Ratiometric two-photon fluorescent probes for mitochondrial hydrogen sulfide in living cells

Article abstract of DOI:10.1021/jo5014838

Hydrogen sulfide (H₂S) is an important signaling molecule with diverse biological roles. Various fluorescent probes for H₂S with biological application have been developed. However, two-photon ratiometric imaging of mitochondrial H

Full text of DOI:10.1021/jo5014838

Article
pubs.acs.org/joc
Ratiometric Two-Photon Fluorescent Probes for Mitochondrial
Hydrogen Sulfide in Living Cells
Xiu-Ling Liu,† Xiao-Jiao Du,‡ Chun-Guang Dai,† and Qin-Hua Song*,†
†Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China
‡School of Life Sciences, University of Science and Technology of China, Hefei 230027, P. R. China
S
* Supporting Information
ABSTRACT: Hydrogen sulfide (H2S) is an important
signaling molecule with diverse biological roles. Various
fluorescent probes for H2S with biological application have
been developed. However, two-photon ratiometric imaging of
mitochondrial H2S is scarce. In this paper, we report two
ratiometric two-photon probes, AcHS-1 and AcHS-2, which
employ 4-amino-1,8-naphthalimide as the fluorophore and 4-
azidobenzyl carbamate as the H2S response site. These probes exhibit high selectivity toward H2S over biothiols and other
reactive species, low detection limits of 50−85 nM, low cytotoxicity, and high stability under physiological conditions.
Furthermore, through cell imaging with one-photon and two-photon microscopy, MCF-7 cells incubated with two probes show a
marked change in emission color from blue to green in response to H2S. Cell images costraining with a mitochondrial dye reveal
that AcHS-2 is a mitochondria-specific two-photon probe for H2S. These results show that AcHS-2 may find useful applications
in biological research such as tracking mitochondrial H2S in living biological specimens.
depth (<100 μm). An attractive approach for the detection of
H2S in organelles deep inside the tissues is through the use of
two-photon microscopy (TPM), which employs near-IR
photons as the excitation source, and it offers many advantages,
including greater penetration depth (>500 μm), localization of
the excitation, and longer observation times.
Furthermore, selective tracking of H2S in an organelle such
as mitochondria is crucial to elucidate the complex contribu-
tions of H2S in the physiological and pathological processes.
However, five ratiometric fluorescent probes for mitochondrial
H2S have been reported,11l,12a,b,14d,e and only one case11l is a
two-photon probe. Therefore, this is still a strong need and
requires challenging work to develop new ratiometric two-
photon probes for mitochondrial H2S.
INTRODUCTION
■
Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling
molecule generated predominately from cysteine by two
pyridoxal-5′-phosphate-dependent enzymes, cystathionine-β-
synthase and cystathionine-γ-lyase, as well as by 3-mercapto-
sulfurtransferase, in the cytosols and mitochondria of
mammalian cells in relatively high concentration (10−100
μM).1,2 The significance of endogenous H2S has been
recognized in a number of physiological and pathological
processes.3,4 H2S plays critical roles in regulating intracellular
redox status and other fundational signaling processes involved
in human health and disease.1,5 Mitochondrial H2S has been
shown to exert protective effects in oxidative stress leading to
dysfunction and cell death,6−8 and has become the focus of
many research endeavors, including pharmacotherapeutic
manipulation.9
To detect H2S in solutions and cells, a number of fluorescent
probes10 have been designed on the basis of reactions of H2S as
a reductant to reduce azide or nitro group on masked
fluorophore11 or as a nucleophile to attack activated electro-
philes,12 precipitate metal salts,13 or others.14 Most of them are
turn-on fluorescent probes, which are difficult to give
quantitative information on the H2S concentration, since
molecular emission intensity can be distinctly affected by
photobleaching, microenvironments, and local probe concen-
tration. Therefore, ratiometric probes for H2S are highly
appealing owing to their ability in quantitative tracking. So far, a
few ratiometric H2S probes have been reported.11d,l,12a,b,14b−e
In addition, under one-photon excitation, short excitation
wavelengths (<500 nm) limit applications of these probes with
regard to deep-tissue imaging due to the shallow penetration
In this work, we developed two ratiometric fluorescent
probes, AcHS-1 and AcHS-2, for detection of H2S. Moreover,
we demonstrated that AcHS-2 is a new mitochondria-specific
probe for H2S, and mitochondrial images can be achieved
under both one-photon and two-photon excitations.
