A ratiometric fluorescent probe for biological signaling molecule H 2S: Fast response and high selectivity

Article abstract of DOI:10.1002/chem.201300455

Selective and fast: A flavylium derivative-based ratiometric fluorescent probe (1) for H₂S is reported. The reaction mechanism (see scheme) is based on the nucleophilic addition of H₂S towards the electrically positive benzopyrylium moiety of 1, which can efficiently differentiate H ₂S from other competitive species. The probe exhibits a fast response toward H₂S, within 10 s, which is superior to most of the reported H₂S probes. Copyright

Full text of DOI:10.1002/chem.201300455

COMMUNICATION
DOI: 10.1002/chem.201300455
A Ratiometric Fluorescent Probe for Biological Signaling Molecule H S:
2
Fast Response and High Selectivity
[
a]
[a]
[a]
[b]
[c]
Jing Liu, Yuan-Qiang Sun, Jingyu Zhang, Ting Yang, Jianbin Cao,
[
a]
[a]
Lishen Zhang, and Wei Guo*
Traditionally, hydrogen sulfide (H S) was considered to be
cient sensitivity, because the physiologically relevant H2S
concentration has been reported to range from nano- to mil-
limolar levels; and the third challenge is fast response under
2
a toxic gas. Later, it was reported that the typical concentra-
[1]
tion of H S in blood is in the range 10–100 mm, suggesting
2
its connection with biological functions. Indeed, recent stud-
mild condition, because H S is metabolized rapidly.
2
ies regard H S as the third gaseous transmitter besides nitric
Previously reported fluorescent probes for H S are mainly
focused on in vitro assay systems. Recently, fluorescent
2
2
[2]
[6]
oxide (NO) and carbon monoxide (CO). For example, H2S
has been recognized to mediate a wide range of physiologi-
cal effects, including regulation of cell growth, vasodilation,
and angiogenesis, mediation of neurotransmission, inhibition
probes that can potentially be used for H S detection in
2
[7]
living systems have become available, and the correspond-
ing design strategies are based on several significant charac-
[3]
[8–10]
of insulin signaling, and regulation of inflammation. More-
over, it acts as an antioxidant or scavenger of reactive
teristic properties of H S, namely dual nucleophilicity,
2
good reducing property towards azide, nitro and hydroxy-
[4]
[11–13]
oxygen species (ROS). Furthermore, studies have shown
that its deregulation is correlated with the symptoms of Alz-
heimerꢀs disease, Downꢀs syndrome, diabetes, and liver cir-
A
H
N
T
E
N
N
amine groups,
high binding affinity towards copper
[14]
[15]
ion, and efficient thiolysis of dinitrophenyl ether. These
unique properties can efficiently differentiate H S from
2
[5]
rhosis. Although H S has been recognized to be linked to
other completive species, in particular, millimolar concentra-
tions of biothiols found inside most cells, thereby enabling
2
various physiological and pathological functions, many of its
underlying molecular events remain unknown. Therefore,
the detection of H S in complicated biological systems.
2
efficient methods to sensitively and selectively detect H S in
However, most of the reported H S probes display a delayed
2
2
living systems are urgently required.
response time (more than 20 min)—except for dansyl
[12a]
[14a]
Fluorescence spectroscopy is a powerful tool for sensing
and imaging trace amounts of samples because of its sim-
plicity, sensitivity, real-time imaging, and especially its non-
destructive detection of target biomolecules in live cells or
tissues. Undoubtedly, our understanding of molecular func-
tion in cell biology has significantly benefited from advance-
ments of fluorescent probes. Generally, the design of fluo-
azide
and HSip-1
(within seconds) reported by Wang
and Nagano, respectively—and, thus, are not satisfactory for
real-time determination of the fluctuations in H S concen-
2
tration in biological systems. This is a problem to be solved.
Ratiometric fluorescent probes can eliminate most or all the
factors that interfere with the signal output, such as instru-
mental efficiency, environmental conditions (pH, polarity,
temperature, and so forth), and the localization of the
probe, by built-in correction of two emission bands, and,
thus, are more favorable in comparison with fluorescence in-
rescent probes for detection of H S in living systems in-
2
volves several substantial challenges: one is to attain suffi-
cient selectivity over other biothiols, including reduced glu-
tathione (GSH, present at levels of about 1–10 mm) and l-
cysteine (l-Cys, about 100 mm); the other is to achieve suffi-
[16]
tensity-based probes. Although a few ratiometric fluores-
[
10,12d,17]
cent probes for H S have been reported,
most of
them still display a delayed response time (more than
0 min) except for CouMC (30 s) reported by Guo and He
2
2
+
+
[
a] J. Liu, Y.-Q. Sun, J. Zhang, L. Zhang, Prof. Dr. W. Guo
School of Chemistry and Chemical Engineering
Shanxi University, 92 Wucheng Road
Taiyuan 030006 (P.R. China)
[17c]
only recently.
Therefore, new methods are required to
meet not only the good selectivity, but also the fast and ex-
cellent ratiometric fluorescence response.
Fax: (+86)351-7011600
E-mail: guow@sxu.edu.cn
Here, we report a new fluorescent probe for the detection
of H S based on a flavylium derivative 1. The probe can effi-
2
[
b] T. Yang
Institute of Biotechnology, Shanxi University (P.R. China)
c] J. Cao
Research Institute of Applied Chemistry
ciently differentiate H S from biothiols and other biological-
2
ly related species based on the selective nucleophilic attack
[
of H S to the electrically positive benzopyrylium moiety of
2
the probe. To the best of our knowledge, such an addition
reaction has never been used to develop a selective fluores-
Shanxi University (P.R. China)
+
[
] These authors contributed equally to this work.
cent probe for H S previously. The value of the probe could
be confirmed by the fast response (within 10 s), the excel-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300455.
2
Chem. Eur. J. 2013, 19, 4717 – 4722
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4717

lent ratiometric fluorescent response (emission shift:
05 nm; 1200-fold enhancement in ratiometric value), and
To test the above-mentioned possibility, we decided to
synthesize and examine the spectra profiles of 1. Compound
2
the high selectivity over other biologically related species in-
cluding biothiols, thereby providing a novel strategy for
1 was easily prepared by condensation of 3-acetyl-7-(dieth
ACHTUNGTRENNUNGy l-
AHCTUNGTREGaNNUN mino)coumarin (2) and 4-(diethylamino)salicylaldehyde (3)
[
19]
probing H S chemistry in biological systems.
in conc. H SO , and its structural identification was con-
2
2
1
4
13
Initially, we focused our attention on searching molecular
firmed by H and C NMR spectroscopy, and ESI-MS (see
the Supporting Information).
probes that are capable of detecting H S over other cellular
2
molecules, in particular, biothiols found inside most cells.
The UV/Vis spectrum of 1 shows the maximum absorp-
tion at 655 nm in PBS buffer (10 mm, pH 7.4, containing
We noted that H S in aqueous solution has a pK of 7.0
2
a
whereas biothiols have higher pK values (Cys, 8.30; GSH,
30% CH CN; Figure 2). With increasing NaHS (a common-
a
3
9
.20). Thus, H S should be a better nucleophile than bio-
2
thiols in physiological media. However, the major challenge
is to find an appropriate receptor so that it can selectively
bind H S over other biothiols based on their different nucle-
2
[8b,9]
[18]
ophilicity, because H2S
and biothiols, such as Cys and
GSH, have been reported to have good reactivity with acti-
vated olefin. To our delight, we found that commercially
available pyronine dye could promptly react with H S in
2
aqueous solution (Figure 1A and Figures S1–S3 in the Sup-
Figure 2. UV/Vis spectra of 1 (10 mm) upon addition of increasing concen-
trations of NaHS (0–20 equiv) in PBS buffer (10 mm, pH 7.4, containing
3
3
0% CH CN). Each spectrum was recorded after 10 s.
ly employed H S donor) concentration (0–20 equiv), the ab-
2
sorption peaks at 655 nm gradually decreased and a new
peak appeared at 415 nm, which is the typical absorption of
Figure 1. The proposed sensing mechanism for H
nine with H S in aqueous solution; B) reaction of 1, a flavylium deriva-
tive with NIR emission with H S.
