O-fluorination of aromatic azides yields improved azido-based fluorescent probes for hydrogen sulfide: Synthesis, spectra, and bioimaging

Article abstract of DOI:10.1002/asia.201402808

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple biological functions. To visualize the endogenous in situ production of H₂S in real time, new coumarin- and boron-dipyrromethene- based fluorescent turn-on probes were developed for fast sensing of H₂S in aqueous buffer and in living cells. Introduction of a fluoro group in the ortho position of the aromatic azide can lead to a greater than twofold increase in the rate of reaction with H₂S. On the basis of o-fluorinated aromatic azides, fluorescent probes with high sensitivity and selectivity toward H₂S over other biologically relevant species were designed and synthesized. The probes can be used to in situ to visualize exogenous H₂S and d-cysteine-dependent endogenously produced H₂S in living cells, which makes them promising tools for potential applications in H₂S biology.

Full text of DOI:10.1002/asia.201402808

FULL PAPER
DOI: 10.1002/asia.201402808
o-Fluorination of Aromatic Azides Yields Improved Azido-Based
Fluorescent Probes for Hydrogen Sulfide: Synthesis, Spectra, and Bioimaging
[
b, c]
[b, c]
[b, c]
[b, c]
[a]
Chao Wei,
Runyu Wang,
Lv Wei,
Longhuai Cheng,
Zhifei Li,
[
b, c]
[a, c]
Zhen Xi,*
and Long Yi*
Abstract: Hydrogen sulfide (H S) is an
ortho position of the aromatic azide
can lead to a greater than twofold in-
sensitivity and selectivity toward H2S
over other biologically relevant species
were designed and synthesized. The
probes can be used to in situ to visual-
2
endogenously produced gaseous signal-
ing molecule with multiple biological
functions. To visualize the endogenous
crease in the rate of reaction with H S.
2
On the basis of o-fluorinated aromatic
in situ production of H S in real time,
azides, fluorescent probes with high
ize exogenous H S and d-cysteine-de-
2
2
new coumarin- and boron-dipyrrome-
thene-based fluorescent turn-on probes
were developed for fast sensing of H2S
in aqueous buffer and in living cells. In-
troduction of a fluoro group in the
pendent endogenously produced H2S
in living cells, which makes them prom-
ising tools for potential applications in
Keywords: bioimaging
pigments fluorescent probes
FRET · hydrogen sulfide
·
dyes/
·
·
H S biology.
2
Introduction
H S has been recognized to be linked to numerous physio-
logical and pathological processes, many of its underlying
molecular events remain unknown. Therefore, research to
2
Hydrogen sulfide (H S) is an important endogenous signal-
2
ing molecule (gasotransmitter) along with nitric oxide (NO)
develop efficient methods for fast detection of H S in living
biological systems is of significant value.
2
[1]
and carbon monoxide (CO). In vivo, H S can be enzymati-
2
cally generated in many organs (heart, liver, kidney, brain,
ileum, uterus, etc.) and tissues (connective tissues, adipose
Traditionally, the major methods for H S detection are
2
colorimetry, electrochemical assay, gas chromatography, and
[2]
[5]
tissues, etc.). The production of endogenous H S and exog-
sulfide precipitation. However, these methods are destruc-
2
enous administration of H S have been demonstrated to
tive and require tedious preparation sequences. Fluores-
cence-based methods have recently emerged as a highly de-
sirable and sensitive approach for in situ and real-time visu-
2
exert protective effects in many pathologies, including pres-
ervation of mitochondrial function, relaxation of vascular
smooth muscles, inhibition of apoptosis, intervention of neu-
ronal transmission, regulation of inflammation, and stimula-
[6–11]
alization of H S in living biological systems.
These
2
probes, which are largely based on specific H S-induced re-
2
[3]
[6–8]
tion of angiogenesis. Studies have shown that the endoge-
actions, include reduction-based probes,
probes based on
[9]
nous concentration of H S is correlated with numerous dis-
precipitation of metal sulfides,
and nucleophile-based
2
[10]
eases, including the symptoms of Alzheimerꢀs disease,
probes. Previously, we developed several H S probes that
2
[4]
Downꢀs syndrome, diabetes, and liver cirrhosis. Although
can be used to in situ visualize the chiral-sensitive cysteine-
[
11]
dependent H S production in living cells.
Despite the
2
[
6–11]
great success of these fluorescent probes for H S biology,
2
most of them showed
a delayed response (typically
[
a] Z. Li, Prof. Dr. L. Yi
State Key Laboratory of Organic-Inorganic Composites
Beijing University of Chemical Technology (BUCT)
1
5 Beisanhuan East Road, Beijing 100029 (P. R. China)
E-mail: yilong@mail.buct.edu.cn
[
b] C. Wei, R. Wang, Dr. L. Wei, L. Cheng, Prof. Dr. Z. Xi
State Key Laboratory of Elemento-Organic Chemistry and Depart-
ment of Chemical Biology
National Pesticide Engineering Research Center (Tianjin)
Nankai University, 94 Weijin Road, Tianjin 300071 (P. R. China)
E-mail: zhenxi@nankai.edu.cn
[
c] C. Wei, R. Wang, Dr. L. Wei, L. Cheng, Prof. Dr. Z. Xi,
Prof. Dr. L. Yi
Collaborative Innovation Center of Chemical Science and Engineer-
ing (Tianjin)
Scheme 1. Schematic drawing showing a) the fluorescent turn-on mecha-
nism based on the reduction of azide to amine with H S and b) the reac-
tivity of azido-based compounds toward H S.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201402808.
2
2
Chem. Asian J. 2014, 00, 0 – 0
1
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

www.chemasianj.org
Long Yi, Zhen Xi et al.
>
20 min) toward H S. Herein, we intended to develop fast-
NaOH, and the resulting free amine 4 was transformed into
azido-based coumarin 5 by means of a Sandmeyer reaction.
