarbazide
2 was reacted with
NaOCl at room temperature, and the reaction product was
isolated and purified through a silica gel column (CH2Cl2/
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Fluorescent Detection of Hypochlorous Acid
FULL PAPER
dized to sulfonic acid,[21] and NaOCl can transfer N1-acyl-N3-
cyanoguanidines to 1,3,4-oxadiazoles through a carbodi-
and gradually increased in intensity, implying that intramo-
lecular FRET occurred due to the HOCl-induced formation
of rhodamine-1,3,4-oxadiazole. Notably, 1b exhibited an
outstanding emission spectral feature with the two emission
peaks well resolved (a large separation of 121 nm), which
can ensure the highly accurate determination of the emis-
sion intensities and ratios. The changes in the absorption
spectra (Figure S6, Supporting Information) are in good
agreement with the variations in the fluorescence spectra.
Consistently, the emission color of the probe showed a dra-
matic change from blue to orange–red upon addition of
NaOCl (Figure S7, Supporting Information). In addition,
like probe 1b, probe 1a displayed the drastic spectral varia-
tions in the presence of HOCl (Figure S8, Supporting Infor-
mation). The ratiometric probe 1b showed a good linearity
between the ratios (I594/I473) and the NaOCl concentration in
the range 1.0ꢂ10À7 to 1.0ꢂ10À4 m (Figure 2B) with a detec-
tion limit of 5.2ꢂ10À8 m (S/N=3), indicating that probe 1b is
potentially useful for the quantitative determination of
HOCl. The free probe is stable over a wide span of pH from
3.0–11.0 (Figure S10A, Supporting Information). The ratio-
metric response of the probe to HOCl is pH-dependent,
with the maximal ratio signal at pH 6.0. As the pKa of
HOCl is 7.6, we reasoned that the probe senses HOCl in-
stead of OClÀ, in agreement with the previous reports.[14b,c]
The time-course studies indicate that the probe has a very
rapid ratiometric response to HOCl (Figure S11A, Support-
ing Information). Furthermore, the properties of probe 1a
are quite similar to those of 1b (Figures S9, S10B, and
S11B, Supporting Information). As shown in Figure 3 and
ACHTUNGTRENNUNG
imide intermediate.[22] On the basis of these literature re-
ports and our findings from the studies of the control com-
pound 8, a possible reaction mechanism is proposed as
shown in Scheme S7 in the Supporting Information. Al-
though the detailed reaction mechanism of the HOCl-pro-
moted transformation of thiosemicarbazides to oxadiazoles
remains to be investigated in the future, the turn-on sensing
mechanism of rhodamine-thiosemicarbazide 2 is evident.
The free rhoACHTUNGTRENNUNGdamine-thiosemicarbazide 2 is non-fluorescent
as it exists as the ring-closed form of the rhodamine. How-
ever, upon treatment with HOCl, rhodamine-thiosemicarb-
ACHTUNGTRENNUNG
the rhoACHTUNGTRENNUNGdamine. The structure of rhodamine-1,3,4-oxadiazole
1
7 is well corroborated by the H NMR, 13C NMR, ESI-MS,
HRMS, and absorption and emission spectra (Figures S2–S4
in the Supporting Information).
Ratiometric responses of probes 1a and 1b to HOCl: As
shown in Figure 2A, upon excitation at 414 nm, the free ra-
tiometric probe 1b showed the featured emission of the cou-
marin moiety at 473 nm but no characteristic emission of
the rhodamine moiety at 594 nm, indicating that there is no
intramolecular FRET in the free probe, as the rhodamine
component has the ring-closed spirolactam structure. How-
ever, upon addition of NaOCl, the coumarin emission at
473 nm gradually decreased, and a new emission band corre-
sponding to the rhodamine emission at 594 nm appeared
Figure 3. Emission intensity ratio (I594/I473) of 1b (5 mm) in the presence of
various biologically relevant analytes (5 equiv). 1) blank; 2) hydrogen
peroxide; 3) hydroxyl radical; 4) superoxide; 5) HOCl; 6) CH3COOOH;
À
7) Fe3+; 8) Cu2+; 9) Co2+; 10) Zn2+; 11) NO2À; 12) NO3À; 13) HCO3
;
14) Val; 15) cytosine; 16) thymine; 17) adenine; 18) glucose; 19) GSH;
20) vitamin C. The data were collected from the emission spectra excited
at 414 nm.
Figure S12 of the Supporting Information, 1b and 1a exhib-
ited high selectivity for HOCl over other biologically rele-
vant species represented by hydrogen peroxide, hydroxyl
radicals, superoxide, CH3COOOH, Fe3+, Cu2+, Co2+, Zn2+
,
À
À
À
NO2 , NO3 , HCO3 , Val, cytosine, thymine, adenine, glu-
cose, GSH, and vitamin C, indicating that these ratiometric
fluorescent probes are potentially useful for biological appli-
cations.
Figure 2. a) Fluorescence titration spectra of 1b (5 mm) in PBS/DMF
(pH 7.4, 1:1) upon gradual addition of NaOCl (0–30 equiv). Excitation at
414 nm. b) The emission ratio (I594/I473) of probe 1b (5 mm) to various con-
centrations of NaOCl (1.0ꢂ10À7–1.0ꢂ10À4 m). Excitation was provided at
414 nm, and the ratios of emission intensities at 594 and 473 nm were
measured.
Living cell imaging studies: In order to be useful as ratio-
metric imaging agents, ratiometric fluorescent probes should
have low cytotoxicity. Thus, we investigated the potential
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W. Lin et al.
toxicities of 1a and 1b against a representative cell line:
RAW264.7 macrophages. The living cells were incubated
with various concentrations (5–200 mm) of the new ratiomet-
ric probes for 24 h, and then the cell viability was deter-
mined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) assays;[23] the results in-
dicated that the ratiometric probes do not exhibit marked
cytotoxicity at concentrations below 100 mm (Figure S13,
Supporting Information).
nously produced HOCl. When stimulated by lipopolysac-
charide (LPS) and 12-myristate-13-acetate (PMA), macro-
phages may produce endogenous HOCl.[24,25] The RAW264.7
macrophage cells loaded with only 1a (5 mm) displayed a
strong blue emission (Figure 5B) and a slight red emission
To demonstrate the capability of these novel probes for
HOCl ratiometric fluorescence imaging in living cells, 1a
and 1b were incubated with three different types of cell
lines: living RAW 264.7 macrophages, Bel 7702 cells, and
HeLa cells. As exhibited in Figure S14 of the Supporting In-
formation, the living RAW 264.7 macrophages incubated
with 1a (5 mm) for 0.5 h at 378C provided a strong blue fluo-
rescence in the coumarin emission window (Figure S14B,
Supporting Information), but almost no red fluorescence in
the rhodamine emission window (Figure S14C, Supporting
Information). By contrast, the living RAW 264.7 macro-
phages treated with 1a and then with NaOCl gave a marked
decrease in the blue emission (Figure S14E, Supporting In-
formation) and a significant increase in the red emission
(Figure S14F, Supporting Information), consistent with the
HOCl-induced ratiometric fluorescent response. A similar
ratiometric response was observed when 1b was incubated
with live HeLa cells in the presence of NaOCl (Figure S15,
Supporting Information). Furthermore, Bel 7702 cells incu-
bated with only 1b (5 mm) for 30 min gave an average emis-
sion ratio value (I605Æ75/I450Æ35) of 0.09 (Figures 4A and C).
By contrast, treatment of 1b-loaded cells with NaOCl
(10 mm) for 30 min elicited a significant increase in the emis-
sion ratio (I605Æ75/I450Æ35) to about 0.59 (Figures 4B and C),
in good agreement with the HOCl-mediated transformation
of thiosemicarbazides to oxadiazoles. Thus, these data estab-
lish that 1a and 1b are cell-membrane permeable and suita-
ble for the ratiometric imaging of HOCl in the living cells.
The promising results of the above ratiometric imaging of
externally added HOCl in living cells encourage us to evalu-
ate the feasibility of 1a further for the detection of endoge-
Figure 5. Images of endogenously produced HOCl in living RAW 264.7
macrophages treated with probe 1a. a) DIC image of the RAW 264.7
macrophages incubated with only 1a (5 mm) for 60 min; b) fluorescence
image of (a) from blue channel; c) fluorescence image of (a) from red
channel; d) fluorescence image of RAW 264.7 macrophages treated with
LPS (1 mgmLÀ1) for 12 h, and then further co-incubated with PMA
(1 mgmLÀ1) and 1a (5 mm) for 60 min; e) fluorescence image of (d) from
blue channel; f) fluorescence image of (d) from red channel. For a color
version, see Figure S18 in the Supporting Information.
(Figure 5C). However, after the macrophage cells were in-
cubated with LPS (1 mgmLÀ1) for 12 h, and then further co-
incubated with PMA (1 mgmLÀ1) and 1a (5 mm) for 60 min,
a marked decrease in the blue emission (Figure 5E) and a
dramatic enhancement in the red emission (Figure 5F) were
observed. These data indicate that 1a is capable of the ratio-
metric fluorescent imaging of endogenously produced
HOCl. To the best of our knowledge, this represents the
first report of the ratiometric fluorescent imaging of endoge-
nously produced HOCl in macrophage cells.
Conclusion
In summary, we have described the HOCl-promoted cycliza-
tion of rhodamine-thiosemicarbazides to rhodamine-oxadia-
zoles, which was then exploited as a novel design strategy
for the development of a new fluorescence turn-on HOCl
probe, 2. On the basis of the FRET signaling mechanism, 2
was further transformed into 1a and 1b, which, to our best
knowledge, represent the first paradigm of FRET-based ra-
tiometric HOCl probes. The outstanding features of 1a and
1b include the well-resolved emission peaks, high sensitivity,
high selectivity, good functionality at physiological pH,
rapid response, low cytotoxicity, and good cell-membrane
permeability. Furthermore, these excellent attributes enable
us to demonstrate, for the first time, the ratiometric imaging
of endogenously produced HOCl in living cells by using
these novel ratiometric probes. We expect that 1a and 1b
Figure 4. Confocal ratio images (I605Æ75/I450Æ35) of HOCl in Bel 7702 cells
with lex =408 nm. A) ratio image (I605Æ75/I450Æ35) of the cells incubated
with 1b (5 mm) only; B) ratio image (I605Æ75/I450Æ35) of 1b-loaded cells
after treatment with NaOCl (10 mm). C) Average ratios (I605Æ75/I450Æ35) of
the cells incubated with 1b (5 mm) only (a) and 1b-loaded cells after
treatment with 10 mm NaOCl (b). For a color version, see Figure S17 in
the Supporting Information.
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Fluorescent Detection of Hypochlorous Acid
FULL PAPER
were acquired using an Olympus inverted fluorescence microscope
equipped with a cooled CCD camera (Figure S14, Supporting Informa-
tion). For the detection of endogenously produced HOCl, the living
RAW 264.7 macrophages were treated with LPS (1 mgmLÀ1) for 12 h,
and then further co-incubated with PMA (1 mgmLÀ1) and 1a (5 mm) for
60 min. Prior to imaging, the cells were washed three times with PBS
(1 mL), and the fluorescence images were acquired using an Olympus
fluorescence microscope equipped with a cooled CCD camera (Figure 5).
will be useful molecular tools for studies of HOCl biology.
In addition, the HOCl-promoted cyclization of rhodamine-
thiosemicarbazides to rhodamine-oxadiazoles should be
widely applicable for the development of a variety of fluo-
rescent HOCl probes.
Bel 7702 cells confocal fluorescence imaging using probe 1b: Bel 7702
cells were cultured in DMEM supplemented with 10% FBS in an atmos-
phere of 5% CO2 and 95% air at 378C. One day before imaging, the
cells were passed and plated on 18 mm glass coverslips coated with poly-
l-lysine (50 mgmLÀ1, Sigma). After 24 h, the cells were incubated with
5 mm 1b (in the culture medium containing 1% DMF) for 0.5 h at 378C,
and then washed three times with PBS. After incubation with 10 mm
NaOCl for another 0.5 h at 378C, the cells were rinsed three times with
PBS. The glass coverslips were attached to slides before imaging. Confo-
cal fluorescence images of intracellular NaOCl in the cells were recorded
using a Nikon 90i C1 Si laser scanning confocal microscope (Figure 4).
The excitation wavelength of the laser was 408 nm. Emissions were cen-
tered at 450Æ35 nm and 605Æ75 nm (double channels).
Experimental Section
Synthesis of compound 7: A solution of NaOCl (18.8 mg, 0.25 mmol) in
H2O (1 mL) was added slowly to a solution of 2 (30.0 mg, 0.05 mmol) in
C2H5OH/H2O (9:1, 40 mL). Subsequently, the reaction mixture was
stirred at room temperature for 2 h, and then the solvents were removed
under reduced pressure. The resulting residue was purified on a silica gel
column (CH2Cl2/C2H5OH=10:1) to afford compound 7 as a dark purple
solid (14.1 mg, isolated yield: 50.6%). M.p. 166–1688C; 1H NMR
(400 Hz, CDCl3): d=1.30 (t, 12H), 3.53–3.58 (q, 8H), 6.72 (d, J=2.0 Hz,
2H), 6.76–6.78 (d, J=9.6 Hz, 2H), 6.85–6.89 (t, 1H), 7.11–7.13 (d, J=
9.2 Hz, 2H), 7.14–7.18 (t, 2H), 7.24–7.26 (d, J=7.6 Hz, 1H), 7.60–7.64 (t,
1H), 7.67–7.70 (d, J=8.0 Hz, 3H), 8.33–8.35 (d, J=8.0 Hz, 1H),
10.50 ppm (bs, 1H); 13C NMR (100 Hz, CDCl3): d=12.64, 46.04, 96.45,
113.92, 114.15, 117.97, 121.80, 123.62, 128.63, 129.16, 129.89, 129.99,
130.22, 130.75, 131.50, 138.62, 155.51, 157.76, 158.11 ppm; ESI-MS m/z
558.2 [M]+; HRMS (EI) m/z calcd for C35H36O2N5 ([M]): 558.2864;
Acknowledgements
À
found: 558.2844; m/z calcd for C35H35O2N5 ([M H]): 557.2785; found:
557.2784.
This research was supported by NSFC (20872032, 20972044, 21172063),
NCET (08-0175), and the Doctoral Fund of Chinese Ministry of Educa-
tion (20100161110008).
Synthesis of 1a: Phenylisothiocyanate (12.7 mg, 0.09 mmol) was added
dropwise to a solution of compound 5a (50.4 mg, 0.06 mmol) in DMF
(1 mL) with stirring. Subsequently, the reaction mixture was heated to
508C and stirred at that temperature for 12 h. After being cooled to
room temperature, the solvent was removed under reduced pressure. The
resulting residue was purified on a silica gel column (CH2Cl2/C2H5OH=
50:1) to give compound 1a as a yellow powder (45.3 mg, yield: 77.6%).
M.p. 220–2248C; 1H NMR (400 Hz, CDCl3): d=1.12–1.16 (t, 12H), 1.20–
1.23 (t, 6H), 3.31–3.90 (20H), 6.30 (s, 2H), 6.46–6.51 (s, 5H), 6.60–6.62
(d, J=8.4 Hz, 1H), 7.02–7.04 (d, J=7.6 Hz, 2H), 7.08–7.10 (d, J=7.6 Hz,
1H), 7.14–7.18 (2H), 7.24 (s, 1H), 7.30–7.34 (m, 2H), 7.70–7.72 (2H),
7.92 (s, 1H), 8.05 ppm (s, 1H); 13C NMR (125 Hz, CDCl3): d=12.19,
12.32, 44.17, 44.75, 67.17, 96.55, 98.01, 103.11, 107.48, 108.21, 109.32,
114.94, 122.19, 124.91, 125.16, 125.79, 127.55, 127.99, 129.18, 129.90,
132.98, 136.11, 137.43, 145.83, 149.15, 151.37, 151.67, 153.97, 157.12,
159.05, 165.13, 165.99, 168.89, 182.20 ppm; ESI-MS m/z 947.3 [M+H]+;
HRMS (ESI) m/z calcd for C54H59N8O6S ([M+H]+): 947.4273; found:
947.4258.
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Synthesis of 1b: 1b was prepared from pure compound 5b according to
the above method for the synthesis of 1a. M.p. 175–1788C; 1H NMR
(500 Hz, CDCl3): d=1.16 (t, 12H), 1.21–1.23 (t, 6H), 3.34–3.80 (20H),
6.29 (s, 2H), 6.46 (s, 5H), 6.59–6.61 (dd, J=9.0, 2.0 Hz, 1H), 7.03–7.05
(d, J=8.0 Hz, 2H), 7.10–7.13 (t, 2H), 7.17–7.20 (t, 2H), 7.30 (d, J=
2.0 Hz, 2H), 7.50 (s, 1H), 7.64 (s, 1H), 7.87 (s, 1H), 8.08 ppm (s, 1H);
13C NMR (125 Hz, CDCl3): d=12.40, 12.53, 44.46, 44.99, 67.46, 96.92,
107.72, 108.63, 109.53, 113.94, 115.24, 125.08, 126.12, 127.38, 127.78,
128.33, 130.03, 130.37, 137.60, 150.56, 151.98, 154.29, 157.42, 159.18,
166.16, 168.99, 182.66 ppm; ESI-MS m/z 947.1 [M+H]+; HRMS (ESI)
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RAW 264.7 macrophage imaging using probe 1a: Raw 264.7 murine mac-
rophages were obtained from Xiangya hospital and cultured in Dulbec-
coꢀs Modified Eagle Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) in an atmosphere of 5% CO2 and 95% air at 378C.
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washed with phosphate-buffered saline (PBS) buffer solution. Subse-
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378C, and then washed three times with PBS. After incubation with
10 mm NaOCl for another 30 min at 378C, the Raw 264.7 murine macro-
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Received: June 22, 2011
Revised: August 31, 2011
Published online: January 23, 2012
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