2
S. A) Reaction of pyro-
7-diethylaminocoumarin. Simultaneously,
a well-defined
2
isosbestic point was noted at 455 nm, indicative of the for-
AHCTUNGTRENNUNG
2
mation of the 1–HS adduct as well as the clean chemical
transformation. Noteworthy is that time-dependent UV/Vis
spectra of 1 in the presence of NaHS (Figure S5 in the Sup-
porting Information) revealed that the reaction is considera-
bly fast, and can be finished within 10 s, indicative of the
fast reaction kinetics. Indeed, the response time is superior
porting Information), and, more importantly, displayed ex-
cellent selectivity for H S over biothiols (Figure S4 in the
2
Supporting Information). Although an undesired fluorescent
turn-off response was obtained due to the interruption of p-
conjugation of pyronine ring, the reaction inspired us to test
the reactivity of 1, a flavylium derivative with near-infrared
to most of the reported H S probes, and comparable to that
2
[12a]
[14a]
of dansyl azide
and HSip-1
(within seconds). In addi-
tion, the probe is stable in a wide pH region (1–9), and dis-
[19]
(
NIR) emission, towards H S (Figure 1B), because there
plays the best response for H S in the physiological pH
2
2
is a similar structural characteristic between 1 and pyronine.
range (Figure S6 in the Supporting Information).
We envisioned that the nucleophilic attack of H S toward
In addition, the 1–HS adduct could be easily obtained by
2
the benzopyrylium moiety of 1 will interrupt the p-conjuga-
tion, thereby leading to the decrease of the NIR emission.
Also, since the adduct contains an intact 7-diethylaminocou-
marin fluorophore the reaction was expected to induce an
enhancement of the blue emission from this moiety. There-
fore, two well-separated emission peaks before and after the
treating 1 with 10 equiv NaSH in CH CN/PBS buffer
3
(10 mm, pH 7.4, 1:1, v/v) with good yield (see the Supporting
Information). It was found that the adduct is stable enough
in solid state, but gradually decomposed to its starting mate-
rial 1 in organic solution, suggesting that the chemical reac-
tion is reversible. Although it was difficult for us to conduct
a structural identification of the adduct by NMR spectrosco-
py due to its instability, the HRMS spectrum of 1 in the
presence of NaHS showed a major signal at m/z 449.1903
(calcd: 449.1899; Figure S7 in the Supporting Information),
addition of H S could be obtained due to the distinct emis-
2
sion between dye 1 and 7-diethylaminocoumarin, enabling
the detection of H S with the efficient ratiometric fluores-
2
cence strategy.
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Chem. Eur. J. 2013, 19, 4717 – 4722

A Ratiometric Fluorescent Probe for H2S
COMMUNICATION
À
1
which could be assigned as [1+HS-1] . Further, H NMR ti-
not only contributes to the accurate measurement of the in-
tensities of the two emission peaks, but also results in a
huge ratiometric value. In fact, in the presence of 20 equiv
NaHS, an approximately 1200-fold enhancement in ratio-
metric value of I485/I690 (from 0.07 to 83.90) is achieved with
respect to the NaHS-free solution.
tration studies showed that almost all aromatic proton sig-
nals shifted to up-field upon addition of HS to the solution
À
of 1; this is consistent with the proposed product in which
the cationic benzopyrylium moiety is changed into a neutral
one (Figure S8 in the Supporting Information). We also ob-
served the saturated ÀCHÀ proton adjacent to the SH group
Next, we examined the fluorescence spectra changes of 1
¯ ¯
¯
¯
¯
¯
of the proposed adduct, where two signals (3.7 and 3.8 ppm)
(10 mm) incubated with various anions (F, Cl, B r¯ , I, AcO, N ,
3
2À
2À
3À
for the ÀCHÀ proton could be found because the adduct re-
NO , SCN, SO , CO3 , PO4 , and CN; 40 equiv of each),
ROS (H O , COH, ClO, O , NO , NO; 20 equiv of each), and
¯
¯
3
4
¯ ¯
¯
action results in a chiral C-4 center. On the basis of the
above results, the proposed reaction mechanism shown in
Figure 1B is strongly supported.
2 2 2
2
biothiols (Cys, 1 mm; GSH, 5 mm) in PBS buffer (10 mm,
pH 7.4, containing 30% CH CN; Figure 4 and Figures S10
3
To examine the ratiometric fluorescence response of 1 to
H S, the probe was titrated with different concentration of
2
NaHS in the same conditions. As shown in Figure 3, the free
probe displayed an NIR emission peak with the maximum
Figure 4. Fluorescence intensity ratio (F485/F690) of 1 (10 mm) in PBS
buffer (10 mm, pH 7.4, containing 30% CH CN) in the presence of vari-
3
À
À
À
À
À
À
3
ous ionic species. 1: 1 only, 2: F , 3: Cl , 4: Br , 5: I , 6: AcO , 7: N , 8:
À
À
2À
2À
3À
À
NO
each); 14: H
3
, 9: SCN , 10: SO
4
, 11: CO
, 15: COH, 16: ClO , 17: O
of each); 20: Cys (1 mm), 21: GSH (5 mm), 22: HS (20 equiv). Data
were recorded after 1 min; lex =450 nm; slit: 5/10 nm.
3
, 12: PO
4
, 13: CN (40 equiv of
À
À
À
2
O
2
2
, 18: NO
2
, 19: NO (20 equiv
À
Figure 3. Emission spectra of 1 (10 mm) upon addition of increasing con-
centrations of NaHS (0–20 equiv) in PBS buffer (10 mm, pH 7.4, contain-
and S11 in the Supporting Information). As can be seen,
these competitive species did not lead to any significant flu-
ing 30% CH
slit: 5/10 nm.
3
CN). Each spectrum was recorded after 10 s; lex =450 nm;
orescence changes of 1, and only H S elicited a dramatic ra-
2
tiometric fluorescence change. We also examined several
other nucleophiles, such as l-cysteine methyl ester hydro-
chloride, amines, ammonium, and thiosulfate. Similarly,
these species induced no obvious change of emission of 1
(Figure S12 in the Supporting Information). In addition, the
at 690 nm (l =450 nm). Its quantum yield was determined
ex
to be 0.035 in this condition, with itself (F=0.34, in
[19]
CH CN)
as reference. Upon addition of NaHS (0–
3
20 equiv), the emission intensity at 690 nm gradually de-
high selectivity of 1 towards H S is observable by the naked
2
creased with the simultaneous appearance of a new blue
emission peak centered at 485 nm, indicating that the chemi-
cal reaction interrupted the p-conjugation of 1, after which
the fluorescence of 7-diethlyaminocoumarin recovered. The
observation is in good agreement with the aforementioned
design concept. Essentially, these changes in the fluores-
cence spectrum stopped and the ratio of the emission inten-
sities at 485 and 690 nm (I485/I690) became constant when the
amount of NaHS added reached 15 equiv (F=0.085 with
eye. When probe 1 was excited at 365 nm with a UV lamp
in the presence of various anions, only H S caused an obvi-
2
ous color change from red to blue (Figure 4, inset). These
results indicate that the structural motif of 1, unlike the
[18]
common Michael receptors,
is considerably efficient in
differentiating H S from biothiols.
2
To explore the practical application of probe 1, we also
repeated the above experiments in pure PBS buffer (10 mm,
pH 7.4; Figures S13–S16 in the Supporting Information). It
was found that, in this condition, 1 also displayed good ra-
tiometric fluorescent response (emission shift: 195 nm; 294-
fold enhancement in ratiometric value; Figure S14 in the
Supporting Information), excellent selectivity (Figure S15 in
the Supporting Information), and fast response time (20 s;
fluorescein as reference). The detection limit for H S was
2
determined to be 0.14 mm based on signal-to-noise ratio (S/
N)=3 (Figure S9 in the Supporting Information). Especially
important is that the difference in the two emission wave-
lengths is very large (emission shift: DF=205 nm), which
Chem. Eur. J. 2013, 19, 4717 – 4722
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4719

W. Guo et al.
Figure S16 in the Supporting Information) for H S, whereas
2
decreased fluorescent intensity was observed. In fact, for
many fluorescence probes, the decreased reactivity and fluo-
rescent intensity in water solution could commonly be ob-
served due to the strong solvation and the efficient quench-
ing of highly polar water molecules. In addition, to avoid
cellular autofluorescence, excitation at wavelengths longer
than 400–460 nm is essential. Basically, when 1 is excited at
Figure 5. A) Resonance structures of the cationic benzopyrylium moiety
of 1. B) The proposed electrostatic repulsion interaction between the cat-
ionic benzopyrylium moiety of 1 and the cationic ammonium group of
Cys.
460 or 470 nm instead of the initial 450 nm, a good ratiomet-
ric fluorescent response could still be obtained (Figure S17
in the Supporting Information).
and the results showed that the probe at a concentration of
10 mm has only minimal cytotoxicity after 24 h (Figure S18 in
the Supporting Information). Thus, the probe at 10 mm was
selected for imaging experiments in living cells. HeLa cells
were incubated with 1 (10 mm) in culture medium for 30 min
at 378C. Probe 1 was found to be cell permeable, and the
cells showed strong fluorescence in the red channel (Fig-
ure 6A) and weak fluorescence in the green channel (Fig-
In fact, the high selectivity of 1 towards H S is impressive.
2
Especially, given that millimolar concentrations of Cys/GSH
elicited almost no fluorescence changes of 1 even in a
lengthy time of 30 min (Figure S12 in the Supporting Infor-
mation). We initially attributed this to the lower pK value
a
of H S compared to Cys/GSH, as mentioned before. Howev-
2
er, it is surprising that mercaptoethanol, which has a pKa
value of 9.6, could also react
with 1 and elicited a ratiometric
fluorescence response (Fig-
ure S15 in the Supporting Infor-
[20]
mation),
although less effec-
tively than H S. Therefore, it
2
seems not convincing enough to
attribute the high selectivity of
1
towards H S over Cys/GSH to
2
the big difference in their pKa
values. However, we noted that
the binding group in 1 is a cati-
onic benzopyrylium moiety, and
Cys or GSH also have a cation-
ic ammonium group. Therefore,
the electrostatic repulsion is
probably the main reason for
Figure 6. A) Fluorescence image of HeLa cells incubated with probe 1 (10 mm) from the red channel; B) fluo-
rescence image of (A) from the green channel; C) overlay of (A) and (B); D) bright field image; E) fluores-
the poor reactivity of Cys/GSH cence image of HeLa cells incubated with 1 for 30 min, and further incubated with NaHS (200 mm) for 30 min,
from the red channel; F) fluorescence image of (E) from the green channel; G) overlap of (E) and (F);
H) bright-field image.
with 1, because it could prevent
the effective collisions of the
two reactants (Figure 5). In
fact, this hypothesis could be
partly supported by the fact that probe 1 was also inert to l-
cysteine methyl ester hydrochloride, which bears a cationic
ammonium group (Figure S12 in the Supporting Informa-
ure 6B). In a control experiment, the cells were pretreated
with 1 for 30 min, and further incubated with NaHS
(200 mm) for 30 min, eliciting an obvious fluorescence de-
crease in the red channel (Figure 6E) and strong fluores-
cence in the green channel (Figure 6F); this is consistent
tion), but was active to the electrically neutral mercapto-
À
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
ethanol as well as anionic HS (Figure S15 in the Supporting
À
Information). In addition, the reactivity in the order HS >
with the H S-induced ratiometric fluorescence response. The
2
Cys/GSH is also understandable on the basis of steric-hin-
drance effects.
above results reveal that 1 is biocompatible, and could be
employed for ratiometric fluorescence imaging of H S in
2
Overall, the above results suggest that the new ratiometric
fluorescent probe is promising for applications in biological
systems. Moreover, the hydrophilicity of the electrically pos-
itive benzopyrylium and the lipophilicity of 7-diethylamino-
coumarin moiety in 1 mean that 1 has the advantage of
proper amphipathicity, which is an important factor for both
cell permeability and intracellular fluorescent imaging. Sub-
living cells. In addition, we evaluated probe 1 in human
serum with NaHS. The blood samples were treated accord-
[9a]
ing the reported literature procedure.
We found that
probe 1 still retains good ratiometric fluorescence response
to NaHS (Figure S19 in the Supporting Information), and
the reaction could be completed in 20 s at room tempera-
ture, indicating its potential for biological application.
In summary, we have developed a novel fluorescent probe
sequently, we tested the capability of 1 to image H S in
2
living cells. First, the MTT assay for probe 1 was conducted,
1 to detect H S with fast response and high selectivity under
2
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ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A Ratiometric Fluorescent Probe for H2S
COMMUNICATION
physiological conditions based on the novel nucleophilic ad-
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2
5
40–545; b) R. Hy sˇ pler, A. Ticha, M. Indrova, Z. Zadak, L. Hys-
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determination of H S with the attractive ratiometric fluores-
2
cence method, through which a huge H S-induced change in
2
the intensity ratio and a big emission shift could be obtained
before and after addition of H S. The obvious changes in
2
the fluorescence color can be observed by eye. Preliminary
confocal laser scanning microscopy experiments show that 1
has potential for practical application in living cells. These
results could trigger the development of new ratiometric flu-
orescence probes for the important biological signaling mol-
1
9, 1196–1198.
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1977.
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[
4] C. Yang, Z. Yang, M. Zhang, Q. Dong, X. Wang, A. Lan, F. Zeng, P.
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1
mmol) and 3-acetyl-7-(diethylamino)coumarin (2; 0.259 g, 1 mmol)
were dissolved in conc. H SO (5 mL) and stirred at 908C for 6 h. After
being cooled to room temperature, ice (10 g) then 70% perchloric acid
0.5 mL) were added; then the solution was filtered, and washed with
water to afford the crude product. The crude product was purified by
silica gel flash chromatography by using CH Cl /CH OH (10:1 to 20:1) as
eluent to afford 1 as a purple solid (yield, 92%). H NMR (DMSO,
2
4
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(
2
2
3
1
6
1
1
7
00 MHz): d=9.08 (s, 1H), 8.65 (d, J=8.4 Hz, 1H), 8.27 (d, J=8.4 Hz,
H), 7.95 (d, J=9.6 Hz, 1H), 7.73 (d, J=9 Hz, 1H), 7.42 (q, J=2.4 Hz,
H), 7.25 (s, 1H), 6.99 (q, J=1.8 Hz, 1H), 6.73 (s, 1H), 3.70 (q, J=
.2 Hz, 4H), 3.59 (q, J=7.2 Hz, 4H), 1.27 (t, J=7.2 Hz, 6H), 1.20 ppm (t,
1
3
6
J=7.2 Hz, 6H); C NMR (150 MHz, [D ]DMSO): d=163.5, 159.0, 158.5,
1
1
4
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Keywords: addition reaction · biosensors · fluorescence
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Received: December 18, 2012
Revised: February 5, 2013
Published online: March 4, 2013
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Chem. Eur. J. 2013, 19, 4717 – 4722