Cascade reaction and FRET-based fluorescent probe for the colorimetric and ratiometric signaling of hydrogen sulfide

Article abstract of DOI:10.1016/j.tetlet.2015.04.054

A novel addition-elimination cascade reaction and FRET-based fluorescent probe for the colorimetric and ratiometric signaling of hydrogen sulfide was designed. Employing of 2-formylbenzoic acid as the trapper, the probe was highly selective and sensitive to H₂S over other biologically relevant species to give color change from colorless to bright orange for naked eye observation. A linear response to H₂S in the range of 0-200 μM indicated that it could detect H₂S quantitatively with a detection limit as low as 0.39 μM. Furthermore, fluorescent inverted microscope imaging experiments demonstrated that the probe could assess intracellular H₂S level change.

Full text of DOI:10.1016/j.tetlet.2015.04.054

Accepted Manuscript
Cascade reaction and FRET-based fluorescent probe for the colorimetric and
ratiometric signaling of hydrogen sulfide
Kunzhu Huang, Meihui Liu, Xiaobo Wang, Dongsheng Cao, Feng Gao, Wei
Wang, Wenbin Zeng
PII:
S0040-4039(15)00691-7
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.04.054
TETL 46193
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 March 2015
10 April 2015
14 April 2015
Please cite this article as: Huang, K., Liu, M., Wang, X., Cao, D., Gao, F., Wang, W., Zeng, W., Cascade reaction
and FRET-based fluorescent probe for the colorimetric and ratiometric signaling of hydrogen sulfide, Tetrahedron
Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.04.054
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Cascade reaction and FRET-based fluorescent probe Leave this area blank for abstract info.
for the colorimetric and ratiometric signaling of
hydrogen sulfide
Kunzhu Huanga, Meihui Liua, Xiaobo Wanga, Dongsheng Caoa, Feng Gaob, Wei Wangb and Wenbin Zenga,*.

1
Tetrahedron Letters
journal homepage: www.els evier.com
Cascade reaction and FRET-based fluorescent probe for the colorimetric and
ratiometric signaling of hydrogen sulfide
Kunzhu Huanga, Meihui Liua, Xiaobo Wanga, Dongsheng Caoa, Feng Gaob, Wei Wangb and Wenbin
Zenga,*.
aSchool of Pharmaceutical Sciences, bThe Third Xiangya Hospital, Central South University, 172 Tongzipo Road, Changsha, 410013, P. R. China.
ARTICLE INFO
ABSTRACT
Article history:
Received
A novel addition-elimination cascade reaction and FRET-based fluorescent probe for the
colorimetric and ratiometric signaling of hydrogen sulfide was designed. Employing of 2-
formylbenzoic acid as the trapper, the probe was highly selective and sensitive to H2S over other
biologically relevant species to give color change from colorless to bright orange for naked eye
observation. A linear response to H2S in the range of 0-200 µM indicated that it could detect H2S
quantitatively with a detection limit as low as 0.39 µM. Furthermore, fluorescent inverted
microscope imaging experiments demonstrated that the probe could assess intracellular H2S
levels change.
Received in revised form
Accepted
Available online
Keywords:
Hydrogen sulfide
2009 Elsevier Ltd. All rights reserved.
Cascade reaction and FRET
Colorimetric and ratiometric fluorescent probe
Live cells’ imaging
Hydrogen sulfide, along with nitric oxide and carbon
monoxide, has recently emerged as an important endogenous
signaling molecule. 1−2 It is well accepted that the physiological
level of H2S regulated a number of processes in cardiovascular,3
immune,4 endocrine,5 as well as the gastrointestinal6 and central
nervous system.7 On the other hand, H2S levels are abnormal in
diseases ranging from Alzheimer’s disease,8 and chronic kidney
disease,9 to liver cirrhosis10 and Down’s syndrome.11 Thus, to
better understand the physiological and pathological functions of
H2S, selective tracking and quantifying this small molecule is
increasingly crucial and has attracted a wide research interest.
When compared with sophisticated techniques for H2S
detection, fluorescence techniques are preferred by virtue of their
high sensitivity/selectivity, high spatiotemporal resolution and
non-destructive advantages.12 Since 2011, several types of
fluorescent probes have been designed. Among them, fluorescent
probes displaying dual functions with both colorimetric and
ratiometric properties could be more useful in convenient visual
sensing by the “naked eye” and in quantitative fluorometric
detection via their internal calibration of dual emissions.13−18
Currently, several dual-function colorimetric and ratiometric
fluorescent probes for H2S have been reported. However, there
are still some limitations. For example, long response time is
often required, especially for most of those probes based on the
nitro reduction approach.14 Besides, the selective detection of
H2S is still challenging due to the interference of other
biothiols.17 In addition, the defects of the probe itself could also
restrict its wide application such as small Stokes’ shift,16 high
detection limits,14 excitation wavelength in the UV region18 or
employment of labile cyanine dyes and zido fluorogens.15
Therefore, it is quite necessary and important to exploit new
probes with improved properties. Herein, we reported an
addition-elimination cascade reaction and FRET-based dual-
function colorimetric and ratiometric fluorescent probe CR-FBA
(as shown in Scheme 1), which featured good selectivity, high
sensitivity and excellent photostability. More importantly, it was
successfully applied to detect H2S in living cells.
The synthesis of probe was outlined as shown in Scheme 2.19
Treatment of 4-(diethylamino)-2-hydroxybenzaldehyde with
diethyl malonate in the presence of piperidine afforded 2, which
was then reacted with Sodium hydroxide to yield 7. Compound 4
and 6 could easily provide the rhodamine/fluorescein acceptor
building block 8. The key intermediate FRET dyad CR was
readily synthesized by condensation reaction of carboxyl
compound 7 with amide 8 in the presence of HBTU and DIEA in
good yield. The target compound CR-FBA was achieved through
the esterification reaction from CR and 2-formylbenzoyl
chloride. The structural characterization of the probe was
characterized by standard 1H-NMR, 13C-NMR, and HRMS.
Initially, colorimetric responses of probe (10 µM) to a range of
biological species were investigated. As we expected, a distinct
visible color changing from colorless to bright orange was
Scheme 1. The proposed mechanism of probe for H2S detection.

