A lysosome-targetable fluorescent probe for imaging hydrogen sulfide in living cells

Article abstract of DOI:10.1021/ol400973v

In this work, a 1,8-naphthalimide-derived fluorescent probe for H ₂S based on the thiolysis of dinitrophenyl ether is reported. This probe exhibits turn-on fluorescence detection of H₂S in bovine serum and lysosome-targetable fluorescent imaging of H₂S with excellent selectivity.

Full text of DOI:10.1021/ol400973v

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
A Lysosome-Targetable Fluorescent
Probe for Imaging Hydrogen Sulfide
in Living Cells
Tianyu Liu,†,‡ Zhaochao Xu,*,†,‡ David R. Spring,*,§ and Jingnan Cui*,†
State Key Laboratory of Fine Chemicals, Dalian University of Technology,
Dalian 116012, China, Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, Dalian 116023, China, and Department of Chemistry,
University of Cambridge, Lensfield Road, Cambridge, U.K.
zcxu@dicp.ac.cn; jncui@dlut.edu.cn; spring@ch.cam.ac.uk
Received April 8, 2013
ABSTRACT
In this work, a 1,8-naphthalimide-derived fluorescent probe for H2S based on the thiolysis of dinitrophenyl ether is reported. This probe exhibits
turn-on fluorescence detection of H2S in bovine serum and lysosome-targetable fluorescent imaging of H2S with excellent selectivity.
Hydrogen sulfide (H2S) is well-known as a toxic gas with
the characteristic smell of rotten eggs, but is now also
considered the third most important gasotransmitter for
regulating cardiovascular, neuronal, immune, endocrine,
and gastrointestinal systems, along with nitric oxide and
carbon monoxide.1 H2S is produced endogenously in
mammalian systems from L-cysteine in reactions catalyzed
mainly by two pyridoxal-50-phosphate-dependent enzymes,
cystathionine β-synthase (CBS) and cystathionine γ-lyase
(CSE).2 As a signal molecule, it modulates neuronal
transmission,2 relaxes smooth muscle,3 regulates the release
of insulin,4 and is involved in inflammation.5 The
endogenous levels of H2S are believed to be related with
some diseases such as Alzheimer’s disease,6 Down’s
syndrome,7 diabetes,8 and liver cirrhosis.9 Inhibitors of
H2S, and H2S donors, in animal disease models have
shown potential for therapeutic exploitation of H2S.10
Thus, visualization of the distribution and concentration
of H2S in living systems would be very important and
helpful to elucidate the biological roles of H2S.
Small molecule fluorescent probes offer high sensitivity,
real-time imaging, and high spatiotemporal resolution and
have excellent potential as useful tools.11,12 A few fluor-
escent probes for H2S have been reported.13 These probes
† Dalian University of Technology.
(6) Eto, K.; Asada, T.; Arima, K.; Makifuchi, T.; Kimura, H.
Biochem. Biophys. Res. Commun. 2002, 293, 1485.
(7) Kamoun, P.; Belardinelli, M.-C.; Chabli, A.; Lallouchi, K.;
Chadefaux-Vekemans, B. Am. J. Med. Genet. A 2003, 116A, 310.
(8) Yang, W.; Yang, G.; Jia, X.; Wu, L.; Wang, R. J. Physiol. 2005,
569, 519.
‡ Chinese Academy of Sciences.
§ University of Cambridge.
(1) Kimura, H. Amino Acids 2011, 41, 113.
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2011, 51, 169.
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Rizzo, G.; Distrutti, E.; Shah, V.; Morelli, A. Hepatology 2005, 42, 539.
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r
10.1021/ol400973v
XXXX American Chemical Society

can image H2S in blood samples and living cells but
without location specificity, in particular subcellular loca-
lization. The distribution of H2S producing enzymes in
tissues is known to a first approximation.14 For example,
CSE is distributed in smooth muscle cells, liver, and
pancreas, whereas CBS is found in the brain, liver, kidney,
and pancreas.14 CBS is also found in the endosomal-
lysosomal system.15 However, the distribution and func-
tion of H2S in different organelles are still unclear. There-
fore, organelle-specific fluorescent probes for H2S are
especiallyrequiredtohelp understand the detailed network
of H2S biology in cells.
introducing different functional groups to the aromatic
‘naphthalene’ moiety and the ‘N-imide site’.27ꢀ36 In our
work, we introduced a dinitrophenyl ether group into the
4-position of 1,8-naphthalimide, which acts as the H2S
reactive site,26 and a 4-(2-aminoethyl)morpholine, which is
a lysosome-targetable group,37 onto the N-imide termus,
thereby, efficiently yielding the fluorescent probe Lyso-
NHS (Scheme 1). To our best knowledge, Lyso-NHS is
the first fluorescent probe that can image H2S in lysosomes
of living cells in minutes.
The design of fluorescent probes for H2S is mainly based
on specific chemical reactions by taking advantage of the
reducing or nucleophilic properties of H2S. For example,
Chang16 and Wang17 et al. pioneered an approach of using
the reduction of azide with H2S to amine to sense H2S,
which has been expanded to design azide-containing fluor-
escent probes by altering fluorophores.18ꢀ23 A ratiometric
fluorescent probe was developed in terms of this strategy.20
Xian and co-workers constructed a H2S probe through a
nucleophilic substitution reaction between H2S and the
disulfide moiety.24 He et al. used the nucleophilic attack of
H2S on the aldehyde functionality to design a fluorescent
probe to sense H2S.25 Lin et al. reported a near-infrared
fluorescent probe for H2S based on thiolysis of dinitro-
phenyl ether.26 However, most of these probes require
complex synthesis and display a response time of ∼1ꢀ2 h.
1,8-Naphthalimide is a cell-permeable fluorophore posses-
sing a visible emission wavelength, high photostability, and
facile synthesis of various fluorescent probes by easily
Scheme 1. Mechanism of H2S Sensing by Lyso-NHS
The pH value of lysosomes is in the range of 4.0ꢀ6.0.38
To monitor H2S in lysosomes, the fluorescent probe
should first have the ability to survive in this acidic
environment and display no fluorescence response. In
order to verify the workability of Lyso-NHS within this
pH range, the influence of pH on the fluorescence of Lyso-
NHS was first determined by fluorescence titration. The
fluorescenceat 555 nm of Lyso-NHS remains unaffected at
pH 8.2ꢀ4.2 and then gradually increases from pH 4.2 to
2.03 due to the inhibition of the photoinduced electron
transfer (PET) process from the morpholine nitrogen to
the fluorophore (Figure 1). The pKa value of Lyso-NHS is
3.12. Therefore, the fluorescence of Lyso-NHS will not
change in lysosomes, which makes Lyso-NHS fit the
purpose.
The emission spectra and fluorescence titration experi-
ments of Lyso-NHS with H2S were then recorded in
aqueous solution (CH3CN/PBS = 1:9, pH = 7.4)
(Figure 2a). The free Lyso-NHS displays quite weak
fluorescence. When H2S was added progressively from 0
to 10 equiv to the solution of Lyso-NHS (NaHS was used
as a hydrogen sulfide source), the fluorescence intensity of
the emission band centered at 555 nm increased in in-
tensity significantly (42-fold) due to the thiolysis of the
dinitrophenyl ether by H2S (Scheme 1).
(14) Yamamoto, J.; Sato, W.; Kosugi, T.; Yamamoto, T.; Kimura,
T.; Taniguchi, S.; Kojima, H.; Maruyama, S.; Imai, E.; Matsuo, S.;
Yuzawa, Y.; Niki, I. Clin. Exp. Nephrol. 2013, 17, 32.
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T. EMBO J. 2011, 30, 2140.
(16) Lippert, A. R.; New, E. J.; Chang, C. J. J. Am. Chem. Soc. 2011,
133, 10078.
(17) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.;
Lefer, D. J.; Wang, B. Angew. Chem., Int. Ed. 2011, 50, 9672.
(18) Das, S. K.; Lim, C. S.; Yang, S. Y.; Han, J. H.; Cho, B. R. Chem.
Commun. 2012, 48, 8395.
(19) Montoya, L. A.; Pluth, M. D. Chem. Commun. 2012, 48, 4767.
(20) Wan, Q.; Song, Y.; Li, Z.; Gao, X.; Ma, H. Chem. Commun.
2013, 49, 502.
(21) Wu, Z.; Li, Z.; Yang, L.; Han, J.; Han, S. Chem. Commun. 2012,
48, 10120.
(22) Yu, F.; Li, P.; Song, P.; Wang, B.; Zhao, J.; Han, K. Chem.
Commun. 2012, 48, 2852.
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134, 9589.
The formation of compound 1 was confirmed by MS
analysis (Figure S1) and the HPLC retention time com-
pared with those of independently synthesized 1, which
is responsible for the fluorescence emission and enhance-
ment at 555 nm (Figure S2). Moreover, the product was
purified and characterized with 1H and 13C NMR, which is
(24) Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.;
Whorton, A. R.; Xian, M. Angew. Chem., Int. Ed. 2011, 50, 10327.
(25) Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S.-Y.; Zhu, H.-L.;
Banerjee, R.; Zhao, J.; He, C. Nat. Commun. 2011, 2, 495.
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B
Org. Lett., Vol. XX, No. XX, XXXX