RESULTS AND DISCUSSION
■
1. Design and Synthesis of Probes. 4-Amino-1,8-
naphthalimide with intramolecular charge transfer (ICT) has
been used as a fluorophore in H2S fluorescent probes,11e,g,12k
but all these probes are the turn-on mode, as shown in Scheme
1. By modifying an ICT-state fluorophore to change the ICT
property, the modified fluorophore could display a ratiometric
spectral variation. If certain molecules such as H2S can induce a
Received: July 3, 2014
© XXXX American Chemical Society
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stirred at room temperature for 3 h, and purified to give target
products AcHS-1 and AcHS-2.
Scheme 1. Reported Probes with a Turn-On Response
Mode11e,g
2. Spectral Response to H2S in Aqueous Solutions.
H2S is a weak acid in aqueous solutions (pKa1 = 7.04, pKa2
=
11.96),22 equilibrating mainly with HS− at physiological pH.
For example, at pH 7.4 and temperature of 37 °C, 18.5% of free
hydrogen sulfide exists as H2S molecule and the remainder is
almost all hydrosulfide anion (HS−) with a negligible
contribution of S2−.23 As the ion distribution of Na2S is the
same with that of H2S in a neutral buffer solution, Na2S was
employed to replace H2S in this work.
Parts a and c of Figure 1 show time-dependent UV/vis
absorption spectra of 10 μM AcHS-1 and 10 μM AcHS-2 in the
presence of 1 mM Na2S, respectively. The absorption band in
the range of 400−500 nm appears and increases, accompanying
the decrease in the absorption peak at 370 nm. This change
results in an isosbestic point at 400 nm, indicating the sensing
reaction is a single transformation. The final absorption spectra
show a larger red shift of 65 nm for AcHS-1 and 63 nm for
AcHS-2 over those11l of two probes reported (25 nm for SHS-
M1 and 40 nm for SHS-M2). The color change of the solution
from colorless to yellow can be observed by the naked eye
(inset of Figure 1a).
The time-dependent fluorescent response of AcHS-1 or
AcHS-2 to H2S was also observed. The fluorescence spectra of
AcHS-1 and AcHS-2 have a wide emission band (400−600
nm) with a peak at 468 nm for both AcHS-1 and for AcHS-2.
With addition of Na2S, the cleavage reaction of AcHS-1 or
AcHS-2 would trigger and release the green fluorescent 3 or 4
with a stronger ICT-state emission (Φf(3) = 0.13, Φf(4) = 0.12
listed in Table 1). Consequently, the fluorescence emission
intensity in the short wavelength region gradually decreases,
accompanying appearance of a new emission band (475−650
nm) with a peak at 530 nm, exhibiting a ratiometric change
with a large red-shift over 62 nm for both AcHS-1 and AcHS-2
as shown in Figure 1b,d. Fluorescence photos of AcHS-1
solutions without and with Na2S are shown in the inset of
Figure 1b. The ratio of F530/F468 increases from 0.35, 0.28 to
3.37, 2.02 for AcHS-1 and AHS-2, respectively, after incubation
for 2 h.
sensing reaction to remove this modifying group, the modified
fluorophore would become a ratiometric two-photon fluo-
rescent probe for the molecule. Based on this design strategy of
ratiometric two-photon fluorescent probes, we designed two
ratiometric probes for H2S, using 4-amino-1,8-naphthalimide as
the fluorophore and 4-azidobenzyl carbamate as a H2S response
site, shown in Scheme 2. As the azide is reduced to amine,
probes may undergo a cleavage of the carbamate and release of
the amino group. The 4-amino-1,8-naphthalimides with ICT
states have large two-photon absorption cross sections.15
Through introducing an electron-withdrawing carbamate
group to convert the 4-amino donor into a weak donor, the
ICT effect of the fluorophore is weakened to result in a blue-
shift of fluorescence. On this basis that the thiolate-triggered
reaction with the azide group would cleave the carbamate
linkage and liberate the amine group,16,17 we expect that a
similar cleavage and release occurs for the new probes in the
presence of H2S.
The synthetic procedure for probes AcHS-1 and AcHS-2 is
outlined in Scheme 3. Using 4-azidophenylmethyl bromide as a
starting material, 4-azidophenylmethanol (5)18 was prepared. n-
Butylamine or aminoethanol was quickly added to a cloudy
solution of 4-nitro-1,8-naphthalic anhydride19 in ethanol. After
being refluxed for 6 h under N2, the solution was filtered,
washed, and purified to give compound 6a20 or 6b.21 A stirred
cloudy solution of 6a or 6b in ethanol was added to the
solution of SnCl2 in concentrated hydrochloric acid at room
temperature to afford compound 320 or compound 4.21
A
Two-photon absorption cross sections (δ) were obtained by
determining the two-photon absorption action spectra of
probes and their sensing products 3 and 4, listed in Table 1.
The δmax values for AcHS-1 and AcHS-2 were 152 and 119
mixture of 3 or 4 and DMAP in toluene was added to a solution
of triphosgene in toluene, heated to reflux for 3 h, diluted with
CH2Cl2, and filtered. To the filtrate was added compound 5,
Scheme 2. Proposed Sensing Mechanism of Probes, AcHS-1 and AcHS-2
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Scheme 3. Synthetic Route of Probes AcHS-1 and AcHS-2
Figure 1. Time-dependent UV/vis absorption (a, c) and fluorescence spectra (b, d) of 10 μm AcHS-1 or AcHS-2 in EtOH/PBS (v/v1:4, pH 7.0)
response toward Na2S (1 mM), respectively, excitation at 410 nm. Insets in (a) and (b): photograph for the solution color and the fluorescent
changes of AcHS-1 without and with Na2S in the buffer solution, respectively.
GM, respectively, and lower than those of 3 and 4 (207 and
218 GM, respectively). The higher two-photon absorption
cross-sections for 3 and 4 over the probes can be attributed to
their enhanced donor, 4-amino group.
sensing reaction of AcHS-1 with Na2S could occur via the
cleavage of azido-based carbonate moiety and release of 3.
Furthermore, the main H2S-induced product is confirmed to be
compound 3 by coinjection HPLC analysis. As shown in Figure
3, with increasing reaction time, the chromatogram peak at 19.5
min of AcHS-1 decreases with a simultaneous increase for a
new peak at 3.2 min, which is assigned to compound 3.
4. Sensitivity and Selectivity. Detection Limits. The
fluorescence spectra of 0.2 μM AcHS-1 in EtOH/PBS (v/v1:4,
pH 7.0) in the presence of various concentrations of Na2S from
0 to 6 μM were collected and are shown in Figure S1
(Supporting Information). A linear regression curve was then
3. Sensing Mechanism. To further confirm the validity of
the proposed sensing mechanism, the sensing reaction of
AcHS-1 to Na2S in DMSO-d6 was analyzed by means of NMR
1
spectroscopy, with partial H NMR spectra shown in Figure 2.
As shown in Figure 2a, a characteristic single peak at 5.48 ppm
was assigned to the proton (Hg) of AcHS-1, and the single peak
1
was not observed from the H NMR spectrum of the reaction
mixture of AcHS-1 and Na2S (Figure 2b). In this field region,
1
most of the emerging peaks for H NMR spectrum of the
fitted according to the ratio of fluorescence intensity at F530
/
reaction mixture match with those of compound 3. Hence, the
F465 in the range of [Na2S] from 0 to 6 μM. The detection limit
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a
Table 1. Photophysical Data for AcHS-1, AcHS-2, 3, and 4
b
c
d
e
f
compd
λabs(10−4ε)
λfl
Φ
λmax
δmax
AcHS-1
370 (1.34)
435 (1.20)
373 (1.34)
436 (1.18)
468
530
468
530
0.15
0.13
0.10
0.12
740
820
730
800
152
207
119
218
3
AcHS-2
4
a
Measurements were performed in EtOH/PBS (v/v1:4, 100 mM, pH
b
7.0), unless otherwise noted. λmax of the one-photon absorption
spectra in nm. The values in parentheses are molar extinction
c
d
coefficients in M−1 cm−1. Fluorescence maxima. Fluorescence
e
quantum yields,
10%. λmax of the two-photon absorption action
f
spectra in nm, measured in acetonitrile. Peak two-photon absorption
action cross sections in GM (1 GM = 10−50 m4 s photon−1), 10%,
measured in acetonitrile.
Figure 3. Time-dependent HPLC profiles of AcHS-1 (10 μM) in the
presence of Na2S (1 mM) in methanol/water (1:1, v/v). The eluent is
a methanol/water solvent mixture (80:20, v/v), monitored at 254 nm
(a, 0 min; b, 5 min; c, 10 min; d−j, interval of 20 min; k, 4 h; l, neat
compound 3). Retention times: 3.2 min (product 3), 19.5 min (AcHS-
1).
(3N/k) was determined to be 0.05 μM, where N is the standard
deviation of the intercept and k is the slope of the fitting
straight line in the inset of Figure S1 (Supporting Information).
Similarly, the detection limit of AcHS-2 was obtained, 0.085
μM (Figure S2, Supporting Information).