2
response H S probes based on the reduction of aromatic
azides by H S. To this end, we rationally designed azido-
based H S fluorescent probes with improved reaction rate
by introduction of a fluoro group in the ortho position of ar-
omatic azides (Scheme 1). The developed coumarin- and
boron-dipyrromethene (BODIPY)-based fluorescent probes
2
[6a]
To improve the cell permeability of the probe, a piperazi-
dine unit was coupled with 5 to provide 7, which can be fur-
ther modified through coupling reactions with the piperazi-
dine linker. The structures of probes 7a and 7b were con-
2
2
1
19
13
firmed by H, F, and C NMR spectroscopy and high-reso-
lution mass spectrometry (HRMS).
are highly selective and show fast response to H S in buffer
2
and in living cells.
Spectroscopic Characterization of Coumarin-Based Probes
Results and Discussion
The fluorescence spectra of probes 7a, 7b and their reaction
with H S (Na S was used as an equivalent) were investigat-
2
2
Rational Design and Synthesis of Coumarin-Based Probes
ed. As shown in Figure 1, both 7a and 7b did not display
H S probes functioning through the reduction of azido
noticeable fluorescence in phosphate-buffered saline (PBS,
2
[6,7]
groups to amines are known in the literature;
however,
20 mm, pH 7.4). On treatment of the probes with H S, more
2
[6a]
most of them, based on aromatic azides, do not respond
than 100-fold fluorescence enhancement was obtained for
both 7a and 7b, which implies that azide moieties were re-
[7]
fast enough for real-time H S detection. Sulfonyl azide can
2
react with H S very fast but suffers from relatively low se-
duced by H S to produce strongly fluorescent compound 8
2
2
[6b]
lectivity in living biological systems.
Therefore, we pro-
(Scheme 2). The mass spectrum also indicated that the reac-
+
posed that electron-withdrawing groups in the ortho position
tion produced 8 (ESI-MS [M+H] : found 306.19; calcd
of the aromatic azide could improve its reaction rate with
306.12).
H S (Scheme 1).
To obtain the kinetic parameters, the fluorescence signal
at 450 nm was plotted as a function of time (Figure 1c). This
revealed that the fluorescence intensity reached the steady
state after about 40 and 100 min at 258C for 7b and 7a, re-
2
Firstly, we chose coumarin as model fluorophore because
of its good quantum yield, water solubility, and easy prepa-
ration. As shown in Scheme 2, treatment of commercially
available 3-aminophenol (1a) and 3-amino-4-fluorophenol
spectively (7a: F=0.002, F=0.72 after reaction with H S;
2
(
1b) with ethyl chloroformate led to the formation of carba-
7b: F=0.012, F=0.75 after reaction with H S). The
2
mate 2. Coumarin 3 was formed by a Pechmann reaction of
carbamate 2 with diethyl-1,3-acetone dicarboxylate in 70%
pseudo-first-order rate constant k was found to be 10.4ꢂ
obs
À4
À4 À1
10 and 4.82ꢂ10
s
for 7b and 7a, respectively, by fitting
[12]
H SO .
Compound 3 was hydrolyzed in refluxing 0.1m
the fluorescence intensity data with a single-exponential
function. The reaction rates k2
2
4
under the test conditions were
calculated
as
4.16
and
À1 À1
1
.93m s
for 7b and 7a, re-
spectively. Thus, introduction
of a fluoro group in the ortho
position of the aromatic azide
led to an approximately 2.2-
fold improvement of the reac-
tion rate with H S. Since the
2
pH in mitochondria normally
lies in the weakly alkaline
[13]
range (ca. 8.0),
gated the reaction kinetics of
b with H S in a pH 8.0 buffer.
we investi-
7
2
The reaction half-time of
.35 min (Figure 1d) implies
8
that the probe could be used
for fast H S detection in mito-
2
[11c]
chondria in living cells.
To
the best of our knowledge, this
is the first investigation of the
reactivity of o-fluorinated aro-
matic azides toward H S.
2
In solution, probe 7b exhib-
ited a noticeable absorbance at
Scheme 2. Synthesis of fluorescent probes 7a and 7b for H
Chem. Asian J. 2014, 00, 0 – 0
2
S detection.
&
&
2
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

www.chemasianj.org
Long Yi, Zhen Xi et al.
A major challenge of H S detection in biological systems
2
is to develop highly selective probes that exhibit distinct re-
sponses to H S over other cellular molecules. To investigate
2
the selectivity of probe 7b, various biologically relevant spe-
cies were incubated with probe 7b in PBS and their fluores-
cence responses measured (Figure 3). These biologically rel-
Figure 1. Time-dependent fluorescence spectra of a) 7a (2.5 mm) and
b) 7b (2.5 mm) on reaction with H S (250 mm) at 258C. Excitation at
50 nm. c) The relationship between fluorescence intensity at 450 nm and
reaction time of 7 (2.5 mm) on treatment with H S (250 mm) at 258C.
d) The relationship between fluorescence intensity at 450 nm and reac-
tion time of probe 7b (2.5 mm) on treatment with H S (250 mm) at 378C
Figure 3. Relative fluorescent response of probe 7b (2.5 mm) to various
biologically relevant species (1 mm) and GSH (5 mm) in PBS (pH 7.4) for
2
3
30 min. 1, probe 7b alone; 2, MgSO
NaOAc; 7, NaN ; 8, NaHCO ; 9, Na
Zn(OTf) ; 13, NaNO ; 14, ascorbic acid; 15, H
nol; 17, GSH; 18, Cys; 19, GSH+Na S; 20, Cys+Na
4
; 3, CaCl
2
; 4, NaF; 5, NaI; 6,
SO ; 11, NaHSO ; 12,
; 16, 2-mercaptoetha-
S; 21, Na S. F/F is
2
3
3
2
C
2
O
4
; 10, Na
2
3
3
A
H
U
G
R
N
U
G
2
2
2 2
O
2
and pH 8.0. The solid line represents the best fit to a single-exponential
function.
2
2
2
0
the emission intensity ratio of the test sample to probe 7b alone (excita-
tion at 350 nm, emission at 450 nm).
335 nm, which shifted to 350 nm after treatment with H2S
(
Supporting Information, Figure S1). A well-defined isosbes-
evant species included reactive sulfur species (glutathione
2À
tic point at 341 nm was observed, indicative of a clean chem-
ical reaction between 7b and H S.
(GSH), cysteine (Cys), HSO3 ), reactive oxygen species
À
(H O ), reactive nitrogen species (NO ), other anions
2
2
2
2
2+
À
À
2+
2+
We investigated the dependence of the fluorescence signal
(HCO , I , etc.), and cations (Zn , Ca , Mg ). The fluo-
3
of probe 7b on H S concentration (Figure 2). As expected,
rescence increases of the test molecules, however, were far
2
a strong emission peak at 450 nm could be detected when
the reaction mixture was excited at 350 nm. Further data
analysis revealed a good linear relationship (r=0.99) be-
tween the fluorescence signal at 450 nm and the concentra-
lower than that of H S. Small-molecules thiols such as GSH
2
and Cys in concentrations of 5 and 1 mm, respectively, did
not trigger significant fluorescence enhancement. Thus, ac-
cording to fluorescence spectra, probe 7b is highly selective
tion of H S (0–150 mm). The detection limit was determined
to be 0.61 mm by setting the signal-to-noise ratio to 3:1.
toward H S over other biologically relevant thiols and
anions.
2
2
[11]
The results demonstrated that probe 7b could react with
H S both qualitatively and quantitatively.
2
Bioimaging Based on 7b
To demonstrate the biological applicability of probe 7b in
vivo, we investigated whether it can be used to image H S in
2
living cells. HEK293 cells were treated with probe 7b (5 mm)
for 30 min and then washed with PBS to remove excess 7b.
The 7b-loaded cells were incubated with different concen-
trations of Na S (50–250 mm) for another 30 min. The cells
2
were subsequently imaged by confocal fluorescence micros-
copy (Supporting Information Figure S2). The addition of
both 7b and Na S resulted in brighter fluorescence than the
2
addition of higher concentration of Na S. These results
2
clearly indicated that probe 7b can be used for imaging H2S
in living cells in a concentration-dependent fashion.
Figure 2. Fluorescence spectra of 7b (2.5 mm) on reaction with different
concentrations of H
rescence intensity at 450 nm and H
2
S (0–150 mm). The linear relationship between fluo-
S concentration is shown in the inset.
Synthesis and Characterization of BODIPY-Based Probes
2
Error bars are plus/minus standard deviation. The solid linear line repre-
sents the best fit. The fluorescence intensity was acquired at 378C after
The emission of the coumarin fluorophore in probe 7b is
incubation of the probe with H
2
S for 30 min (excitation at 350 nm).
centered in the blue region. We aimed to use fluorescence
Chem. Asian J. 2014, 00, 0 – 0
3
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

www.chemasianj.org
Long Yi, Zhen Xi et al.
1
3
19
C, and F NMR spectroscopy
19
and HRMS. The
spectrum of 9b shows two
peaks at and
À146.8 ppm (see Supporting
Information), which are consis-
tent with two kinds of F atoms
in 9b. The fluorescence spectra
of probes 9a, 9b and their re-
F NMR
À129.5
action with H S were investi-
2
gated. As shown in Figure 4,
both 9a and 9b show weak
fluorescence of the BODIPY
fluorophore in PBS buffer
(
pH 7.4, containing 10% DMF
Scheme 3. a) Schematic showing a FRET-based turn-on mechanism based on the reduction of azide (R) to
amine by H
2
S and b) the chemical response of FRET-based molecules toward H
2
S.
as cosolvent). On treatment of
the probes with H S, significant
2
fluorescence enhancement of
resonance energy transfer (FRET) to achieve longer-wave-
both coumarin and BODIPY emission was observed for
both 9a and 9b (9a: F=0.040, F=0.21 after reaction with
H S; 9b: F=0.025, F=0.20 after reaction with H S). The
length emission of our H S probes. As shown in Scheme 3a,
2
a general strategy for H S probes could be based on a two-
2
2
2
fluorophore cassette comprised of a reaction-site-containing
FRET donor (here azido-containing coumarin) and a FRET
acceptor linked by a rigid spacer. FRET occurs after remov-
ing the reaction site in the FRET donor, which results in an
increase in fluorescence of the FRET acceptor. We em-
ployed BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-inda-
cene) as FRET acceptor (Supporting Information, Fig-
ure S4), because BODIPY dyes are photostable and have
increase in BODIPY fluorescence should be due to FRET
from the coumarin donor. The kinetics of the reaction of
H S with 9 is similar to that of 7 (Figure 4c), that is, o-fluori-
2
nation of aromatic azides can improve the reaction rate of
azide reduction by H S approximately twofold. The reaction
2
half-time of 9b and H S at 378C at pH 8.0 can reach 7.5 min
2
(Figure 4d), which implies that the probe has potential ap-
plications in fast sensing of H S in biological systems.
2
[14]
high quantum yields.
To investigate the selectivity of probes 9, various biologi-
cally relevant species were incubated with 9 in PBS buffer
(pH 7.4, containing 10% DMF) and their fluorescence re-
sponses measured (Figure 5). The test compounds including
biological thiols did not trigger significant fluorescence en-
hancement of the BODIPY fluorophore for both 9a and 9b.
Thus, the FRET-based probes 9 are highly selective toward
Probes 9a and 9b (Scheme 3b) were synthesized by cou-
1
pling reaction of 7 and BODIPY and characterized by H,
H S according to fluorescence spectra and thus suitable for
2
biological applications.
We also investigated the fluorescence response of probe
9
b to H S at different pH values (Supporting Information
2
Figure S6). This indicated that the probe can function over
a wide range of pH from 6.5 to 8.5.
Bioimaging with a BODIPY-Based Probe
Probe 9b was employed to stain living cells for bioimaging.
The probe-treated cells were incubated with Na S (0–
2
2
50 mm) for 30 min. Compared with cells treated only with
the probe, the addition of both probe and Na S resulted in
2
significant fluorescence increase of the green channel
(
Figure 6). Furthermore, the good morphologies of cells in
Figure 4. Time-dependent fluorescence spectra of a) 9a (5 mm) and b) 9b
bright-field transmission images suggested satisfactory bio-
compatibility of the probe. Therefore, the new FRET-based
(
5 mm) on reaction with H
relationship between fluorescence intensity at 457 nm and reaction time
of probes 9 (5 mm) on treatment with H S (1 mm) at 258C. d) The rela-
tionship between fluorescence intensity at 508 nm and reaction time of
probe 9b (5 mm) on treatment with H S (1 mm) at 378C and pH 8.0. The
2
S (1 mm) at 258C. Excitation at 360 nm. c) The
probe 9b can be used to detect intracellular H S.
2
2
Our further goal was to use BODIPY-based probe 9b to
detect endogenously produced H S from living cells. Our
2
2
solid line represents the best fit to a single-exponential function.
previous work indicated that d-Cys can serve as a precursor
&
&
Chem. Asian J. 2014, 00, 0 – 0
4
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

www.chemasianj.org
Long Yi, Zhen Xi et al.
Figure 7. Confocal microscopy images of d-Cys-dependent endogenous
S production in living cells with probe 9b. HEK293 cells were incubat-
H
2
ed with a) 9b (2 mm) for 30 min, b) 9b (2 mm) and d-Cys (50 mm) for
À1
3
0 min, and c) dl-PPG (50 mgmL ) for 20 min and then 9b (2 mm) and
d-Cys (50 mm) for 30 min. The bright-field images are shown below. Scale
bar: 50 mm.
Figure 5. Relative fluorescent responses of probes 9 (5 mm) to various bio-
logically relevant species (1 mm) and GSH (5 mm) in PBS (pH 7.4, con-
taining 10% DMF) for 30 min. a) Test with 9a. b) Test with 9b. 1, probe
production (Figure 7b,c), which should be due to enzyme in-
activation by the inhibitor.
9
alone; 2, MgSO
NaHCO ; 9, Na
2 2
NaNO ; 14, ascorbic acid; 15, H O
4
; 3, CaCl
2
; 4, NaF; 5, NaI; 6, NaOAc; 7, NaN
SO ; 11, NaHSO ; 12, Zn(OTf) ; 13,
; 16, 2-mercaptoethanol; 17, GSH; 18,
S; 20, Cys+Na S; 21, Na S. Excitation at 360 nm,
3
; 8,
3
2
C
2
O
4
; 10, Na
2
3
3
A
H
U
T
E
U
G
2
2
Cys; 19, GSH+Na
2
2
2
emission at 508 nm.
Conclusions
To visualize biological H S in living cells, we have developed
2
new coumarin- and BODIPY-based fluorescent probes for
H S. Introduction of a fluoro group in the ortho position of
2
the aromatic azide improved the reaction rate with H2S
more than twofold. This strategy should enable relatively
facile tuning of the H S reaction rate by means of probe
2
design. For example, one can add two or more fluoro sub-
stituents to the aromatic azide to achieve faster sensing of
H S than with the single fluoro group in this work. We be-
2
lieve that extension of this work by such a strategy could
produce fast azido-based fluorescent probes for real-time
H S bioimaging in living cells. On the other hand, the
2
probes developed in this work are highly sensitive and selec-
Figure 6. Confocal microscopy images of exogenous H
2
S in living cells
tive toward H S over other biologically relevant species.
2
with probe 9b. HEK293 cells were incubated with 9b (2 mm) for 30 min
Confocal imaging indicated that the probes could monitor
(
a) and with 9b (2 mm) for 30 min and then Na
2
S (50–250 mm) for 30 min
changes in intracellular H S levels. We further employed
2
(
b–d). The bright-field images are shown below. Scale bar: 50 mm.
a BODIPY-based probe for bioimaging of d-Cys-dependent
endogenous H S production in living human cells, which
2
suggests potential applications of our probes in H S biology.
2
that can be converted to H S through enzymatic H S biosyn-
2
[
2
11c]
thesis in living human cells.
In this work, cells were co-in-
cubated with 50 mm d-Cys and probe 9b for 30 min. The
cells showed clearly enhanced fluorescence responses com-
pared to those without the addition of Cys (Figure 7a,b).
The fluorescence triggered by 9b could be attributed to en-
Experimental Section
Materials and Methods
All chemicals and solvents used for synthesis were purchased from com-
mercial suppliers and used without further purification. The progress of
reactions was monitored by TLC on precoated silica plates (Merck 60F-
zymatically generated endogenous H S from d-Cys in living
2
cells. Inhibition experiments were performed by adding dl-
propargylglycine (dl-PPG), an analogue of cysteine, as
2
54, 250 mm in thickness), and spots were visualized with UV light.
Merck silica gel 60 (70–200 mesh) was used for purification by column
[11c]
a known inhibitor for H S biogenesis.
The fluorescence
1
13
2
chromatography. H and C NMR spectra were recorded on a Bruker
400 spectrometer. Chemical shifts are reported in parts per million rela-
signal change implied the suppression of endogenous H2S
Chem. Asian J. 2014, 00, 0 – 0
5
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

www.chemasianj.org
Long Yi, Zhen Xi et al.
tive to TMS as internal standard or residual solvent peaks (CDCl
3
:
chromatography on silica to give a light yellow solid (947 mg, yield
1
7
.26 ppm, DMSO: 2.50 ppm). High-resolution mass spectra (HRMS)
36%). TLC (CH
[D ]DMSO): d=7.69 (d, J=11.7 Hz, 1H), 7.39 (d, J=7.2 Hz, 1H), 6.49
(s, 1H), 3.90 ppm (s, 2H); C NMR (100 MHz, [D
159.7, 151.7, 150.3, 149.6, 149.3, 131.8, 116.8, 116.3, 113.2, 113.0, 109.6,
2 2 f
Cl :MeOH 40:1): R =0.50; H NMR (400 MHz,
were obtained on a Varian 7.0 T FTICR-MS. Spectroscopic measure-
ments were performed in PBS (20 mm, pH 7.4) buffer. Probes were dis-
solved in DMSO to prepare stock solutions with a concentration of 1–
6
1
3
6
]DMSO): d=170.8,
1
9
1
0 mm. The UV/Vis spectra were recorded on a CARY 100 Bio (Varian,
37.4 ppm; F NMR (376.5 MHz, [D
6
]DMSO): d=À131.13 ppm; HRMS
+
USA). Fluorescence studies were carried out with a Varian Cary Eclipse
calcd for [M+H] : 264.0421; found: 264. 0419.
spectrophotometer at 258C.
Fluoro-Containing Coumarin Boc-Amine Derivative 6b
Synthesis
HATU (932 mg, 2.45 mmol) and DIPEA (437 mL, 2.50 mmol) were
added to a CH
2 2
Cl solution (20 mL) of coumarin acid 5b (526 mg,
Ethyl 3-hydroxy-6-fluorophenylcarbamate (2b)
2
.00 mmol) and mono-tBoc-piperazine (456 mg, 2.45 mmol) at room tem-
4
-Amino-3-fluorophenol (1b, 5.0 g, 39.3 mmol) in EtOAc (50 mL) was
perature. The solution was stirred at room temperature overnight and
then poured into 200 mL of ice/water. The precipitate was filtered off to
give the desired produce as a light pink solid. The product 6b was puri-
heated to reflux for 30 min. Ethyl chloroformate (3 mL, 31.5 mmol) was
added to the refluxing mixture in drops over 30 min. The reaction mix-
ture was further heated to reflux for 1 h. The mixture was filtered while
hot and the filtrate was evaporated to dryness. Propan-2-ol was added to
the off-white residue, and the dissolved mixture was kept at 48C over-
fied by column chromatography on silica to give a yellow powder
1
(733 mg, yield 85%). TLC (CH
(400 MHz, CDCl
(s, 1H), 3.75 (s, 2H), 3.65–3.53 (m, 2H), 3.43 (m, 6H), 1.42 ppm (s, 9H);
2
Cl
2
:MeOH 40:1): R
f
=0.4; H NMR
3
): d=7.33–7.13 (m, 1H), 6.95 (d, J=7.0 Hz, 1H), 6.21
night to obtain 2b as precipitate in a yield of 75% (4.71 g). TLC
1
13
(
CH
2
Cl
2
:MeOH 40:1): R
f
=0.47; H NMR (400 MHz, [D
6
]DMSO): d=
C NMR (100 MHz, CDCl
3
): d=166.0, 159.5, 154.3, 152.3, 150.3, 149.9,
9
1
3
1
2
.35 (s, 1H), 8.99 (s, 1H), 7.28 (d, J=3.9 Hz, 1H), 6.94 (t, J=9.7 Hz,
148.5, 132.2, 132.1, 116.3, 112.0, 111.8, 109.0, 80.5, 45.9, 41.8, 37.1,
1
9
H), 6.61–6.33 (m, 1H), 4.11 (q, J=7.0 Hz, 2H), 1.20 ppm (t, J=7.1 Hz,
28.3 ppm; F NMR (376.5 MHz, CDCl
for [M+H] : 432.1683; found: 432.1679.
3
): d=À129.67 ppm; HRMS calcd
1
3
+
H); C NMR (100 MHz, [D
6
]DMSO): d=154.1, 153.7, 148.8, 146.4,
+
26.9, 115.8, 110.78, 110.2, 60.8, 14.6 ppm; HRMS calcd for [M+H] :
Fluoro-Containing Coumarin Amine Derivative 7b
00.0723; found: 200.0715.
Compound 7b was obtained by treatment of 6b (431 mg, 1.00 mmol)
Ethyl 2-(6-fluoro-7-(ethoxycarbonylamino)-2-oxo-2H-chromen-4-yl)acetic
acid (3k)
2 2
with TFA:CH Cl (1:1) solution. The product 7b was purified by column
chromatography on silica to give a faintly yellow powder (315 mg, yield
1
7
0% H
2
SO
4
(20 mL) was added dropwise to a mixture of 2b (1.99 g,
95%). TLC (CH
[D ]DMSO): d=7.64 (d, J=10.8 Hz, 1H, Ar), 7.41 (d, J=6.0 Hz, 1H,
Ar), 6.37 (s, 1H, Ar), 3.97 (s, 2H), 3.41 (brs, 5H), 2.76 (s, 2H), 2.66 ppm
2 2 f
Cl :MeOH 20:1): R =0.20; H NMR (400 MHz,
1
0.0 mmol) and diethyl-1,3-acetone dicarboxylate (1.81 mL, 10.0 mmol).
6
After stirring overnight, TLC showed no starting material, and then the
solution was poured into 200 mL of ice/water. The precipitate was fil-
tered to give the desired produce as a light pink solid. The product 3b
was purified by column chromatography on silica to give a yellow
1
3
(s, 2H); C NMR (100 MHz, [D
6
]DMSO): d=166.2, 159.2, 151.0, 149.7,
148.7, 131.1, 116.8, 115.4, 112.9, 112.7, 108.9, 46.3, 45.5, 45.1, 42.2,
1
9
35.9 ppm; F NMR (376.5 MHz, [D
6
]DMSO): d=À131.42 ppm; HRMS
+
powder (2.62 g, yield 85%). TLC (CH
2
Cl
]DMSO): d=9.88 (s, 1H), 7.86 (d, J=6.8 Hz,
H), 7.60 (d, J=11.5 Hz, 1H), 6.43 (s, 1H), 4.19 (q, J=7.1 Hz, 2H), 3.88
2
:MeOH 40:1):
R
f
=0.30;
calcd for [M+H] : 332.1153; found: 332.1155.
1
H NMR (400 MHz, [D
6
Coumarin-BODIPY Probe 9a
1
1
3
(
s, 2H), 1.26 ppm (t, J=7.1 Hz, 3H); C NMR (100 MHz, [D
6
]DMSO):
HATU (932 mg, 2.45 mmol) and N,N-diisopropylethylamine (DIPEA,
d=170.9, 160.1, 153.9, 150.7, 150.1, 149.8, 148.2, 130.9, 130.8, 115.5, 114.7,
4
37 mL, 2.50 mmol) were added to a CH
in acid 7a (626 mg, 2.00 mmol) and BODIPY-COOH (737 mg,
.00 mmol), at room temperature. The solution was stirred at room tem-
2 2
Cl solution (20 mL) of coumar-
1
9
1
11.9, 111.7, 109.2, 61.6, 37.4, 14.8 ppm;
]DMSO): d=À129.62 ppm; HRMS calcd for [M+H] : 310.0727;
found: 310.0727.
F NMR (376.5 MHz,
+
[
D
6
2
perature overnight. TLC showed no starting material, and then the solu-
tion was poured into 200 mL of ice/water. The precipitate was filtered off
to give the desired produce as a light pink solid. The product 9a was pu-
2
-(6-Fluoro-7-amino-2-oxo-2H-chromen-4-yl)acetic acid (4b)
Compound 3b (3.09 g, 10.0 mmol) was added to 20 mL of sodium hy-
droxide solution (4.00 g, 100 mmol). The mixture was stirred under reflux
for 3 h and then cooled to 08C. The pH was adjusted to 2.0 by dropwise
addition of conc. H SO . A light yellow precipitate was observed. The re-
2 4
action mixture was cooled to 08C and the precipitate was filtered off to
give the desired produce as a light yellow solid 4b (1.66 g, yield 70%).
rified by column chromatography on silica to give a yellow powder
1
(1.12 g, yield 85%). TLC (CH
(400 MHz, CDCl
7.38 (d, J=7.6 Hz, 2H), 6.96–6.92 (m, 2H), 6.21 (s, 1H), 5.98 (s, 2H),
2
Cl
2
:MeOH=100:1): R
f
=0.2; H NMR
3
): d=7.57 (d, J=7.6 Hz, 2H), 7.51 (d, J=6.8 Hz, 1H),
1
3
3.84 (s, 2H), 3.70–3.63 (m, 8H), 2.53 (s, 6H), 1.36 ppm (s, 6H); C NMR
(100 MHz, CDCl ): d=169.8, 166.5, 159.8, 156.0, 154.8, 148.7, 144.3,
3
4
b was pure enough for use in the next reaction without further purifica-
142.6, 140.0, 137.2, 135.5, 131.1, 128.6, 128.1, 126.1, 121.5, 116.0, 115.5,
1
19
tion. TLC (CH
400 MHz, [D ]DMSO): d=7.35 (d, J=12.1 Hz, 1H), 6.63 (d, J=7.6 Hz,
H), 6.25 (s, 2H), 6.09 (s, 1H), 3.76 ppm (s, 2H); C NMR (100 MHz,
2
Cl
2
:MeOH 5:1 with one drop AcOH): R
f
=0.30; H NMR
115.1, 107.3, 46.0, 42.0, 38.6, 37.1, 29.6, 14.6 ppm; F NMR (376.5 MHz,
À
(
1
6
CDCl
662.2476,.
3
): d=À146.1 ppm (m); HRMS calcd for [MÀH] : 662.2499; found:
1
3
[
1
D
6
]DMSO): d=170.6, 160.4, 151.5, 150.1, 148.3, 145.9, 141.2, 111.4,
1
9
Fluoro-Containing Coumarin-BODIPY Probe 9b
07.3, 101.7, 37.7 ppm;
F NMR (376.5 MHz, [D ]DMSO): d=
6
+
À138.29 ppm; HRMS calcd for [M+H] : 238.0515; found: 238. 0508.
-(6-Fluoro-7-azido-2-oxo-2H-chromen-4-yl)acetic acid (5b)
A solution of NaNO (2.07 g, 30.0 mmol) in water (7 mL) was added
HATU (932 mg, 2.45 mmol) and DIPEA (437 mL, 2.50 mmol) were
added to a CH
2 2
Cl solution (20 mL) of coumarin acid 7b (662 mg,
2
2
.00 mmol) and BODIPY-COOH (737 mg, 2.00 mmol) at room tempera-
2
ture. The solution was stirred at room temperature overnight. TLC
showed no starting material, and then the solution was poured into
200 mL of ice/water. The precipitate was filtered off to give the desired
product as a light pink solid. The product 9b was purified by column
dropwise to a cooled solution of amino acid 4b (2.37 g, 10.0 mmol) in
2
5
0% H
2
SO
4
(100 mL). The reaction temperature was maintained at 0–
(2.60 mg, 40.0 mmol) in
8C. After stirring for 30 min, a solution of NaN
3
water (10 mL) was added dropwise into the mixture at 08C. The resulting
reaction mixture was stirred overnight, after which it was extracted with
EtOAc (3ꢂ100 mL). The organic layer was washed with brine and then
dried with anhydrous sodium sulfate. After filtration, the organic phase
was concentrated under vacuum. The product 5a was purified by column
chromatography on silica to give a yellow powder (1.17 g, yield 85%).
1
TLC (CH
2
Cl
2
:MeOH 100:1): R
f
=0.2; H NMR (400 MHz, CDCl
3
): d=
7.59 (d, J=8.0 Hz, 2H), 7.40 (d, J=7.6 Hz, 2H), 7.27 (d, J=9.6 Hz, 1H),
7.03 (d, J=6.8 Hz, 1H), 6.28 (s, 1H), 5.99 (s, 2H), 3.81 (brs, 2H), 3.71–
1
3
3.66 (m, 8H), 2.55 (s, 6H), 1.38 ppm (s, 6H); C NMR (100 MHz,
&
&
Chem. Asian J. 2014, 00, 0 – 0
6
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

www.chemasianj.org
Long Yi, Zhen Xi et al.
CDCl
3
): d=169.8, 166.1, 159.5, 156.0, 152.4, 150.4, 150.0, 148.1, 142.7,
B. L. Predmore, D. J. Lefer, B. H. Wang, Angew. Chem. Int. Ed.
2011, 50, 9672–9675; Angew. Chem. 2011, 123, 9846–9849.
1
4
39.9, 137.3, 135.5, 132.5, 131.1, 128.6, 128.1, 121.5, 116.3, 111.7, 109.2,
6.0, 42.1, 38.6, 37.2, 29.7, 14.6 ppm; F NMR (376.5 MHz, CDCl
1
9
3
): d=
[7] a) L. A. Montoya, M. D. Pluth, Chem. Commun. 2012, 48, 4767–
4769; b) S. Chen, Z. J. Chen, W. Ren, H. W. Ai, J. Am. Chem. Soc.
2012, 134, 9589–9592; c) S. K. Das, C. S. Lim, S. Y. Yang, J. H. Han,
B. R. Cho, Chem. Commun. 2012, 48, 8395–8397; d) B. F. Chen, W.
Li, C. Lv, M. M. Zhao, H. W. Jin, H. F. Jin, J. B. Du, L. R. Zhang,
X. J. Tang, Analyst 2013, 138, 946–951; e) W. Sun, J. L. Fan, C. Hu,
J. F. Cao, H. Zhang, X. Q. Xiong, J. Y. Wang, S. Cui, S. G. Sun, X. J.
Peng, Chem. Commun. 2013, 49, 3890–3892; f) V. S. Lin, A. R. Lip-
pert, C. J. Chang, Proc. Natl. Acad. Sci. USA 2013, 110, 7131–7135;
g) G. D. Zhou, H. L. Wang, H. L. Ma, X. Q. Chen, Tetrahedron 2013,
À
À129.5, À146.1 ppm (m). HRMS calcd for [MÀH] : 680.2404; found:
6
80.2391.
Cell Culture and Bioimaging
2
HEK-293 cells were cultured at 378C and 5% CO in high-glucose Dul-
becco’s modified Eagle’s medium (GIBICO) supplemented with 10%
À1
À1
fetal bovine serum, 100 UmL penicillin, 100 mgmL streptomycin, and
mm l-glutamine. The cells were maintained in exponential growth and
4
4
then seeded in a glass-bottom 35 mm plate at a density of about 2ꢂ10
per well. Cells were passaged every 2–3 d and used between passages 3
and 10. Cells were imaged on a confocal microscope (Olympus FV1000
UPLSAPO40X) with a 40ꢂ objective lens. All images were analyzed
with Olympus FV1000-ASW. Emission was collected in the blue or green
channel with 405 nm excitation.
6
9, 867–870; h) A. R. Lippert, J. Inorg. Biochem. 2014, 133, 136–
1
42; i) G. J. Mao, T. T. Wei, X. X. Wang, S. Y. Huan, D. Q. Lu, J.
Zhang, X. B. Zhang, W. Tan, G. L. Shen, R. Q. Yu, Anal. Chem.
013, 85, 7875–7881.
2
[
8] a) F. B. Yu, P. Li, P. Song, B. S. Wang, J. Z. Zhao, K. L. Han, Chem.
Commun. 2012, 48, 2852–2854; b) Q. Q. Wan, Y. C. Song, Z. Li,
X. H. Gao, H. M. Ma, Chem. Commun. 2013, 49, 502–504; c) S. K.
Bae, C. H. Heo, D. J. Choi, D. Sen, E. H. Joe, B. R. Cho, H. M. Kim,
J. Am. Chem. Soc. 2013, 135, 9915–9923; d) L. Zhang, S. Li, M.
Hong, Y. Xu, S. Wang, Y. Liu, Y. Qian, J. Zhao, Org. Biomol. Chem.
Acknowledgements
2
014, 12, 5115–5125.
This work was supported by the MOST (2010CB126102), NSFC
[9] a) K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T.
Komatsu, T. Ueno, T. Terai, H. Kimura, T. Nagano, J. Am. Chem.
Soc. 2011, 133, 18003–18005; b) J. L. Long, L. P. Wang, D. Xie,
Y. W. Zhan, J. Lumin. 2013, 139, 40–46.
(
21332004 and 21402007), SRF for ROCS (SEM), the Fundamental Re-
search Funds for the Central Universities (YS1401), and Public Hatching
Platform for Recruited Talents of Beijing University of Chemical Tech-
nology.
[10] a) C. R. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman, A. R.
Whorton, M. Xian, Angew. Chem. Int. Ed. 2011, 50, 10327–10329;
Angew. Chem. 2011, 123, 10511–10513; b) J. Liu, Y. Q. Sun, J. Y.
Zhang, T. Yang, J. B. Cao, L. S. Zhang, W. Guo, Chem. Eur. J. 2013,
19, 4717–4722; c) X. W. Cao, W. Y. Lin, K. B. Zheng, L. W. He,
Chem. Commun. 2012, 48, 10529–10531; d) Y. C. Chen, C. C. Zhu,
Z. H. Yang, J. J. Chen, Y. F. He, Y. Jiao, W. J. He, L. Qiu, J. J. Cen,
Z. J. Guo, Angew. Chem. Int. Ed. 2013, 52, 1688–1691; Angew.
Chem. 2013, 125, 1732–1735; e) Z. Xu, L. Xu, J. Zhou, Y. F. Xu,
W. P. Zhu, X. H. Qian, Chem. Commun. 2012, 48, 10871–10873;
f) Y. Qian, J. Karpus, O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee,
J. Zhao, C. He, Nat. Commun. 2011, 2, 495; g) X. Wang, J. Sun, W.
Zhang, X. Ma, J. Lv, B. Tang, Chem. Sci. 2013, 4, 2551–2556; h) Y.
Qian, L. Zhang, S. T. Ding, X. Deng, C. He, X. E. Zheng, H. L. Zhu,
J. Zhao, Chem. Sci. 2012, 3, 2920–2923; i) Z. J. H, S. S. Ding, D. H.
Yu, F. H. Huang, G. Q. Feng, Chem. Commun. 2014, 50, 9185–9187;
j) L. Yuan, Q. P. Zuo, Chem. Asian J. 2014, 9, 1544–1549.
[
[
[
1] a) C. Szabꢃ, Nat. Rev. Drug Discovery 2007, 6, 917–935; b) L. Li, P.
Rose, P. K. Moore, Annu. Rev. Pharmacol. Toxicol. 2011, 51, 169–
1
87.
2] a) B. Predmore, D. Lefer, G. Gojon, Antioxid. Redox Signaling 2012,
1
1
7, 119–140; b) M. Whiteman, P. K. Moore, J. Cell. Mol. Med. 2009,
3, 488–507; c) H. Kimura, Exp. Physiol. 2011, 96, 833–835.
3] a) G. D. Yang, L. Y. Wu, B. Jiang, W. Yang, J. S. Qi, K. Cao, Q. H.
Meng, A. K. Mustafa, W. T. Mu, S. M. Zhang, S. H. Snyder, R.
Wang, Science 2008, 322, 587–590; b) L. F. Hu, M. Lu, Z. Y. Wu,
P. T. Wong, J. S. Bian, Mol. Pharmacol. 2009, 75, 27–34; c) K. H.
Kulkarni, E. M. Monjok, R. Zeyssig, G. Kouamou, O. N. Bongmba,
C. A. Opere, Y. F. Njie, S. E. Ohia, Neurochem. Res. 2009, 34, 400–
4
6
06; d) L. Li, M. Bhatia, P. K. Moore, Curr. Opin. Pharmacol. 2006,
, 125–129; e) H. Kimura, Amino Acids 2011, 41, 113–121.
[
4] a) P. Kamoun, M. C. Belardinelli, A. Chabli, K. Lallouchi, B. Chade-
faux-Vekemans, Am. J. Med. Genet. Part A 2003, 116, 310–311; b) S.
Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G.
Rizzo, E. Distrutti, V. Shah, A. Morelli, Hepatology 2005, 42, 539–
[11] a) C. Wei, Q. Zhu, W. W. Liu, W. B. Chen, Z. Xi, L. Yi, Org.
Biomol. Chem. 2014, 12, 479–485; b) C. Wei, L. Wei, Z. Xi, L. Yi,
Tetrahedron Lett. 2013, 54, 6937–6939; c) L. Wei, L. Yi, F. B. Song,
C. Wei, B. F. Wang, Z. Xi, Sci. Rep. 2014, 4, 4521.
5
48.
[12] S. Kellner, S. Seidu-Larry, J. Burhenne, Y. Motorin, M. Helm, Nucle-
ic Acids Res. 2011, 39, 7348–7360.
[13] J. Llopis, J. M. Mccaffery, A. Miyawaki, M. G. Farquhar, R. Y. Tsien,
Proc. Natl. Acad. Sci. USA 1998, 95, 6803–6808.
[
5] a) D. Jimꢄnez, R. Martinez-Manez, F. Sancenon, J. V. Ros-Lis, A.
Benito, J. Soto, J. Am. Chem. Soc. 2003, 125, 9000–9001; b) W. Lei,
P. K. Dasgupta, Anal. Chim. Acta 1989, 226, 165–170; c) D. G.
Searcy, M. A. Peterson, Anal. Biochem. 2004, 324, 269–275; d) N. S.
Lawrence, J. Davis, L. Jiang, T. G. J. Jones, S. N. Davies, R. G.
Compton, Electroanalysis 2000, 12, 1453–1460; e) J. Radford-
Knoery, G. A. Cutter, Anal. Chem. 1993, 65, 976–982.
[14] A. Loudet, K. Burgess, Chem. Rev. 2007, 107, 4891–4932.
Received: July 9, 2014
Published online: && &&, 0000
[
6] a) A. R. Lippert, E. J. New, C. J. Chang, J. Am. Chem. Soc. 2011,
1
33, 10078–10080; b) H. J. Peng, Y. F. Cheng, C. F. Dai, A. L. King,
Chem. Asian J. 2014, 00, 0 – 0
7
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

FULL PAPER
Fluorescent Probes
Chao Wei, Runyu Wang, Lv Wei,
Longhuai Cheng, Zhifei Li, Zhen Xi,*
Long Yi*
&&&& — &&&&
o-Fluorination of Aromatic Azides
Yields Improved Azido-Based Fluores-
cent Probes for Hydrogen Sulfide:
Synthesis, Spectra, and Bioimaging
Just add fluorine! On the basis of
introducing fluoro substituents into the
ortho position of aromatic azides,
which more than doubles their rate of
oped for real-time bioimaging of H S.
These probes can be used for in situ
visualization of d-cysteine-dependent
2
endogenously produced H S in living
2
reaction with H S, new BODIPY-
cells (see figure).
2
based fluorescent probes were devel-
&
&
Chem. Asian J. 2014, 00, 0 – 0
8
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!