Figure 1. Influence of pH on the fluorescence of Lyso-NHS in
aqueous solution (CH3CN/PBS = 1:9). Excitation wavelength
is 450 nm. [Lyso-NHS] = 10 μM.
identical to compound 1 (see the Supporting Information
forspectraldata). The pH-dependentfluorescenceofLyso-
NHS with the addition of H2S confirmed the applicability
of Lyso-NHS in lysosomes (Figure S3). The time-depen-
dent fluorescence responses were next detected with the
addition of 10equivofH2S, and theresults showedthatthe
reaction was completed within 20 min (Figure 2bꢀc).
Notably, the background fluorescence of Lyso-NHS is
very weak, and within minutes a high fluorescence increase
is observed which relays the reaction of Lyso-NHS with
H2S (Figure 2c); therefore, the time scale allows Lyso-NHS
to sense H2S in real-time intracellular imaging. There was
good linearity between the fluorescence intensity and the
concentrations of H2S in the range 0 to 100 μM with a
detection limit of 0.48 μM (Figure S4). The absorption
titration of Lyso-NHS with H2S was subsequently per-
formed and also reflected the thiolysis of the dinitrophenyl
ether. Compound Lyso-NHS exhibits maximum absorp-
tion at 360 nm. On addition of 0ꢀ10 equiv of H2S to the
solution of Lyso-NHS, the absorbance at 360 nm de-
creased sharply to its limiting value, while an absorption
band at 440 nm developed which induced the color change
from colorless to yellow (Figures 2d, S5).
Figure 2. (a) Fluorescent emission spectra of 10 μM compound
Lyso-NHS in the presence of 0ꢀ10 equiv of H2S in aqueous
solution (CH3CN/PBS = 1:9, pH = 7.4, 37 °C) (NaHS was
dissolved in water at a concentration of 10 mM). Excitation at
450 nm. (b) Time dependence of fluorescence profiles of Lyso-
NHS (10 μM) with 10 equiv of H2S. (c) Time dependence
of fluorescence intensity of Lyso-NHS (10 μM) at 555 nm with
10 equiv of H2S. (d) UVꢀvis absorption spectra of 10 μM
compound Lyso-NHS in the presence of 0ꢀ10 equiv of H2S in
an aqueous solution (CH3CN/PBS = 1:9, pH = 7.4, 37 °C).
The fluorescence titration of Lyso-NHS with various
analytes was conducted to examine the selectivity. As
shown in Figure 3, the addition of 100 equiv of Naþꢀ,
S2O32ꢀ, S2O42ꢀ, S2O52ꢀ, SO3ꢀ, N3ꢀ, CO32ꢀ, CH3COOꢀ,
OHꢀ, SO42ꢀ, H2O2, HSO4ꢀ, homocysteine, and ascorbic
acid produced a nominal change in the fluorescence spec-
tra of Lyso-NHS. Othertestedanalytesincluding 100 equiv
of Zn2þ, cysteine, and glutathione induced fluorescence
but with much smaller enhancement. Therefore the probe
Lyso-NHS has a very high selectivity for H2S.
Ca2þ, Kþ, Mg2þ, HCO3ꢀ, Fꢀ, Clꢀ, Brꢀ, Iꢀ, NO3
,
Figure 3. Fluorescence responses of 10 μM Lyso-NHS to various
analytes in aqueous solution (CH3CN/PBS = 1:9, pH = 7.4, 37 °C).
Excitation at 450 nm. Bars represent the final fluorescence
intensity of Lyso-NHS with 1 mM analytes over the original
emission of free Lyso-NHS. (1) Free Lyso-NHS; (2) Zn2þ; (3)
Naþ; (4) Ca2þ; (5) Kþ; (6) Mg2þ; (7) HCO3ꢀ; (8) Fꢀ; (9) Clꢀ; (10)
Brꢀ; (11) Iꢀ; (12) NO3ꢀ; (13) S2O32ꢀ; (14) S2O42ꢀ; (15) S2O52ꢀ
;
;
2ꢀ
(16) SO3ꢀ; (17) N3ꢀ; (18) CO32ꢀ; (19) CH3COOꢀ; (20) SO4
(21) H2O2; (22) HSO4ꢀ; (23) homocysteine; (24) ascorbic acid;
(25) cysteine; (26) glutathione; (27) NaHS.
The tests in buffer solutions have shown the potential
utility of Lyso-NHS inbiological samples. Wefirst checked
the fluorescence response of Lyso-NHS with H2S in bovine
serum. The background fluorescence of bovine serum
sample is relatively weak. With the addition of NaHS,
the fluorescence intensity of emission of the bovine serum
sample withLyso-NHS increases significantly. It should be
noted that the fluorescence enhancement is observed
immediately with the addition of NaHS and reaches the
maximum value in minutes (Figure S6a). The concentra-
tion-dependent fluorescence responses of Lyso-NHS with
NaHS were next detected, and a linear relationship for the
fluorescence intensity of Lyso-NHS versus hydrogen sul-
phide concentration was exhibited. As seen in Figure S6b,
Org. Lett., Vol. XX, No. XX, XXXX
C

an excellent linear correlation between the added NaHS
concentration and the fluorescence intensity of Lyso-NHS
at 555 nm was observed. The fast responses and excellent
linear relationship provided a real-time quantitative detec-
tion method for hydrogen sulfide in biological samples.
The fluorescence titration of Lyso-NHS in bovine serum
with various analytes was also conducted, which showed
Lyso-NHS to have a high selectivity for H2S (Figure S7).
We then sought to examine whether Lyso-NHS can
localize to the lysosome and sense H2S in living cells. The
cell permeability of Lyso-NHS was first investigated.
MCF-7 cells were incubated with 5 μM Lyso-NHS for
30 min and exhibited no fluorescence (Figure 4a). Then the
cells were incubated with 20 μM NaHS and after 5 min
displayed enhanced green fluorescence (Figure 4b). After
10 min of incubation with NaHS, a higher turn-on fluor-
escence response can be observed (Figure 4c). These
experiments indicate Lyso-NHS can be used to detect
H2S in living cells. The cytotoxicity of Lyso-NHS was
examined toward MCF-7 cells cells by an MTT assay
(Figure S8). The results showed that >90% MCF-7 cells
survived after 12 h (5.0 μM Lyso-NHS incubation), and
after 24 h the cell viability remained at ∼80%, demonstrat-
ing Lyso-NHS to be minimally toxic toward cultured cell
lines.
Figure 5. Lyso-NHS colocalizes to lysosomes in MCF-7 cells. (a)
5.0 μM Lyso-NHS with 20 μM of H2S incubated 10 min at 37 °C
(Channel 1: λex = 450 nm, λem = 520ꢀ560 nm). (b) 5.0 μM NR
(Channel 2: λex = 559 nm, λem = 565ꢀ610 nm). (c) Merged
images of (a) and (b). (d) Bright field image. (e) Intensity profile
of regions of interest (ROI) across MCF-7 cells. (f) Intensity
correlation plot of dyes Lyso-NHS and NR. Scale bars = 20 μm.
dinitrophenyl ether, known as Lyso-NHS. Due to rapid
conversion to the fluorescent compound 5 by H2S, a large
fluorescence increase is obtained with emission centered at
555 nm in aqueous solution. Concomitantly, the solution
color changes from colorless to yellow with the conveni-
ence and aesthetic appeal of a colorimetric assay. The
probe has a high selectivity for H2S over competitive
analytes. This probe is applicable to H2S detection in
bovine serum and live cell imaging and has the ability to
detect intracellular H2S in lysosomes. The successful ap-
plication of our probe to detect lysosomal H2S will help to
study the biological role of H2S in lysosomes and encour-
age the appearance of new H2S probes suitable for other
organelle localizations.
Figure 4. Time-dependent exogenous H2S released from NaHS
(20 μM) in MCF-7 cells stained with Lyso-NHS (5.0 μM) at
37 °C: (a) 0 min; (b) 5 min; (c) 10 min; (d) merged images of (c)
and bright field. Scale bars = 20 μm.
The fluorescence localization was examined by costain-
ing cells with a commercially available lysosome-specific
dye Neutral Red (NR) (5 μM) (Figure 5aꢀb). As shown in
Figure 5c, the fluorescence patterns of Lyso-NHS and the
NR signals overlapped perfectly, indicating that the fluor-
escence response of Lyso-NHS to H2S was localized at the
lysosome. The intensity profiles of the linear regions of
interest across MCF-7 cells stained with Lyso-NHS and
NR also display close synchrony (Figure 5e). The high
Pearson coefficient and overlap coefficient are 0.885 and
1.419, respectively (Figure 5f). These experiments indicate
Lyso-NHS can specifically localize in lysosomes and be
used to detect H2S in lysosomes of living cells.
Acknowledgment. We are thankful for financial sup-
port from the National Natural Science Foundation of
China (21276251), Ministry of Human Resources and
Social Security of PRC, the 100 talents program funded
by the Chinese Academy of Sciences, State Key Labora-
tory of Fine Chemicals of China (KF1105), and the
Herchel Smith Fund. D.R.S. is supported by the EPSRC,
ERC, BBSRC, MRC, and Wellcome Trust.
Supporting Information Available. Synthesis, charac-
teristics and spectroscopic data of Lyso-NHS. This ma-
terial is available free of charge via the Internet at http://
pubs.acs.org.
In conclusion, we have reported a 1,8-naphthalimide-
derived fluorescent probe for H2S based on thiolysis of
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX