Chemoselective reduction and self-immolation based FRET probes for detecting hydrogen sulfide in solution and in cells

Article abstract of DOI:10.1039/c4ob00923a

Hydrogen sulfide (H₂S) has been regarded as the third gaseous transmitter. Based on the mechanism of chemoselective azido reduction and self-immolation, five fluorescence resonance energy transfer (FRET) probes for the detection of H₂S were designed and synthesized. The effect of functional substitution of the self-immolative moiety on azido reduction and quinone-methide rearrangement were investigated. Their fluorescence responses and chemoselectivity for H₂S detection were evaluated in solutions and in cells. This strategy may provide a general route for designing H ₂S probes with many commercially available FRET pairs. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00923a

Organic &
Biomolecular Chemistry
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Chemoselective reduction and self-immolation
based FRET probes for detecting hydrogen sulfide
in solution and in cells†
Cite this: Org. Biomol. Chem., 2014,
12, 5629
Bifeng Chen,‡a Peng Wang,‡a Qingqing Jina and Xinjing Tang*a,b
Hydrogen sulfide (H2S) has been regarded as the third gaseous transmitter. Based on the mechanism of
chemoselective azido reduction and self-immolation, five fluorescence resonance energy transfer (FRET)
probes for the detection of H2S were designed and synthesized. The effect of functional substitution of
the self-immolative moiety on azido reduction and quinone-methide rearrangement were investigated.
Their fluorescence responses and chemoselectivity for H2S detection were evaluated in solutions and in
cells. This strategy may provide a general route for designing H2S probes with many commercially avail-
able FRET pairs.
Received 5th May 2014,
Accepted 4th June 2014
DOI: 10.1039/c4ob00923a
www.rsc.org/obc
pitation of heavy metal sulfide: fluorescence quenching of Cu2+
from Cu2+-coordinated organometallic complexes was elimi-
Introduction
Although H2S has been recognized as a well-known toxic pollu- nated by the formation of CuS;13 (c) nucleophilic reaction: the
tant gas with an unpleasant smell, it has now been recognized nucleophilic sulfide is utilized to react with fluorescent probes
as an important gaseous transmitter in addition to nitric oxide through Michael addition or nucleophilic substitution.14
(NO) and carbon monoxide (CO).1 It is involved in a variety of
Although much progress has been achieved on the develop-
physiological and pathological processes in biological systems, ment of fluorescent probes for H2S, the sensitive moieties for
such as vasodilation,2 angiogenesis,3 neuromodulation,4 apo- H2S (such as azido) are directly located on the reporter moie-
ptosis5 and inflammation.6 The endogenous H2S is syn- ties which caused the limited options for available fluoro-
thesized in mammalian systems mainly with involvement of phores and/or difficult and laborious synthesis of fluorescent
pyridoxal-5′-phosphate-dependent enzymes, such as cystathio- probes. A general strategy with a universal H2S sensitive
nine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercapto- moiety and many commercially available fluorophores will
pyruvate sulfur transferase (3-MST).7 Due to the quick greatly expand the scope of fluorescent probes for H2S detec-
metabolism of H2S in vivo, it was difficult to detect this transi- tion. With this idea in hand, we designed a series of fluore-
ent gaseous signaling molecule selectively and sensitively. scent probes based on fluorescence resonance energy transfer
Several approaches, including colorimetric assay,8 electrochemi- (FRET). These FRET probes contained the azido moiety as the
cal assay,9 gas chromatography10 and sulfide precipitation,11 sensor, and a fluorophore and a quencher as the FRET pair
have been developed for the detection of H2S. However, they are linked by a self-immolative linker which can dissociate
usually time consuming and destructive with relatively high cost through quinone-methide rearrangement after the reduction
and not suitable for applications in biological samples.
of the azido moiety,15 as shown in Scheme 1.
Recently, several kinds of fluorescent probes for H2S detec-
tion have been reported. In general, these probes were designed
based on the different mechanisms of specific H2S-involved
reactions: (a) chemical reduction: the reduction of azide, nitro,
hydroxyl amine group, disulfide or selenoxide;12 (b) preci-
Results and discussion
Synthesis and evaluation of FRET-based H2S fluorescent probes
The FRET quencher–fluorophore pairs have been widely used
in oligonucleotide probes and peptide probes for detecting
nucleic acid mutation and enzymatic activities.16 Fluorescein
isothiocyanate (FITC) and [4′-(N,N′-dimethylamino)phenylazo]
benzoyl (DABCYL), as an excellent FRET pair, were chosen as
the fluorophore and quencher moieties due to their easy com-
mercial availability and wide applications in sensors. Different
aState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing, 100191, China
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, China.
E-mail: xinjingt@bjmu.edu.cn; Tel: +86-010-82800535
†Electronic supplementary information (ESI) available: Synthetic details, NMR,
MS and additional figures are provided. See DOI: 10.1039/c4ob00923a
‡These authors have equal contributions.
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Scheme 1 The structures of FRET probes.
immolative linkers were developed, as shown in Scheme 1. For
FRET-P1 (the synthetic route is shown in Scheme S1†), a p-azido-
benzyl moiety was first linked to 2,6-bis(hydroxymethyl)-p-
cresol whose two hydroxyl groups were then selectively coupled
with a DABCYL and fluorescein through ester and carbomate
formation respectively. With the existence of H2S, the azido
moiety could be reduced to the amine moiety that could
further trigger self-immolation through quinone-methide
rearrangement, as shown in Scheme 2; 1,4 or 1,6-quinone-
methide rearrangement induced the cleavage of the chemical
bond C–O and release the DABCYL. FITC was then excited to
emit fluorescence thanks to the release of DABCYL. Initial
evaluation of its response to the existence of H2S indicated that
a relatively slow increase in fluorescence intensity was observed
with the excitation at 488 nm (Fig. S1†). This slow response may
be due to the dual immolation of 1,6 and 1,4-quinone-methide
rearrangement (Scheme 2). To shorten the two-step immolation,
another two probes, FRET-P2 and FRET-P3, were designed and
synthesized (Schemes S2 and S3†).
For these two probes, only one 1,4-quinone-methide
rearrangement was needed to break the immolative linker and
release the fluorescein moiety. Before the synthesis of FRET-P2
and FRET-P3, a preliminary experiment on the azide reduction
of methyl 3-methyl-2-azide benzoate in the presence of H2S
was carried out. A quick response to H2S was observed with
the introduction of the electron acceptor on the phenyl moiety
(Fig. S2†). Further coupling with DABCYL and fluorescein to
this linker afforded two probes, FRET-P2 and FRET-P3.
However, their responses to H2S were still quite slow (Fig. S3
Scheme 2 The self-immolation mechanism of FRET-P1 for the detec-
tion of H2S.
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and S4†). Although the electron acceptor group on the phenyl
moiety made the azido group easier to be reduced, it actually
slowed down the 1,4-quinone-methide rearrangement. This
may led to even slower responses of these two probes to H2S.
Based on the above observations, we further developed
another two FRET probes, FRET-P4 and FRET-P5 (Schemes S4
and S5†). These two probes have a 4-azidophenylhydroxyl
acetic acid moiety. Once the azido moiety was reduced to the
amine group, only one 1,6-quinone-methide rearrangement
was needed to break the linkage of the fluorophore and the
quencher. Time dependence of increase in fluorescence inten-
sity indicated that FRET-P4 showed much faster response to
H2S than FRET-P1 (Fig. S5†). According to a previous literature
report,17 introducing an electron donor group on the ortho- or
para- of the azidobenzyl moiety could promote the intramole-
cular electron transfer and 1,4 or 1,6-elimination. FRET-P5 con-
taining the methoxy group on the phenyl moiety further
enhanced the kinetics of H2S triggered fluorescence emission, as
shown in Fig. 1. By further simple separation and mass spectra
analysis, we confirmed the existence of the product [4′-(N,N′-di-
methylamino)phenylazo]benzoyl acid (DABSYL acid) (Fig. S9†).
After the evaluation of these FRET based probes, FRET-P5
showed the best sensitivity for the response of H2S with a
12-fold enhancement in fluorescence intensity with 100 μM
NaHS in 60 min, as shown in Fig. 1 and S6.† However, only
2.5, 1.3, 1.3 and 2.5 fold enhancements were observed for
FRET-P1, FRET-P2, FRET-P3 and FRET-P4, respectively, with
relative slower response under the same conditions.
Fig. 2 Fluorescence responses of 100 μM FRET-P5 to relevant RSS
species. Bars represent the mean fluorescence responses at 1 h after the
addition of 100 μM NaHS or 1 mM of all other relevant RSS species
(reduced GSH and L-Cys set at 5 mM or 10 mM). All measurements were
done in fluorometer. Data were acquired at 37 °C in 10 mM degassed
solution (PBS–DMF, v : v = 6 : 4, pH 7.4) with excitation at 488 nm.
lytes and 6.4–8.7 folds over the presence of all other RSS
species with high concentrations (1 mM for NaHSO3, Na2S2O3
and Na2S2O5, and 5 mM for reduced GSH and L-Cys) in 60 min
at room temperature. Even with higher concentration up to
10 mM of reduced GSH and L-Cys, FRET-P5 still showed good
selectivity for H2S. In addition to reactive sulfur species, we
also evaluated the probe over other anions, reactive oxygen
species (ROS), reactive nitrogen species (RNS) and 10% FBS in
RPMI-1640. All these species and medium did not trigger fluo-
rescence emission and had no obvious effect on the detection
of H2S with the probe (Fig. S7†).
Selectivity of FRET-P5 for the detection of H2S
Due to the outstanding fluorescence response of FRET-P5 for
the detection of H2S, it was selected for further evaluation.
Fig. 2 shows the selectivity for sulfide over biologically relevant
Concentration dependence of FRET-P5 for fluorescence
reactive sulfur species (RSS), such as reduced GSH, L-Cys, detection of H2S
NaHSO3, Na2S2O3 and Na2S2O5. With the inclusion of 100 μM
FRET-P5, the relative fluorescence intensity in the presence of
100 μM NaHS was 12 folds over probe solution without ana-
The concentration dependence of the triggered fluorescence
intensity on H2S was also evaluated, as shown in Fig. 3. The
Fig. 3 Linear correlation of fluorescence intensity toward H2S concen-
tration using fluorospectrophotometer: FRET-P5, 100 μM; NaHS, 0, 1,
10, 25, 50, 100, μM, data were acquired at 37 °C in 10 mM degassed
solution (PBS–DMF, v : v = 6 : 4, pH 7.4, diamond) and (commercial fetal
bovine serum–DMF, v : v = 6 : 4, pH 7.4, square) with excitation at
488 nm. Emission was collected 60 min after the addition of NaHS.
Fig. 1 Fluorescence response of 100 μM FRET-P5 to 100 μM NaHS.
Data were acquired at 37 °C in 10 mM degassed solution (PBS–DMF, v : v
= 6 : 4, pH = 7.4) with excitation at 488 nm. Emission was collected from
500 to 650 nm at 0, 5, 10, 15, 20, 30, 40, 50 and 60 min after the
addition of 100 μM NaHS.
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probe FRET-P5 was treated with H2S in degassed buffer and
fetal bovine serum at various concentrations. The fluorescence
enhancement of FRET-P5 solutions (100 μM) was monitored
using a fluorescence microplate reader at 37 °C. Standard
curves for FRET-P5 in degassed buffer and fetal bovine serum
were obtained between fluorescence signal at 538 nm and the
concentrations (μM) of H2S for FRET-P5 in 60 min incubation.
The regression equations were FEx/Em(488/538 nm) = 0.692 [H2S] +
6.39 with R2 = 0.997 and FEx/Em(488/538 nm) = 0.395 [H2S] + 9.47
with R2 = 0.989 for degassed buffer and fetal bovine serum
respectively. The relatively low slope for fetal bovine serum
may be due to the fact that proteins presented in plasma are
scavengers for H2S.18
Detection of H2S in HeLa cells using a high content analyzer
We then tested the potential application of FRET-P5 for the
detection of H2S in HeLa cells. First, the cell viability was
tested with FRET-P5. As indicated in Fig. S8,† no obvious toxi-
city was observed with the probe concentration up to 100 μM.
HeLa cells were first incubated with 10 μM FRET-P5 PBS buffer
for 30 min in a 96-well plate and then washed with PBS buffer.
Cells were further incubated without or with 100 μM NaHS for
another 60 min. After final washing, the cells were then sub-
jected to imaging and analysis by a High Content Analyzer
(Operetta, Perkin Elmer). As shown in Fig. 4, the addition of
H2S induced intense intracellular fluorescence emission than
that without H2S addition. Reduced glutathione was previously
used as the substance by CBS for enzymatic production of
endogenous H2S.14b When HeLa cells were treated with
reduced glutathione (250 μM), we also observed the increase in
fluorescence emission of cells which indicated more gene-
ration of endogenous H2S by the enzyme in cells. Further ana-
lysis indicated that the addition of exogenous H2S (100 μM)
induced about 2 fold enhancement in fluorescence emission,
and the endogenous product of H2S by glutathione induced
about 1.6 fold enhancement in fluorescence emission.
Fig. 4 Visualization (I) and quantification (II) of exogenous and
endogenous H2S with FRET-P5 in HeLa cells by a high content analyzer.
A: cells were incubated with FRET-P5 (10 μM, 20 min) followed by PBS
washing and further incubated for 60 min; B: cells pre-stimulated by
PMA (1 μg ml−1, 30 min) were treated with NaHS (100 μM, 60 min) after
incubation with FRET-P5 (10 μM, 20 min); C: cells were treated with
glutathione (250 μM, 60 min) after the incubation with FRET-P5 (10 μM,
20 min); D, E, F: the images of A, B, C with Hoechst staining at 4 °C,
respectively; scale bars represent 100 μm.
Attachment of an electron donor (methoxy) on the aromatic
moiety (FRET-P5) further increased the kinetics of the azido
reduction by H2S. Further evaluation of FRET-P5 showed its
high sensitivity and selectivity for the detection of H2S over
other biologically relevant RSS, ROS, RNS and common ions.
The linear relationships were well obeyed between the fluo-
rescence intensity of released fluorescein and the concen-
trations of H2S in both degassed buffer and fetal bovine
serum. The study indicated that a general designing strategy
may be used for sensors with many commercially available
fluorophores. And the structure/function relationship provides
more insights into designing more chemoprobes based on
FRET fluorophores and self-immolative linkers.
Conclusions
We designed and synthesized a series of fluorescent probes
(FRET-P1, FRET-P2, FRET-P3, FRET-P4 and FRET-P5) for the
detection of H2S based on FRET. Azido functionalized self-
immolative linkers were used for the linkage of fluorescein
and DABCYL. With the reduction of the azido moiety by H2S, a
1,4 or 1,6-quinone-methide rearrangement triggered the break-
age of linkers and the release of fluorescence, and induced the
increase in fluorescence intensity. And quinone-methide
rearrangement is the rate-limiting step for the quick response
to H2S with these FRET probes. An electron acceptor group on
the aromatic moiety (FRET-P2 and FRET-P3) enhanced the
Acknowledgements
reduction of the azido moiety by H2S, but decreased the This work was supported by the National Basic Research
quinone-methide rearrangement, which led to less efficient Program of China (973 Program; grant no. 2013CB933800 and
fluorescence detection of H2S. Shortening two steps (FRET-P1) 2012CB720600), the National Natural Science Foundation of
to one step (FRET-P4) of quinone-methide rearrangement China (grant no. 21372018) and the State Key Laboratory of
improved the kinetics of H2S triggered fluorescence emission. Pharmaceutical Biotechnology (grant no. KF-GN-201305).
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