Fluorescent probes based on nucleophilic substitution-cyclization for hydrogen sulfide detection and bioimaging

Article abstract of DOI:10.1002/chem.201303757

The design, synthesis, properties, and cell imaging applications of a series of 2-pyridyl disulfide based fluorescent probes (WSP1, WSP2, WSP3, WSP4 and WSP5) for hydrogen sulfide detection are reported. The strategy is based on the dual-nucleophilicity of hydrogen sulfide. A hydrogen sulfide mediated tandem nucleophilic substitution-cyclization reaction is used to release the fluorophores and turn on the fluorescence. The probes showed high sensitivity and selectivity for hydrogen sulfide over other reactive sulfur species, including cysteine and glutathione. Copyright

Full text of DOI:10.1002/chem.201303757

DOI: 10.1002/chem.201303757
Full Paper
&
Cell Imaging
Fluorescent Probes Based on Nucleophilic Substitution–
Cyclization for Hydrogen Sulfide Detection and Bioimaging
Bo Peng,[a] Wei Chen,[a] Chunrong Liu,[a] Ethan W. Rosser,[a] Armando Pacheco,[a] Yu Zhao,[a]
Hector C. Aguilar,[b] and Ming Xian*[a]
Abstract: The design, synthesis, properties, and cell imaging
applications of a series of 2-pyridyl disulfide based fluores-
cent probes (WSP1, WSP2, WSP3, WSP4 and WSP5) for hy-
drogen sulfide detection are reported. The strategy is based
on the dual-nucleophilicity of hydrogen sulfide. A hydrogen
sulfide mediated tandem nucleophilic substitution-cycliza-
tion reaction is used to release the fluorophores and turn on
the fluorescence. The probes showed high sensitivity and se-
lectivity for hydrogen sulfide over other reactive sulfur spe-
cies, including cysteine and glutathione.
Introduction
post-translational modification. It provides a possible mecha-
nism by which H2S alters the functions of a wide range of cel-
lular proteins and enzymes.[11–16] It is possible that many more
important reactions of H2S are to be discovered. Nevertheless,
the production of endogenous H2S and the exogenous admin-
istration of H2S have been demonstrated to exert protective ef-
fects in many pathologies.[1–8] It is important, therefore, to un-
derstand the chemistry and properties of H2S and to develop
effective and convenient methods for the detection of H2S in
biological systems.
Hydrogen sulfide (H2S), although historically known as a toxic
gas, has been recently classified as a critical cell signalling mol-
ecule, like nitric oxide (NO).[1–6] Results published in the past
few years increasingly suggests that H2S is a mediator of many
physiological and/or pathological processes, especially in cardi-
ovascular systems.[1–6] The production of H2S in mammalian
systems has been attributed to at least three endogenous en-
zymes:[5–10] cystathionine b-synthase, cystathionine g-lyase, and
3-mercaptopyruvate sulfur-transferase. These enzymes use cys-
teine or cysteine derivatives as the substrates and convert
them into H2S within different organs and tissues.
Fluorescence-based assays can be very useful in this field
owing to the high sensitivity and convenience. Fluorescence
methods are suitable for non-destructive detection of bio-tar-
gets in live cells or tissues with readily available instruments. In
2011 our laboratory and several other groups reported the first
reaction-based fluorescent probes for H2S detection.[17–21] These
works have inspired many researchers to develop new H2S flu-
orescent probes and a number of publications have appeared
in the past two years.[22,23] One of the main challenges for cur-
rent fluorescent probes is the high selectivity for H2S versus
other biothiols. The strategy used in the design of our first
generation H2S probe was based on a unique H2S-mediated
To date, the exact mechanisms of action for H2S are still
under active investigation. As a highly reactive molecule, H2S is
known to react with a number of biological targets, and these
reactions may be responsible for the biological functions of
H2S. For example, H2S reacts readily with methemoglobin to
form sulfhemoglobin, which might act as a metabolic sink for
H2S. H2S is a powerful reducing agent that is likely to react
with endogenous oxidants, such as peroxynitrite, superoxide,
and hydrogen peroxide. H2S can also cause protein S-sulfhydra-
tion (that is, converting cysteine residues ÀSH into persulfides nucleophilic addition followed by an intramolecular cyclization
ÀSÀSH). Although the detailed mechanism of S-sulfhydration is
to turn on the fluorescence signals.[18] This method is highly
specific for H2S. We have expanded this strategy and a series
of new probes have been prepared and evaluated. This article
is to report the design and synthesis of these probes, the opti-
mization of the detection conditions, as well as the applica-
tions of the probes in cell imaging.
still unclear, this process has been considered as an important
[a] B. Peng,+ Dr. W. Chen,+ Dr. C. Liu, E. W. Rosser, A. Pacheco, Y. Zhao,
Prof. M. Xian
Department of Chemistry, Washington State University
Pullman, WA 99164 (USA)
Fax: (+1)509-335-8867
E-mail: mxian@wsu.edu
[b] Prof. H. C. Aguilar
Results and Discussion
Paul G. Allen School for Global Animal Health
Washington State University
Pullman, WA 99164 (USA)
Design and synthesis of probes
[+] These authors contributed equally to this work.
H2S can be considered as a reactive nucleophile in biological
systems and it should undergo nucleophilic substitution or ad-
dition reactions easily. To selectively detect H2S from the bio-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303757.
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logical thiol pool, the key is to differentiate H2S from other bio-
thiols, such as cysteine (Cys) and glutathione (GSH). H2S is
a non-substituted thiol that can undergo nucleophilic reaction
twice; other thiols such as Cys and GSH are mono-substituted
thiols that can only pursue nucleophilic reaction one time.
Based on this property, we believe that compounds containing
dual-electrophilic centers are useful reagents for H2S detection.
As shown in Scheme 1, H2S should react with the most electro-
tried the reaction with H2S (using NaHS as the equivalent in
a buffer system; Scheme 2). In this model compound we envi-
sioned 2,2’-dipyridyl disulfide to be an effective electrophile for
trapping H2S and benzene to be an appropriate linker. The ri-
gidity of benzene 1,2-substitutions should facilitate the pro-
posed intramolecular cyclization. As expected, the reaction
went well and the desired cyclization product 2 and phenol
were obtained in good yield (83%).
Based on the structure of the
model compound, we believed
the introduction of pseudo fluo-
rophores through the ester link-
age should result in selective flu-
orescent probes for H2S. There-
fore a series of probes were syn-
thesized (shown in Scheme 3).
The common intermediate, 2-(2-
pyridinyldithio)-benzoic acid 3,
was readily prepared from
a mixed-disulfide formation be-
tween 2-mercaptobenzoic acid
and 2,2’-dipyridyl disulfide. Com-
pound 3 was then coupled with
Scheme 1. General design of the nucleophilic substitution–cyclization-based fluorescent probes for H2S.
ÀOH-containing fluorophores to
philic component of probe A to form a free SH containing in-
termediate B1. If another electrophilic reaction center is pres-
ent at a suitable position, such as the ester group shown in
B1, the SH group should undergo a spontaneous cyclization to
release the fluorophore and form product P. This strategy not
only can capture H2S as a stable and analyzable product P, but
Scheme 2. Model reaction between compound 1 and H2S.
also allows the visualation of H2S signals by convenient and
sensitive fluorescence measure-
ments. It should be noted that
substrate A may also react with
biothiols (RSH). However, prod-
uct B2 should not undergo the
cyclization to release the fluoro-
phore. Furthermore, if appropri-
ate electrophiles are employed,
it is possible for the first reaction
with biothiols to be reversible or
non-productive so that the
probes will not be consumed by
biothiols. Another advantage of
this strategy is that we can easily
modify the structures of all three
subunits of the template (elec-
trophile, linker, and fluorophore).
Therefore
a large library of
probes can be prepared and
tested. This has great potential
to produce ideal fluorescent
probes with optimized proper-
ties.
To test this idea, we first pre-
pared a model compound 1 and Scheme 3. Preparation of fluorescent probes WSP1–WSP5.
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give the probes WSP1–5 (WSP=Washington State probes). We
chose methoxy fluorescein, 7-hydroxycoumarin, resorufin, and
2-methyl TokyoGreen as the fluorophores because of their
readily availability, excellent fluorescence properties, and easy
fluorescence quenching by hydroxy group substitution.[24] For
WSP5, two reaction centers were introduced to the core struc-
ture of fluorescein. Upon reaction with H2S, it should produce
highly fluorescent species.
