Communication
To examine the stability of the probes, a control experiment
was performed in which each probe was incubated in the ab-
sence of any analyte in PBS buffer (Figure S5). The results indi-
cated that the fluorescence change of these probes within 1 h
was much smaller than that in the presence of H2S. In order to
evaluate the specificity of these fluorinated coumarins for H2S,
they were incubated with various biologically related species
in PBS buffer, and the fluorescence change was measured ac-
cordingly (Figure 3, Figure S6, and Figure S7). Among all the
Figure 4. In vitro continuous CBS activity assay based on probe 1. The time-
dependent emission intensity of probe 1 (20 mm) at 480 nm in the presence
of 5 mmL-Cys upon addition of 0–12 mg CBS enzyme in 1 mL Tris-HCl
(pH 8.0) buffer; excitation, 360 nm.
Cys, l-propargylglycine (PPG),[11] which was also observed with
probe 1 by the continuous fluorophotometric assay (Fig-
ure S10).
To further demonstrate the biological applicability of probes
1 and 2, we examined whether they could be used to detect
H2S in living cells in real time. To this end, HEK293A cells were
first treated with probe 1 or 2 (2 mm) for 30 min and then with
Na2S (200 mm) for different times. The significant fluorescence
increase in the cyan channel (Figures S11 and S12) indicated
that probes 1 and 2 can be used for imaging of H2S in living
cells. Bright-field images show that the cells retained good
morphology after incubation with 1, suggesting the probe has
low cytoxicity at the experimental concentration. Consequent-
ly, we determined the average fluorescence intensity in cells at
different time points. The increase in the intensity saturated
after 5 min for 1 (Figure S12), implying that this probe can be
used for imaging exogenous H2S in living cells at a rapid
speed.
Figure 3. Fluorescent response of probe 1 (1 mm) to various biologically rele-
vant species (100 mm) or H2S (100 mm) or biothiols (Cys or Hcy, 1 mm; GSH,
5 mm) in PBS (50 mm, pH 7.4). Other species comprise SO32À, S2O32À, NO2À
,
H2O2, ClOÀ, Zn2+, and Fe3+. Excitation, 370 nm.
tested molecules, only H2S showed a noticeable fluorescence
response. The fluorescence increase by biothiols (Cys and GSH)
at millimolar concentrations, however, is far below that in-
duced by H2S. These results suggest that multi-fluorinated
azido coumarins are selective to H2S over other biological spe-
cies.
Considering that the H2S-mediated reduction reaction for
probe 1 was the fastest, we predominantly used this probe for
further tests and biological applications. To gain detailed infor-
mation about the sensitivity of 1, the fluorescence intensity
change was closely monitored by addition of various concen-
trations of H2S to the probe (Figure S8). Data analysis revealed
an excellent linear relationship (r=0.9944) between the fluo-
rescence signal at 490 nm and the concentration of H2S (10–
50 mm). The detection limit was determined to be 0.67 mm by
using the 3s/k method.[11] These results demonstrated that
probe 1 could react with H2S with good sensitivity and quanti-
tatively. In addition, we also investigated the fluorescence re-
sponse of probe 1 to H2S at different pH values (Figure S9).
The results indicated that the probe can function over a wide
range of pH from 6.0 to 8.5.
To test whether 1 could detect endogenous production of
H2S, cells were treated with d-Cys or l-Cys and then with 1. As
shown in Figure 5, HEK293A cells incubated with 1 displayed
a fairly weak fluorescence under the confocal fluorescence mi-
croscope (Figure 5a). In the presence of Cys to induce endoge-
nous H2S production,[10f] a significant fluorescent enhancement
can be observed (Figure 5b, 5c), implying that 1 can be used
to visualize cysteine-dependent H2S production in situ in living
cells. The measured average fluorescence indicated that d-Cys
induced a higher H2S production than l-Cys in a time-depen-
dent manner in living cells (Figure 5d and Figures S13–S15).
The above results suggested that 1 was cell-permeable and
could react with intracellular H2S efficiently.
To further demonstrate the biological applicability of the
multi-fluorinated azido coumarins, we used the probe 1 to
monitor the CBS activity in vitro. The enzymatic reaction can
be visualized by the specific fluorescence response of probe 1
to the enzymatically produced H2S. Preliminary studies indicat-
ed that significant fluorescence appeared at 480 nm during
the enzymatic reaction after addition of increased concentra-
tions of CBS and as the concentration of CBS increased
(Figure 4). Particularly noteworthy is the continuous nature of
the assay. The CBS activity can be inhibited by an analogue of
In summary, we rationally designed and synthesized a series
of multi-fluorinated azido coumarins for rapid sensing of H2S in
buffer and in living cells. The kinetic studies indicated that an
increase in fluorine substituents leads to an increased rate of
H2S-mediated reduction reaction, which is also supported by
calculations. The tetra-fluorinated coumarin 1 can respond to
H2S rapidly (t1/2 ꢀ1 min) and selectively. In preliminary studies
probe 1 was applied for continuous enzymatic assay and for
bioimaging in living cells. We believe that this design strategy
Chem. Asian J. 2016, 11, 68 – 71
70
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim