Development of a highly selective fluorescence probe for hydrogen sulfide

Article abstract of DOI:10.1021/ja207851s

Hydrogen sulfide (H₂S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H₂S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry

Full text of DOI:10.1021/ja207851s

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Development of a Highly Selective Fluorescence Probe for
Hydrogen Sulfide
†
†
‡
‡
‡
†
Kiyoshi Sasakura, Kenjiro Hanaoka, Norihiro Shibuya, Yoshinori Mikami, Yuka Kimura, Toru Komatsu,
†
†
‡
,†
Tasuku Ueno, Takuya Terai, Hideo Kimura, and Tetsuo Nagano*
†
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry,
‡
4
-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan
S Supporting Information
b
ABSTRACT: Hydrogen sulfide (H S) has recently been
2
identified as a biological response modifier. Here, we report
the design and synthesis of a novel fluorescence probe for
H S, HSip-1, utilizing azamacrocyclic copper(II) ion com-
2
plex chemistry to control the fluorescence. HSip-1 showed
high selectivity and high sensitivity for H S, and its potential
2
for biological applications was confirmed by employing it for
2
+
fluorescence imaging of H S in live cells.
2
Figure 1. Structures of four macrocyclic fluoresceinꢀCu conjugates:
2+ 2+
2+ 2+
(
A) TACN-AF+Cu , (B) Cyclen-AF+Cu (HSip-1), (C) Cyclam-
AF+Cu , and (D) TMCyclen-AF+Cu .
lthough hydrogen sulfide (H S) is a toxic gas with the cha-
racteristic smell of rotten eggs, recent studies have regarded
H S as the third gaseous transmitter, in addition to nitric oxide
2
2
+
2+
A
(DPA)ꢀfluorescein complex with Cu (DPA-AF+Cu ) showed
1
5
a selective turn-on fluorescence response to sulfide anion. We
2+
2
(
NO) and carbon monoxide (CO). H S appears to be involved
confirmed that DPA-AF+Cu could detect 10 μM H S in an
2
2
in various physiological processes, including relaxation of vascu-
aqueous solution at pH 7.4 (Supporting Information, Figure S1),
but this probe also showed fluorescence enhancement upon
addition of 10 mM GSH. We hypothesized that improvement of
1
2
lar smooth muscles, mediation of neurotransmission, inhibi-
3
tion of insulin signaling, and regulation of inflammation and O
2
4
,5
2+
sensing. There are several methods for selective detection of
the chelatorꢀCu complex stability in the presence of biothiols
H S, among which the most commonly used are the methylene
would result in a good selectivity for H S at low concentrations in
2
2
6,7
blue method and the sulfide ion-selective electrode method.
the presence of high concentrations of GSH. It is well known that
2+
However, these methods are destructive, requiring homogeniza-
tion of samples. Therefore, for detailed studies of the physiological
functions of H S, a new method to measure H S concentration in
azamacrocyclic rings form stable metal complexes with Cu , and
2+
the paramagnetic Cu center has a pronounced quenching effect
16
on fluorophores. On the basis of these facts, we expected that
2
2
2+
cells is required. We focused on fluorescence imaging, because it
Cu would be released from the azamacrocyclic ring when H S
2
2+
is suitable for nondestructive detection of targeted biomolecules
binds to the Cu center, resulting in fluorescence enhancement,
8
2+
in live cells or tissues with readily available instruments. Thus,
whereas the azamacrocyclic Cu complex would retain its stru-
we set out to develop a sensitive and selective fluorescence probe
cture in the presence of high concentrations of GSH, showing no
fluorescence enhancement.
for H S. This involves two substantial challenges. One is to attain
2
sufficient selectivity over other biothiols; i.e., the probe has to be
On the basis of this hypothesis, we designed and synthesized
able to measure endogenous H S without interference from
other biothiols, including reduced glutathione (GSH, present at
four sensor probes based on a fluorescein scaffold conjugated
2
2+
with an azamacrocyclic Cu complex, employing 1,4,7-triazacy-
clononane (TACN), 1,4,7,10-tetraazacyclododecane (Cyclen),
levels of about 1ꢀ10 mM), L-cysteine (L-Cys, about 100 μM),
9
0 00
1,4,8,11-tetraazacyclotetradecane (Cyclam), and N,N ,N -tri-
2+
and thiol-containing proteins. The other challenge is to achieve
sufficient sensitivity, because the concentration of H S required
methylcyclen (TMCyclen) as chelators for Cu instead of DPA
(Figure 1 and Supporting Information, Schemes S1ꢀS4).
Next, we examined the sensitivity and selectivity of the syn-
2
to elicit physiological responses has been reported to be 10
μMꢀ1 mM, although there remains some controversy as to the
intracellular H S levels generated in response to physiological
thesized compounds for H
2
S over GSH by measuring the fluore-
S or 10 mM GSH.
TACN-AF+Cu showed high sensitivity and low selectivity
2
1
ꢀ3,10
stimuli.
scence increment upon addition of 10 μM H
2
2+
A few fluorescence probes for H S have been reported,
2
2+
utilizing sulfide anion-triggered removal of a 2,4-dinitrobenze-
(Figure S2), while Cyclam-AF+Cu (Figure S3) and TMCy-
11ꢀ13
2+
nesulfonyl group or reduction of azide to amine.
However,
clen-AF+Cu (Figure S4) showed low sensitivity and high
the reactions are relatively slow and show poor selectivity for H S
2
ꢀ
over reactive oxygen species (ROS), such as superoxide (O ) or
Received: August 23, 2011
Published: October 14, 2011
2
12,14
Na SO .
Recently, Chang et al. have reported that a dipicolylamine
2
3
r 2011 American Chemical Society
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dx.doi.org/10.1021/ja207851s
|J. Am. Chem. Soc. 2011, 133, 18003–18005

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2
Figure 4. (A) Structure of cysteine-activated H S donor. (B) Time
course of reactions of 1 μM HSip-1 with no addition (red) or addition of
5
1
0 μM H
mM L-Cys (green) in 30 mM HEPES buffer (pH 7.4) with 0.1%
S donor and L-Cys were
added at 180 s, as indicated by an arrow. Ex/Em = 491/516 nm.
2 2
S donor (blue), 1 mM L-Cys (yellow), or 50 μM H S donor +
Figure 2. (A) Time course of reactions of HSip-1 with no addition
tetrahydrofuran as a cosolvent at 37 °C. H
2
(
red) or addition of 10 mM GSH (orange), 100 μM Na
2
S (green), or 10
S and
μM Na
2
S (blue) in 30 mM HEPES buffer (pH 7.4) at 37 °C. Na
2
GSH were added at 180 s. Ex/Em = 491/516 nm. (B) Fluorescence
spectra of 1 μM HSip-1 before (red) and after (blue) reaction with
2
100 μM Na S in 30 mM HEPES buffer (pH 7.4).
2
Figure 5. Visualization of H S in live cells using HSip-1 DA. HeLa cells
were incubated with 30 μM HSip-1 DA in DMEM containing 0.3%
DMSO for 30 min. (A) Differential interference contrast (DIC) and
fluorescence (FL) images were captured after addition of 500 μM Na S in
2
HBSS solution. (B) Average FI20 min/FI0 min intensity ratios in fluores-
Figure 3. Fluorescence enhancement of 1 μM HSip-1 in the presence of
thiols, inorganic sulfur compounds, and sodium ascorbate. Bars repre-
sent relative fluorescence intensity 30 min after addition of thiols,
inorganic sulfur compounds, or sodium ascorbate. The reactions of 1
cence images after addition of 0, 200, or 500 μM Na S in HBSS buffer.
2
The excitation and emission wavelengths were 470ꢀ490 and 515ꢀ
μM HSip-1 with (1) 10 μM Na
mM DL-Hcy, (5) 1 mM 2-ME, (6) 100 μM DTT, (7) 1 mM NaSCN,
8) 1 mM Na SO , (9) 1 mM Na , and (10) 10 mM sodium
ascorbate were performed in 30 mM HEPES buffer (pH 7.4) at 37 °C.
2
S, (2) 10 mM GSH, (3) 1 mM L-Cys, (4)
550 nm, respectively. Scale bar, 10 μm. Representative fluorescence
1
images from replicate experiments (n = 3) are shown. Error bars are (SD.
(
2
3
2 2 3
S O
Xian et al. have reported cysteine-activated H S donors which
2
2
+
mimic the slow and continuous endogenous biosynthesis of H2S
selectivity. Fortunately, Cyclen-AF+Cu (HSip-1 = hydrogen
from L-Cys mediated by enzymes such as cystathionine β-synthase
sulfide imaging probe-1) showed excellent properties as a
fluorescence probe for H S (Absmax/Emmax = 491/516 nm,
Φ = 0.019 in 30 mM HEPES buffer at pH 7.4); i.e., it showed
a large and immediate increment of fluorescence intensity by
17
(
CBS) and cystathionine γ-lyase (CSE). We synthesized this
2
H S donor (Figure 4A) to examine the feasibility of using HSip-1 to
2
fl
monitor pseudo-enzymatic H S production. We could detect time-
2
dependent H S release associated with decomposition of the H S
2
2
5
0-fold upon addition of 10 μM H S, whereas almost no
2
donor by using HSip-1 in the presence of 1 mM L-Cys (Figure 4B).
The spectroscopic properties of HSip-1, as well as its selec-
fluorescence increment was observed upon addition of 10 mM
GSH (Figure 2). In addition, the fluorescence enhancement
induced by H S was retained even in the presence of 10 mM
GSH (Figure S5). HSip-1 also showed high selectivity over other
thiols (1 mM L-Cys, DL-Hcy, 2-mercaptoethanol (2-ME), and
00 μM dithiothreitol (DTT)), inorganic sulfur compounds
tivity for H S, seemed appropriate for cellular application, so we
2
2
next considered the suitability of HSip-1 for fluorescence imaging
of cellular H S. However, this probe is membrane-impermeable
2
because of its hydrophilicity derived from the carboxyl group and
2+
1
he cyclenꢀCu complex moiety. Therefore, we employed a
standard diacetylation approach to improve the membrane per-
meability and synthesized diacetylated HSip-1 (HSip-1 DA); this
was confirmed to be a membrane-permeable precursor that is
hydrolyzed to HSip-1 by intracellular esterases (Scheme S2).
Next, HeLa cells were incubated with 30 μM HSip-1 DA con-
taining 0.3% DMSO as a cosolvent in Dulbecco’s modified
Eagle’s medium (DMEM) for 30 min and then washed with
Hanks’ Balanced Salt Solutions (HBSS), and various concentra-
(
1 mM NaSCN, Na SO , and Na S O ), and a reducing condi-
2
3
2 2 3
tion (10 mM sodium ascorbate) (Figures 3 and S6). Moreover,
HSip-1 did not show any fluorescence enhancement in response
to ROS or reactive nitrogen species (RNS), such as hydro-
gen peroxide (H O ), hydroxyl radical ( OH), peroxynitrite
ONOO ), hypochlorite ( OCl), superoxide (O ), singlet
•
2
2
ꢀ
1
ꢀ
ꢀ
2
(
•
oxygen ( O ), and nitric oxide ( NO). Upon addition of Angeli’s
salt (NO donor), a small fluorescence enhancement was observed
2
ꢀ
(
Figures S7 and S8). Thus, HSip-1 offers high selectivity for H2S
compared with previously reported fluorescence probes utilizing a
,4-dinitrosulfonyl group or an azide group.
tions of Na S (0, 200, or 500 μM) were added to the medium.
2
Upon addition of 500 μM Na S, a large intracellular fluorescence
2
2
enhancement was observed, while no significant fluorescence
1
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dx.doi.org/10.1021/ja207851s |J. Am. Chem. Soc. 2011, 133, 18003–18005

Journal of the American Chemical Society
COMMUNICATION
increment was seen in the absence of Na S (Figure 5). The
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results of CCK-8 assay showed that HSip-1 DA exhibits no
cytotoxicity at concentrations up to 100 μM (Figure S9).
In summary, we have developed a novel fluorescence probe for
1
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RNS and has excellent photophysical properties for biological
applications, arising from its fluorescein scaffold. We con-
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enzymatic H S release in a cuvette and for real-time fluorescence
2
imaging of intracellular H S in live cells. In addition, we also
2
confirmed that HSip-1 could detect H S produced by 3-mercap-
2
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agonists and antagonists, as well as for detailed investigation of a
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AUTHOR INFORMATION
Corresponding Author
tlong@mol.f.u-tokyo.ac.jp
’
ACKNOWLEDGMENT
This work was supported in part by a Grant-in-Aid for JSPS
Fellows (to K.S.) and by a Grant-in-Aid for Scientific Research
(
Specially Promoted Research No. 22000006 to T.N., 21659024
to K.H., and 21750135 to T.T.) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan. K.H. was
also supported by Inoue Foundation for Science, the Research
Foundation for Pharmaceutical Sciences, Konica Minolta Science
and Technology Foundation, and The Asahi Glass Foundation.
’
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