However, when a small amount of surfactant hexadecyltrime-
thylammonium bromide (CTAB) was added into the detection
system, the turn-on rate was significantly increased and the
fluorescence intensity was also significantly enhanced (110-
fold). The effects of CTAB may be attributed to 1) CTAB can in-
crease the solubility of the probe in aqueous buffers, and
2) CTAB is a cationic surfactant, which may absorb sulfide
anion (HSÀ) and facilitate the reaction between sulfide anion
and the probe. These effects were also noted in previous work
on fluorescent probes.[25]
Fluorescence properties and responses of probes WSP1–5 to
H2S
We then applied this optimized condition to other probes.
As shown in Figures 2 and 3, all of them (WSP2, WSP3, WSP4,
and WSP5) exhibited fast fluorescence turn-on toward H2S, and
usually the fluorescence signals can reach a steady state in
a few minutes; the intensities increased 275-, 68-, 20-, and 60-
fold (for WSP2, WSP3, WSP4, and WSP5, respectively). This may
With these probes in hand, we tested their fluorescent proper-
ties. As expected, these probes exhibited very weak fluores-
cence with low quantum yields (Ff <0.1, as shown in Table 1)
owing to the esterification of the hydroxy group of fluoro-
phores. This low background fluorescence is critical for highly
sensitive detection of H2S.
Table 1. Fluorescent properties of probes WSP1–WSP5.
Probes
lex [nm]
lem [nm][a]
Ff[b]
WSP1
WSP2
WSP3
WSP4
WSP5
476
385
550
512
502
516
456
586
531
525
0.003
0.003
0.014
0.088
0.020
[a] The maximal emission of the probes; [b] The fluorescence quantum
yield.
Figure 2. Time-dependent fluorescence changes of the probes (10 mm) in
the presence of NaHS (50 mm). The reactions were carried out for 30 min at
room temperature in PBS buffer (10 mm, pH 7.4) with 1 mm CTAB. Data
&
were acquired at 456 nm with excitation at 385 nm for WSP2 ( ); at 586 nm
*
with excitation at 550 nm for WSP3 ( ); at 531 nm with excitation at 512 nm
We then tested their fluorescence responses to H2S and opti-
mized the fluorescence measurement conditions. WSP1 was
used as the representative species in these studies. As shown
in Figure 1, the fluorescence intensity of WSP1 increased dra-
matically when H2S was present in the solution. We also found
that media plays an important role in this process. When
a mixed CH3CN/PBS buffer (10 mm, pH 7.4, 1:1, v/v) solution
was used (owing to poor water solubility of the probe), the
fluorescence turn-on rate was somewhat slow. The intensity
could reach the maximum in about 30 min (36-fold increase).
~
!
for WSP4 ( ); at 525 nm with excitation at 502 nm for WSP5 ( ).
Figure 3. Fluorescence spectra changes of probes (10 mm) in the absence
(g) or presence (c) of NaHS (50 mm). The reactions were carried out for
5 min at room temperature in PBS buffer (10 mm, pH 7.4) with 1 mm CTAB.
Data were acquired with excitation at 385 nm for WSP2 (a); with excitation
at 550 nm for WSP3 (b); with excitation at 512 nm for WSP4 (c); with excita-
tion at 502 nm for WSP5 (d).
Figure 1. Time-dependent fluorescence changes of 10 mm WSP1 in the pres-
~
~
&
*
*
ence ( and ) or absence ( ) of 50 mm NaHS. ( ) and ( ) data were ob-
&
tained in 10 mm PBS buffer (pH 7.4) containing 1 mm CTAB. ( ) data were
obtained in CH3CN/PBS buffer (10 mm, pH 7.4, 1:1, v/v). Data were acquired
at 516 nm with excitation at 476 nm.
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be very favorable for fast detection of H2S. Upon gradual intro-
duction of H2S, the fluorescence intensity at the maximal emis-
sion wavelength of the probes increased drastically. As can be
seen in Figure S1 in the Supporting Information, good linear
relationships were obtained between the fluorescence intensi-
ty at the maximal emission wavelength of the probes and dif-
ferent hydrogen sulfide concentrations. The detection limits[26]
were determined to be 60, 79, 47, 266, and 47 nm for WSP1,
WSP2, WSP3, WSP4, and WSP5, respectively (Table 2).
pH 10, which might be due to the hydrolysis of the ester link-
age under this pH value.