2
Tetrahedron Letters
Scheme 2. Synthesis of CR-FBA.
Figure 1. Changes in visible color of probe (10 µM) with NaHS
(400 µM) and other various biological species (4 mM). Conditions:
acetonitrile-PBS buffer solution (60 mM, pH 7.4, 1:9 v/v) at 25 oC
for 25 min.
observed upon the addition of NaHS (400 µM) (Figure 1). In
contrast, no significant color change was generated by treatment
−
2−
species (SO32−, HSO3 , and S2O3 ), non-thiol amino acids (Lys,
Glu, Arg) and biothiols (GSH, Cys, and Hcy), even at a high
concentration (4 mM). These observations clearly indicated that
probe exhibited high selectivity for naked-eye detection of H2S
over other biological species.
Fluorescence responses of CR-FBA to different biological
species were also studied as shown in Figure 2. Notably, the
probe revealed no significant responses toward common anions
−
−
−
−
2−
−
(F , I , N3 , and CN ), reactive sulfur species (SO3 , HSO3 , and
2−
S2O3 ), non-thiol amino acids (Lys, Glu, Arg) and biothiol
(GSH). And only two selected biothiols (Cys and Hcy)) induced
small fluorescence changes. By contrast, upon treatment of NaHS
with the probe, the most significant fluorescence changes were
observed, which demonstrated its high selectivity toward H2S.
Figure 3. Fluorescence responses of probe (10 µM) in the presence
of 0-80 equiv of NaHS. Conditions: excitation wavelength is 416
nm, acetonitrile-PBS buffer solution (60 mM, pH 7.4, 1:9 v/v) at 25
oC for 25 min.
The concentration-dependent fluorescence spectra were further
examined. As shown in Figure 3A, in the absence of NaHS, the
emission spectrum of probe exhibited an emission band with
maxima at 474 nm that are typical of the coumarin moiety,
demonstrating that there was no FRET in the free probe. Upon
the gradually addition of NaHS, such emission band gradually
decreased along with a new red-shift emission band at 542 nm
and a well-defined isoemission point at 525 nm. The intensity
ratios (I542/I474) reached the saturation state when adding 40 equiv
NaHS. An obvious increase in I542/I474 changing from 0.16 to
2.65 (Figure 3B) was also observed. These results confirmed the
existence of FRET, as the conversion of rhodamine/fluorescein
Figure 2. Fluorescence spectra of CR-FBA (10 µM) upon the
addition of NaHS (400 µM) and other various biological species (4
mM). Conditions: excitation wavelength is 416 nm, acetonitrile-PBS
buffer solution (60 mM, pH 7.4, 1:9 v/v) at 25oC for 25 min.