We then investigated the selectivity of the probes for H2S,
which is the most important property to justify the effective-
ness of the probes. In these experiments, each probe (10 mm)
was treated with different reactive sulfur species, including
Cys, GSH, Hcy, SO32À, S2O32À (all at 200 mm) under the optimized
conditions, and fluorescence signals were recorded after
mixing for 5 min. As shown in Figure 5, these sulfur-containing
Table 2. Turn-on fold changes and detection limits (DL) of probes WSP1–
WSP5.
Probes
Turn-on folds
DL[a] [nm]
WSP1
WSP2
WSP3
WSP4
WSP5
130
275
68
20
60
60
79
47
266
47
[a] DL is the detection limit (3S/m, in which S is the standard deviation of
blank measurements and m is the slope of the linear equation).[26]
To evaluate the potential applications of the probes in differ-
ent biological environments, we studied pH effects for all of
the five probes (Figure 4). In general these probes worked well
under normal biological pH ranges (ca. 6–9). However, WSP3
and WSP4 showed increased background fluorescence under
Figure 5. Fluorescence intensity of the probes (10 mm) in the presence of
various reactive sulfur species: 1) control; 2) 50 mm NaHS; 3) 200 mm Cys;
4) 200 mm GSH; 5) 200 mm Hcy; 6) 200 mm Na2SO3; 7) 200 mm Na2S2O3;
8) 50 mm NaHS+200 mm Cys; 9) 50 mm NaHS+200 mm GSH. WSP1 (a), WSP2
(b), WSP3 (c), WSP4 (d), and WSP5 (e).
species did not lead to any significant fluorescence responses,
whereas NaHS (50 mm) gave very strong signals for all of the
probes. Therefore all of the probes are shown to have good se-
lectivity for H2S over other reactive sulfur species, including
Cys and GSH. As can be imagined, these disulfide-based
probes may also react with biothiols in biological systems. Al-
though such reactions will not turn on fluorescence, it could
consume the probes and higher loading of the probes may be
needed. This is a potential problem for this type of probes.
However we realized that the fluorescence turn-on rates of our
probes under the optimized conditions were quite fast (within
a few minutes). Therefore it is possible that the probes can
give significant fluorescence signals even when H2S co-exists
with biothiols. To test this, a solution of NaHS (50 mm) and Cys
or GSH (200 mm) was prepared and the probe (10 mm) was
then loaded. Pleasantly, we still observed significant fluores-
cence signals for each probe (Figure 5), although at decreased
levels compared to NaHS only. These results suggested that
Figure 4. Fluorescence intensity changes of the probes (10 mm) at different
~
&
pH values in the absence ( ) or presence ( ) of NaHS (50 mm). The reactions
were carried out for 5 min at room temperature in 10 mm PBS solution with
1 mm CTAB. WSP1 (a), WSP2 (b), WSP3 (c), WSP4 (d), and WSP5 (e).
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the probes were effective for H2S
detection in the presence of bio-
thiols.
After confirming the sensitivity
and selectivity of this series of
probes for H2S in aqueous buf-
fers, we decided to explore their
applications in imaging H2S in
living cells. WSP4 and WSP5
were selected for this study be-
cause of their strong fluorescent
intensity observed when treating
with H2S. Before we conducted
cell imaging experiments, we re-
alized that our probes contain
the fluorophores through an
ester linkage. One concern is
Figure 7. Fluorescence images of H2S in HeLa cells using WSP4 and WSP5. (a–f) Cells on 24-well plate were incu-
bated with WSP4 (30 mm) for 30 min, then washed and subjected to different treatments: (a,b) Control (no NaHS
was added); (c,d) treated with 30 mm NaHS; (e,f) treated with 60 mm NaHS. (g–l) Cells on 24-well plate were incu-
that such a linkage may be labile bated with WSP5 (50 mm) for 30 min, then washed and subjected to different treatments: (g,h) Control (no NaHS
was added); (i,j) treated with 50 mm NaHS; (k,l) treated with 100 mm NaHS. Scale bar: 100 nm.
in the presence of cellular ester-
ases. To address this question,
we tested the stability of the
probes (WSP1, WSP4, and WSP5) in the presence of esterase
(esterase E-0887, from rabbit liver). As shown in Figure 6, the
probes were incubated with the enzyme for 30 min, and no
probe and treated with different concentrations of NaHS. As
shown in Figure 7, we did not observe significant fluorescence
in cells when NaHS was absent. However, strong fluorescence
in the cells was observed after treating with NaHS for 30 min.
Cells treated with 60 mm NaHS displayed obviously stronger
fluorescence than cells treated with 30 mm NaHS. Furthermore,
WSP5 was also tested by using the same procedure and similar
results were observed. We thus concluded that these probes
can be used for the detection of H2S in living cells.
To further explore the applications of the probes in H2S stud-
ies we used them in the evaluation of novel H2S donors. H2S
donors are another type of important research in this field,
and our laboratory has recently developed several different
controllable H2S donors.[27] Among these the perthiol-based
donors are of particularly interest because they have exhibited
promising activity against myocardial ischemia-reperfusion in-
jury.[27a] To monitor H2S generation in cells from this type of
donors, we applied our newly developed fluorescent probes.
Briefly, HeLa cells were first incubated with YZ-4-074, a per-
thiol-based donor, for 30 min. After that, the exo-cellular donor
was removed by washing with buffers. The cells were then in-
cubated with WSP4. As shown in Figure 8, donor-treated cells
showed much enhanced fluorescent signals compared to vehi-
cle-treated cells. We also applied WSP5 in the same procedure,
and similar results were observed (Supporting Information, Fig-
ure S2). These results demonstrated that these probes can be
used to evaluate synthetic H2S donor in cells.
Figure 6. Fluorescence intensity of the probe (10 mm). 1) Control, without
the esterase; 2) incubation with esterase (0.06 UmLÀ1) at room temperature
for 30 min; 3) incubation with 50 mm NaHS for 5 min after (2). WSP1 (a),
WSP4 (b), WSP5 (c).
significant fluorescence increase was observed. Thereafter,
NaHS was added into the mixture and a strong fluorescence
increase was observed. These results suggested that the
probes were stable to esterases.
Conclusion
In summary, we have presented in this study the development
of a series of nucleophilic substitution–cyclization-based fluo-
rescent probes for H2S. Five probes (WSP1–WSP5) were pre-
pared and evaluated. These probes were selective for H2S over
other sulfur-containing species, including Cys and GSH. More-
over, fluorescence “turn-on” is fast. The efficiencies of these
Living cell imaging studies
Having demonstrated the stability of the probes, we then used
them in monitoring H2S in live cells. In brief, freshly cultured
HeLa cells were incubated with probe WSP4 for 30 min. Then
the cells were washed by medium buffer to remove excess
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J1 =7.8 Hz, J2 =1.2 Hz, 1H, HAr), 8.48 ppm (dd, J=4.2, 0.9 Hz, 1H,
HAr); 13C NMR (75 MHz, CDCl3, 258C, TMS) d=110.9, 116.5, 117.2,
118.8, 120.0, 121.4, 125.8, 126.2, 126.5, 129.0, 132.4, 134.5, 137.6,
142.2, 143.1, 149.9, 153.3, 155.0, 158.9, 160.5, 164.4 ppm; IR (thin
film) n˜ =3098.6, 3064.4, 3034.0, 1733.6, 1707.0 cmÀ1; MS (ESI) [2M+
Na]+ calcd for C42H26N2O8S4Na, 837.0; found, 836.7; HRMS [M+H]+
calcd for C21H14NO4S2, 408.0364; found, 408.0362.
WSP3 was obtained as an orange solid (266 mg, 58% yield). M.p.
139–1418C; 1H NMR (300 MHz, CDCl3, 258C, TMS) d=6.37 (d, J=
1.8 Hz, 1H, HAr), 6.90 (dd, J1 =9.6, J2 =1.8 Hz, 1H, HAr), 7.11–7.15 (m,
1H, HAr), 7.31–7.41 (m, 3H, HAr), 7.48 (d, J=9.9 Hz, 1H, HAr), 7.54–
7.63 (m, 3H, HAr), 7.89 (d, J=8.1 Hz, 1H, HAr), 8.02 (d, J=7.5 Hz, 1H,
HAr), 8.32 (dd, J1 =7.8 Hz, J2 =0.9 Hz, 1H, HAr), 8.50 ppm (d, J=
3.9 Hz, 1H, HAr); 13C NMR (75 MHz, CDCl3, 258C, TMS) d=107.2,
109.9, 119.4, 119.9, 121.2, 125.4, 126.0, 126.3, 131.2, 131.4, 132.1,
134.3, 134.8, 135.2, 137.5, 141.9, 144.4, 148.4, 149.2, 149.5, 153.2,
158.5, 163.9, 186.2 ppm; IR (thin film) n˜ =3100.1, 3064.4, 3040.6,
1704.4, 1624.0 cmÀ1; MS (ESI) [2M+Na]+ calcd for C48H28N4O8S4Na,
939.1; found, 938.8; HRMS [M+H]+ calcd for C24H15N2O4S2,
459.0473; found, 459.0487.
Figure 8. Fluorescence images of H2S production from a H2S donor YZ-4-074
in HeLa cells. Cells on a 24-well plate were incubated with or without
YZ-4-074 for 30 min, then washed and incubated with 30 mm WSP4 for
30 min. (a,b) Controls (no donor was added); (c,d) treated with 100 mm
YZ-4-074. Scale bar: 100 nm.
WSP4 was obtained as an orange solid (301 mg, 55% yield). M.p.
114–1178C; 1H NMR (300 MHz, CDCl3, 258C, TMS) d=2.12 (s, 3H;
CH3), 6.47 (d, J=1.8 Hz, 1H, HAr), 6.60 (dd, J1 =9.9 Hz, J2 =1.8 Hz,
1H, HAr), 6.99 (d, J=9.9 Hz, 1H, HAr), 7.10–7.14 (m, 3H, HAr), 7.19–
7.21 (m, 1H, HAr), 7.34–7.47 (m, 4H, HAr), 7.50 (d, J=8.4 Hz, 1H, HAr),
7.54–7.62 (m, 3H, HAr), 8.00 (d, J=8.1 Hz, 1H, HAr), 8.30 ppm (dd,
J1 =7.8 Hz, J2 =1.8 Hz, 1H, HAr), 8.47–8.50 (m, 1H, HAr); 13C NMR
(75 MHz, CDCl3, 258C, TMS) d=19.6(s; CH3), 106.1, 110.4, 118.4,
118.6, 119.6, 120.4, 121.0, 125.3, 125.9, 126.1, 126.2, 129.0, 129.1,
129.6, 130.5, 130.6, 130.9, 131.9, 132.0, 134.2, 136.1, 137.3, 141.9,
148.0, 149.6, 153.0, 154.0, 158.4, 158.4, 163.8, 185.8 ppm; IR (thin
film) n˜ =3053.0, 2946.6, 2916.1, 1726.0, 1596.7 cmÀ1; MS (ESI) [2M+
Na]+ calcd for C64H42N2O8S4Na, 1117.2; found, 1116.9; HRMS [M+
H]+ calcd for C32H22NO4S2, 548.0990; found, 548.0974.
probes were demonstrated in aqueous solution and in cell
imaging. Further development of nucleophilic substitution–
cyclization-based probes and application of these probes in
H2S studies are currently ongoing in our laboratory.
Experimental Section
WSP5 was obtained as a light yellow solid (370 mg, 45% yield).
M.p. 125–1288C; 1H NMR (300 MHz, CDCl3, 258C, TMS) d=6.92–
6.95 (m, 2H, HAr), 7.00–7.03 (m, 2H, HAr), 7.10–7.12 (m, 2H, HAr),
7.23–7.38 (m, 5H, HAr), 7.52–7.61 (m, 6H, HAr), 7.64–7.75 (m, 2H,
HAr), 7.99 (d, J=7.8 Hz, 2H, HAr), 8.07 (d, J=7.2 Hz, 1H, HAr), 8.30 (d,
J=7.2 Hz, 2H, HAr), 8.47 ppm (d, J=4.2 Hz, 2H, HAr); 13C NMR
(75 MHz, CDCl3, 258C, TMS) d=81.6, 110.7, 116.8, 118.0, 119.8,
121.1, 124.1, 125.3, 125.8, 126.0, 126.2, 129.2, 130.2, 132.1, 134.1,
135.4, 137.4, 141.8, 149.6, 151.6, 151.9, 153.0, 158.8, 164.3,
169.2 ppm; IR (thin film) n˜ =3060.6, 2954.2, 2923.7, 1771.6,
1718.4 cmÀ1; MS (ESI) [M+Na]+ calcd for C44H26N2O7S4Na, 845.1;
found, 845.1; HRMS [M+H]+ calcd for C44H27N2O7S4, 823.0701;
found, 823.0714.
Synthesis of the probes
Compound 3 was prepared using the procedure described previ-
ously.[18] General procedure for the synthesis of the probes: Dry
CH2Cl2 (25 mL) was added to a mixture of compound 3 (262 mg,
1.0 mmol), OH-containing fluorophore (1.0 mmol), EDC (192 mg,
1.0 mmol), and DMAP (12.2 mg, 0.1 mmol) in a 50 mL round-bot-
tomed flask at room temperature. The mixture was stirred for 12 h.
Then solvent was removed under reduced pressure and resulted
crude product was purified by fresh column chromatography to
provide the desired product.
Compound 1 was obtained as a white solid (273 mg, 80.5% yield).
1
M.p. 113–1158C; H NMR (300 MHz, [D1]CDCl3, 258C, TMS) d=7.06–
7.11 (m, 1H, HAr), 7.24–7.36 (m, 4H, HAr), 7.41–7.47 (m, 2H, HAr),
7.50–7.57 (m, 3H, HAr), 7.97 (dd, J1 =8.1 Hz, J2 =2.4 Hz, 1H, HAr),
8.30 (dd, J=7.8, 1.5 Hz, 1H, HAr), 8.45–8.47 ppm (m, 1H, HAr);
13C NMR (75 MHz, CDCl3, 258C, TMS) d=119.7, 121.0, 121.7, 125.8,
126.0, 126.2, 126.4, 129.8, 132.0, 133.8, 137.4, 141.4, 149.6, 150.5,
159.0, 164.9 ppm; IR (thin film) n˜ =3999.0, 3072.0, 3041.0, 1707.0,
1562.5 cmÀ1; MS (ESI) [M+Na]+ calcd for C18H13NO2S2Na, 362.0;
found, 362.0; HRMS [M+H]+ calcd for C18H14NO2S2, 340.0466;
found, 340.0468.
General procedure for H2S determination
Unless otherwise noted, all of the measurements were carried out
at room temperature for 5 min in 10 mm PBS buffer (pH 7.4) con-
taining 1 mm CTAB according to the following procedure: in a test
tube, 3.5 mL of 10 mm PBS buffer (pH 7.4) and 40 mL of the stock
solution of CTAB were mixed. A portion of the stock solution
(16 mL) of the probe was then added to this mixture. The resulting
solution was well-mixed, followed by the addition of the requisite
volume of testing species solution. The final volume of the solution
was adjusted to 4 mL with 10 mm PBS buffer (pH 7.4). After mixing
and standing for 5 min at room temperature, 3 mL of the solution
was transferred into a 1 cm quartz cell and fluorescence signal was
recorded.
Data for WSP1 was the same as previously reported.[18]
WSP2 was obtained as a white solid (374 mg, 92% yield). M.p.
165–1678C; 1H NMR (300 MHz, CDCl3, 258C, TMS) d=6.44 (d, J=
9.3 Hz, 1H, HAr), 7.09–7.14 (m, 1H, HAr), 7.23 (d, J=2.1 Hz, 1H, HAr),
7.28–7.29 (m, 1H, HAr), 7.34–7.39 (m, 1H, HAr), 7.52–7.61 (m, 4H,
HAr),7.74 (d, J=9.9 Hz, 1H, HAr), 7.99–8.01 (m, 1H, HAr), 8.31 (dd,
Chem. Eur. J. 2014, 20, 1010 – 1016
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HeLa cells were cultured in Dulbecco’s modified Eagle’s Medium
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Keywords: cell imaging
·
cyclization
·
fluorescence
·
fluorescent probes · hydrogen sulfide
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Received: September 25, 2013
Published online on December 11, 2013
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