3
Figure 6. Fluorescence response of CR-FBA with NaHS in living
H9C2 cells. The cells were pre-treated with the probe (10 µM) for 20
min, and then incubated with NaHS (A) 0 µM, (B) 100 µM, (C) 400
µM for 25 min. Blue excitation (420-485 nm), green emission (> 515
nm), Scale bars: 10 µm.
HPLC-MS measurements were carried out (Figure S7). The
results demonstrated that the reaction between probe and NaHS
proceeded as illustrated in Scheme 1.
We thereafter sought to apply whether CR-FBA can sense H2S
in living cells. The cell line H9C2 was selected as a bioassay
model. Incubation of H9C2 cells with probe (10 µM) for 20 min
o
at 37 C was followed by the addition of NaHS for another 25
Figure 4. Time-dependent fluorescence intensity changes of probe
(10 µM) at 542 nm upon addition of varied concentrations of NaHS.
Conditions: excitation wavelength is 416 nm, acetonitrile-PBS buffer
solution (60 mM, pH 7.4, 1:9 v/v) at 25oC .
min. Subsequently, fluorescence imaging experiments were
carried out by a fluorescent inverted microscope. As shown in
Figure 6, H9C2 cells showed almost no fluorescence with only
probe, while in the presence of probe and NaHS, displayed
enhanced fluorescence. And more, with the increase of NaHS
concentration, the fluorescence intensity increased as well.
Therefore, these experiments results indicated that CR-FBA has
the potential to be used to detect hydrogen sulfide in living cells.
In summary, we have developed a new colorimetric and
ratiometric fluorescent probe CR-FBA for H2S based on FRET
mechanism and a novel addition-elimination cascade reaction.
With the employing of 2-formylbenzoic acid as the trapper, CR-
FBA was highly selective and sensitive to H2S over other
biologically relevant species to give color change from colorless
to bright orange under naked eye observation. A good linearity
between the fluorescence intensity ratios and the concentrations
of NaHS in the range of 0-200 µM suggested that it can detect
H2S quantitatively with a great limit as low as 0.39 µM.
Furthermore, preliminary fluorescence imaging experiments
showed that probe has potential to assess intracellular H2S levels
change in H9C2 cells. In view of the increasing interest for
biological research of H2S, we expect that the probe has great
potential for in vitro and in vivo applications as a functional and
elucidative tool.
from the spirolactam ring to open-ring form. Besides, a good
linearity between the fluorescence intensity ratios and the
concentrations of NaHS was obtained in the range of 0-200 µM
(Figure 3C). According to fluorescence titration data, the limit of
detection was calculated to be 0.39 µM.
The time-dependent fluorescence responses of probe were
monitored (Figure 4). The results showed that probe was quite
stable under the test conditions, and emission intensity at 542 nm
gradually increased against time until reached a plateau at about
10 min when 40 equiv of NaHS was added. Although the longer
time (25 min) was required to meet the signal saturation while
treated with the lower concentration of NaHS, distinct
fluorescence signal changes could be observed within 15 min.
It is well known that the performance of a fluorescent probe is
highly dependent on the pH of the medium, thus, a pH-dependent
fluorescent responses was carried out. As shown in Figure 5,
I542/I474 of probe was hardly affected between broad range of pH
from 1.0 to 11.0. Upon addition of NaHS, it could respond to H2S
with a remarkable I541/I474 enhancement from pH 6.0 to 11.0.
These results indicated the good fluorescence responses of probe
for physiological environment application.
Acknowledgements
The optical changes of CR-FBA in the presence of NaHS
suggested that H2S specifically triggered the nucleophilic
addition-cyclization cascade reaction, simultaneously; the
compound FRET dyad CR was released. To confirm this,
We are grateful to National Natural Science Foundation of
China (81271634), Doctoral Fund of Ministry of Education of
China (No. 20120162110070), and Hunan Provincial Natural
Science Foundation of China (12JJ1012).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi. ·····.
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