Selectivity. The selectivity of probes was determined for H2S
over other biologically relevant species, including reactive
sulfide (cysteine (Cys), glutathione (GSH), SCN−), reductants
(S2O32−, SO32−, ascribic acid (Vc), S2O42−), reactive oxygen
solutions (pH 2.0−10.0) were measured (Figure 5a). The
fluorescence spectra of probes AcHS-1 and AcHS-2 were
recorded at various pH solutions (EtOH/PBS (v/v1:4). The
pH-dependence response was estimated by the fluorescence
intensity ratio at F530/F468 under excitation at 410 nm. Under
neutral or alkaline conditions (pH > 6), probes AcHS-1 and
AcHS-2 can respond to H2S with a remarkable fluorescence
change, while no obvious response was observed in acidic
solutions (pH < 5).
For the pH effect on the stability of the probes, UV/vis
absorption spectra of AcHS-1 and AcHS-2 in various solutions
from pH 2 to 10 were recorded, respectively (Figure 5b). The
stability of probes AcHS-1 and AcHS-2 were estimated by
measuring the absorbance at 370 mn for absorption peak of the
probes and 436 nm for absorption peak of compound 3 in
various pH solutions (Figure 5b). As shown in Figure 5b, no
significant change at the two wavelengths, 370 and 436 nm, was
−
(HClO, H2O2, t-BuOOH), nitrogen species (NO2 ), and metal
ions (Ca2+, Na+, K+). Both AcHS-1 and AcHS-2 displayed a
marked response for H2S over other biological relevant species.
As shown in Figure 4, pronounced fluorescence changes were
observed from the solution with H2S, and no significant change
occurred for solutions with other analytes. The fluorescence
intensity ratios (F530/F468) for Na2S over other analytes were
8−17-fold for AcHS-1 and 5−7-fold for AcHS-2. Meanwhile,
the naked-eye color of probe solutions changed from colorless
to yellow, and the emission color from blue to green only in the
presence of Na2S (inset of Figure 4left).
5. pH Effect on Fluorescent Response and Stability of
Probes. To examine the pH-dependent fluorescent response,
fluorescence intensities of the sensing reaction in different
1
Figure 2. Partial H NMR (400 MHz) spectra of AcHS-1 (a), AcHS-1 and Na2S (molar ratio 1:10) in DMSO-d6 (b).
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Figure 4. Left: Fluorescence responses of AcHS-1 (10 μM) to various analytes (1 mM) at F530/F468 in EtOH/PBS (v/v1:4, pH 7.0). Inset: photos of
naked-eye and fluorescent responses of AcHS-1 to various analytes. Right: fluorescence spectra of AcHS-2 (10 μM) in the presence of various
analytes (1 mM) in the buffer solution. Inset: the corresponding fluorescence response, under excitation at 400 nm, incubation for 2 h.
Figure 5. (a) Plot of the ratio of F530/F468 for AcHS-1 (10 μM) and AcHS-2 (10 μM) vs pH value in the presence of 1 mM Na2S. (b) Absorbance at
370 and 436 nm of 10 μM AcHS-1 and AcHS-2, in different pH solutions, incubation for 2 h.
observed at pH 2−8. This indicates that the probes are stable in
pH 2−8 solutions. In addition, the stability of probes under the
physiological condition was also estimated by tracking their
UV/vis absorption spectra in a PBS buffer (pH 7.4) for 10 h
(Figure S3, Supporting Information). Their absorption spectra
display no significant change. These results show that AcHS-1
and AcHS-2 not only can detect H2S but also are considerably
more stable under physiological conditions (pH 6−8).
6. Fluorescence Imaging of Living Cells. Cell Toxicity of
Probes. In order to detect H2S in living MCF-7 cells, a MTT
experiment was carried out to assess the cytotoxicity of the
probes. In the MTT assays, MCF-7 cells were dealt with probes
in different concentrations from 5 to 20 μM for 24 h. The
results show that the probes have low toxicity to cultured cells
under the experimental conditions (Figure 6) and the cell
viability is 93% and 89% for AcHS-1 and AcHS-2 at 5 μM,
respectively.
Figure 6. MTT assay of MCF-7 cells in the presence of different
concentrations of probes.
Fluorescent Imaging of Cells. The MCF-7 cells were placed
in a 96-well plate and treated with 5 μM AcHS-1 or AcHS-2 for
1 h, and then the cells in one well were incubated with 0.5 mM
Na2S for an additional 1 h. Two types of cells were washed with
PBS three times for confocal fluorescence microscopy (Figures
7 and 8). As shown in Figure 7, cells incubated alone with
AcHS-1 display blue emission collected from the blue channel
(408−500 nm), and cells costained by AcHS-1 and Na2S emit
green fluorescence collected from the green channel (500−650
nm) under one-photon excitation of 405 nm. Upon two-
photon excitation at 750 nm, cells stained alone with AcHS-1
emitted similar fluorescence, shown in Figure S4 (Supporting
Information).
Similarly, cells stained by AcHS-2 with and without Na2S
emit different-color fluorescence (Figure 8a−f). Moreover,
upon two-photon excitation at 750 nm for cells stained alone
with AcHS-2 as shown in Figure S4 (Supporting Information)
and 800 nm for cells costained by AcHS-2 and Na2S (Figure
8g−i), confocal fluorescence microscopy collects similar
emissions from the two channels. These results show that
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to 3.9 and 2.9, respectively. This indicates that two probes can
serve as ratiometric fluorescent probes for H2S in cells.
The cell images above show that the probes could localize at
mitochondria. To further investigate the mitochondria local-
ization of AcHS-2, a commercially available mitochondrial dye
(MitoTracker Red CMXRos)24 was employed for a colocaliza-
tion study. MCF-7 cells were stained with AcHS-2 and the
MitoTracker in succession and observed under the confocal
fluorescence microscopy by one- and two-photon images. As
shown in Figure 10, the green-channel images for AcHS-2 with
and without Na2S merged well with the red-channel images for
MitoTracker dye under one-photon excitation (Figure 10d, h).
The colocalization assay with the mitochondrial dye and AcHS-
2 revealed that fluorescence of AcHS-2 was colocalized with
that of the mitochondrial dye, and the overlap coefficients were
0.79 for the cells without Na2S (Figure 10d) and 0.71 for the
cells with Na2S (Figure 10h). Upon one- and two-photon
excitation at 375 and 750 nm, confocal images of MCF-7 cells
costained by AcHS-2 and anther mitochondrial specific dye
MitoTracker Deep Red FM24 displayed excellent overlap
between the green-channel images for AcHS-2 and the red-
channel images for the MitoTracker dye under one-photon
excitation (Figure S5, Supporting Information). The overlap
coefficients between the green channel and the red channel
were 0.80 for one-photon image (Figure S5d, Supporting
Information), 0.87 for two-photon image (Figure S5h,
Supporting Information). The high overlap coefficients indicate
that AcHS-2 can accumulate in the mitochondria.
Figure 7. Confocal fluorescence images of MCF-7 cells incubated with
5 μM AHS-1 for 1 h (a−c) and then 0.5 mM Na2S for 1 h (d−f).
Images were acquired using 405 nm excitation and emission channels
of 408−500 nm (blue) and 500−650 nm (green). Cells shown are
representative images from replicate experiments (n = 3).
It is well-known that the commercial dyes for mitochondria
are cationic and can accumulate in the mitochondria due to the
negatively charged surface of the mitochondrial membrane. In
this work, however, two probes are electroneutral, and the
difference in structures is N-n-butyl for AcHS-1 and N-(2-
hydroxyethyl) for AcHS-2. By comparing cell images of two
probes, we found that all images of the cells stained with AcHS-
2 were brighter than those stained with AcHS-1. Hence, we
inferred that the hydrogen bonds formed between the hydroxyl
group (and 4-amino) with mitochondrial membrane may be
the reason why the probes, especially AcHS-2, accumulate in
the mitochondria.
CONCLUSION
■
In summary, we developed two ratiometric two-photon
fluorescent probes based on an ICT-state fluorophore, 4-
amino-1,8-naphthalimide. By modifying the fluorophore with
an electron-withdrawing group, 4-azidophenyl methoxy carbon-
yl, to convert the amino group into a weak donor, also as the
site of the sensing reaction to H2S, the resulting probe
molecules display a new ICT property, affording ratiometric
spectral changes and achieving two-photon excitation. The two
fluorescent probes can be used for specific ratiometric sensing
of H2S upon both one-photon and two-photon excitation. The
study provided a design strategy of ratiometric two-photon
fluorescent probes. Furthermore, cell-imaging experiments
reveal that AcHS-2 is a new mitochondria-specific and
emission-ratiometric two-photon probe for H2S. The new
probe is promising to be utilized in a variety of chemical and
biological applications.
Figure 8. Confocal fluorescence images of MCF-7 cells incubated with
5 μM AHS-2 for 1 h (a−c) and then 0.5 mM Na2S for 1 h, under 405
nm excitation (d−f) and 800 nm excitation (g−i). Images were
acquired with emission channels of 408−500 nm (blue) and 500−650
nm (green). Cells shown are representative images from replicate
experiments (n = 3).
AcHS-2 can detect H2S in live cells by cellular imaging under
both one-photon and two-photon excitation.
From the ratiometric fluorescence images shown in Figures 7
and 8, the values of Fgreen/Fblue, the ratio of the relative emission
intensity of 500−650 nm (green) and 408−500 nm (blue),
showed large changes for systems between with and without
Na2S (Figure 9). The values of Fgreen/Fblue for MCF-7 cells
stained alone with AcHS-1 and AcHS-2 were 0.31 and 0.38, and
the values for the cells incubated with additional Na2S increased
EXPERIMENTAL SECTION
■
1
Instrumentation and Methods. H and 13C NMR spectra were
recorded on an NMR spectrometer operating at 400 or 300 and 75
MHz, respectively. FT-IR spectra were carried out with an infrared
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Figure 9. Average intensity ratios (Fgreen/Fblue) for AcHS-1, AcHS-1/Na2S (Figure 7cf), AcHS-2, and AcHS-2/Na2S (Figure 8cf).
where I0 is the input intensity at the focus z = 0, L is the sample length,
α is the linear absorption coefficien, and β is the two-photon
2
absorption coefficient. z0 = πω0 /λ is the Rayleigh diffraction length,
and ω0 is the radius of beam at focus. Thus, once an open-aperture Z-
scan is performed, the nonlinear absorption coefficient β can be
unambiguously deduced. Furthermore, the molecular TPA cross-
section (σ) could be determined by using the following relationship29
δ = hυβ × 10−3 / N d
)
A
(2)
(
) (
where h is the Planck’s constant, υ is the frequency of input intensity,
NA is the Avogadro constant, and d is the concentration of the sample.
Determination of Detection Limits. The detection limit was
calculated on the basis of the method reported in the literature.30 The
fluorescence emission spectra of probes were measured three times
and the standard deviation of a blank measurement was achieved. The
ratio of fluorescence intensity at F530/F465 vs the concentration of Na2S
was plotted. The detection limit was calculated by using the equation
detection limit = 3N/k, where N is the standard deviation of the blank
measurement k is the slope of the plot.
Cell Cultures and MTT Assays. MCF-7 cells were seeded in
DMEM (Dulbecco’s modified Eagle’s medium) supplemented with
10% FBS (fetal bovine serum) in an atmosphere of 5% CO2 and 95%
air at 37 °C. Before the experiment, the cells well placed in a 96-well
plate, followed by addition of various concentrations of probes. The
final concentrations of probes vary from 0 to 20 μM. The cells were
then incubated at 37 °C in an atmosphere of 5% CO2 and 95% air for
24 h, followed by MTT assays (n = 4).
Cell-Imaging Experiments. MCF-7 cells cultured under the
above condition were employed for cell-imaging experiments. The
imaging of MCF-7 cells was performed by a laser scanning confocal
fluorescence microscope (Zeiss LSM 510 Meta NLO). The cells were
treated initially with 5 μM AcHS-2 for 1 h. Then, Na2S (0.5 mM) was
added to the incubation medium, and the cells were incubated for 1 h.
The excitation wavelength was 375 or 405 nm and 750 or 805 nm for
one-photon and two-photon assays, respectively. Fluorescence signals
were collected from the blue channel (420−550 nm) and the green
channel (500−640 nm), respectively.
The colocalization experiment was carried out at the monoemission
mode. MCF-7 cells were stained by AcHS-2 (5 μM, 1 h) and
MitoTracker Red CMXRos (0.5 μM, 30 min) or MitoTracker Deep
Red FM (1 μM, 30 min) in sequence. The images for AcHS-2 were
collected with band paths of 408−500 nm for cells stained by only
AcHS-2 or 500−650 nm for cells stained by AcHS-2 and Na2S in
succession, while the images for two mitochondria dyes were collected
with a band path of 600−705 nm or 665−750 nm upon excitation at
578 or 633 nm, respectively.
Figure 10. Confocal fluorescence images of MCF-7 cells costained by
AcHS-2 (5 μM, 1 h) and MitoTracker Red CMXRos (0.5 μM, 30
min) without (a−d) and with 0.5 mM Na2S (e−h): (a, e) fluorescence
images obtained with band paths of 408−500 nm for cells incubated
by only AcHS-2 and 500−650 nm for incubated by AcHS-2 and Na2S,
upon excitation of AcHS-2 at 405 nm; (b, f) bright-field images; (c, g)
images from a band path of 600−705 nm; (d, h) merged images of (a),
(b) and (c) or (e), (f) and (g).
spectrometer. High-resolution mass spectrometry data were obtained
with a FTMS spectrometer or a LC−TOF MS spectrometer. UV−vis
absorption spectra and fluorescence spectra were recorded at room
temperature on a UV/vis spectrometer and a sprectrofluoropho-
tometer, respectively. The reaction mixtures of the probe with H2S
were analyzed by a HPLC with a C-18 reversed-phase column.
General Procedure for Spectral Measurements. Sample
solutions of AcHS-1 or AcHS-2 (10 μM, 2.5 mL) in a buffer solution
(0.01 M pH 7.0 phosphate buffer/EtOH = 4/1) were prepared in a
quartz cuvette (1 cm × 1 cm). Various 250 mM analytes were
prepared in pH 7.0 phosphate buffer, and a 10 μL of the analyte was
added to sample. Water for sample preparation was purified with a
Millipore system. All pH values were measured on a pH meter. The
fluorescence quantum yields (ΦF) of probes and compound 3 and 4
were determined in spectroscopic-grade solvents. The optical density
of the dilute solution of all compounds (the references AcHS-1 and
AcHS-2 and compounds 3 and 4) was around 0.05 at the excitation
wavelength, using fluorescein (Φf = 0.90 in 0.1 N NaOH)25 solution
and quinine sulfate (Φf = 0.54 in 0.1 N H2SO4)26 as references at
excitation wavelengths of 436 and 370 nm, respectively.
Measurements of Two-Photon Absorption Cross Section (δ).
Two-photon absorption (TPA) experiments were carried out on the
open-aperture Z-scan setup27 using a femtosecond laser with a pulse
duration of 140 fs and 80 MHz repetition rate. The thermal heating of
the sample with high repetition-rate laser pulse was removed by the
use of a mechanical chopper running at 1 kHz.28 Nonlinear absorption
coefficient β was measured by the open-aperture Z-scan technique. For
the open aperture, the normalized transmittance as a function of the
position along the z axis can be written as eq 1
Materials. Unless stated otherwise, all chemical regents were
purchased from commercial sources and used without further
purification. Solvents of technical quality were distilled prior to use.
Water for preparation of solutions was purified with a Millipore water
system.
Synthesis of 4-Azidophenylmethanol (5).18 4-Azidophenyl-
methyl bromide (1.3 g, 0.6 mmol) was suspended in water (5 mL).
Under nitrogen atmosphere, the mixture was stirred overnight at 90
°C and then cooled at room temperature and extracted with ethyl
∞
[−q(z)]m
βI0Leff
Tz =
, where q(z) =
∑
[1 + (z/z0)2]α
(m + 1)3/2
(1)
m=0
G
dx.doi.org/10.1021/jo5014838 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
acetate. The organic phase was dried with anhydrous Na2SO4 and
concentrated to dryness, the residue was purified using flash
chromatography (EtOAc/petroleum ether, 1:4 v/v), and 5 was
obtained as a yellow oil (455 mg, 50%): 1H NMR (CDCl3, 400
MHz) δ 7.35 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 8.1 Hz, 2H), 4.67 (s,
2H) ppm.
4.12 (t, J = 6.5 Hz, 2H), 3.58 (d, J = 5.4 Hz, 2H); 13C NMR (DMSO-
d6, 75 MHz) δ 41.3, 57.9, 107.6, 108.1, 119.3, 121.8, 123.9, 129.2,
129.7, 130.9, 133.9, 152.6, 163.0, 163.9; IR (Nujol) ν 3439 (s), 3352
̅
(s), 3251 (s), 2958 (s), 1632 (m), 1650 (s), 1586 (s), 1380 (s), 774
(s) cm−1; UV/vis (pH 7.0 PBS buffer) λmax (ε) 436 nm (11800 mol−1
dm3 cm−1); FTMS (ESI) calcd for C14H13N2O3 [M+] 257.0921, found
257.0919.
Synthesis of N-n-Butyl-4-nitro-1,8-naphthalimide (6a).20 n-
Butylamine (200 μL, 1.6 mmol) was quickly added to a cloudy
solution of 4-nitro-1,8-naphthalic anhydride19 (340 mg, 1.4 mmol) in
ethanol (7 mL). After being refluxed for 6 h under N2, the reaction was
allowed to cool to room temperature, filtered, and washed with cool
ethanol (2 mL). The residue was purified using flash chromatography
(EtOAc/petroleum ether, 1:10 v/v), and compound 6a was obtained
as a pale yellow solid (180 mg, 43%): 1H NMR (CDCl3, 400 MHz) δ
8.85 (dd, J = 8.7 Hz, J = 1.0 Hz, 1H), 8.74 (dd, J = 7.3 Hz, J = 1.0 Hz,
1H), 8.70 (d, J = 8.0 Hz, 1H), 8.41(d, J = 8.0 Hz, 1H), 7.98 (dd, J =
8.7 Hz, J = 7.3 Hz, 1H), 4.20 (t, J = 7.5 Hz, 2H), 1.72 (m, 2H), 1.46
(m, 2H), 0.99 (t, J = 7.3 Hz, 3H) ppm.
Synthesis of AcHS-2. The preparation process was the same as
that used for AcHS-1. The reaction mixture was purified by flash
column chromatography (EtOAc/petroleum ether, 1:2 v/v) to give
1
AcHS-2 as a pale yellow solid (12 mg, 7.0%): H NMR (CDCl3, 300
MHz) δ = 8.61−8.67 (m, 2H), 8.41 (d, J = 8.4 Hz, 1H), 8.19 (d, J =
8.4 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.45−7.50 (m, 3H), 7.07 (d, J =
8.4 Hz, 2H), 5.27 (s, 2H), 4.56 (t, J = 6.8 Hz, 2H), 3.84 (t, J = 6.8 Hz,
2H) ppm; 13C NMR (CDCl3, 75 MHz) δ 41.7, 41.4, 67.6, 117.0,
117.6, 119.5, 123.1, 123.2, 126.5,126.8,129.2, 130. 6,131.8, 132.1,
133.1, 139.4, 140.9, 153.1, 163.7, 164.3; IR (Nujol) ν 3437 (s), 3290
̅
(s), 3251 (s), 2923 (m), 2115 (s), 1694, (m), 1650 (s), 1538 (s), 1250
(m), 781 (s) cm−1; UV/vis (pH 7.0 PBS buffer) λmax (ε) 373 nm
(13400 mol−1 dm3 cm−1); TOF-MS (ES+) calcd for C22H18N5O5 [M+]
432.1308, found 432.1306.
Synthesis of N-n-Butyl-4-amido-1,8-naphthalimide (3).20
A
stirred cloudy solution of 6a (140 mg, 0.47 mmol) in ethanol (5 mL)
was added dropwise to the solution of SnCl2·2H2O (677 mg, 3 mmol)
in concentrated hydrochloric acid (1 mL) at room temperature for 15
min. The reaction was quenched with aqueous 10% Na2CO3 and
filtered. After being washed with water (3 × 10 mL), the residue was
dried in vacuo to afford compound 3 (98 mg, 78%): 1H NMR
(DMSO-d6, 400 MHz) δ 8.61 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 7.3 Hz,
1H), 8.18−8.20 (m, 1H), 7.43−7.67 (m, 1H), 7.43 (s, 2H), 6.84 (d, J
= 8.4 Hz, 1H), 4.00 (t, J = 7.2 Hz, 2H), 1.54−1.62 (m, 2H), 1.28−1.38
(m, 2H), 0.93 (t, J = 7.6 Hz, 3H) ppm; 13C NMR (DMSO-d6, 75
MHz) δ 13.7, 19.8, 29.8, 40.0, 107.5, 108.1, 119.3, 121.8, 123.9, 129.2,
ASSOCIATED CONTENT
■
S
* Supporting Information
Data for detection limits, stability of probes and confocal
fluorescence images of cells, and NMR spectra of related
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
129.6, 130.9, 133.9, 152.6, 162.9, 163.7; IR (Nujol) ν 3431 (s), 3355
̅
AUTHOR INFORMATION
(s), 2954 (m), 1636 (s), 1576 (s), 2372 (s), 771 cm−1(s); UV/vis (pH
7.0 PBS buffer) λmax (ε) 435 nm (12000 mol−1 dm3 cm−1); FTMS
(ESI) calcd for C16H17N2O2 [M+] 269.1284, found 269.1281.
■
Corresponding Author
*E-mail: qhsong@ustc.edu.cn. Fax: (+86) 0551-63601592. Tel:
(+86) 0551-63607992.
Synthesis of AcHS-1. A mixture of 3 (134 mg, 0.5 mmol) and
DMAP (192 mg, 1.50 mmol) in toluene (7.5 mL) was added dropwise
to a solution of triphosgene (152 mg, 0.5 mmol) in toluene. The
resulting solution was heated to reflux for 3 h. After being cooled to
room temperature, the reaction mixture was diluted with CH2Cl2 (6
mL) and filtered. To the filtrate was added 5 (100 mg, 0.55 mmol),
and the solution was stirred at room temperature for 3 h. The reaction
was then concentrated and purified by flash column chromatography
(EtOAc/petroleum ether, 1:2 v/v) to afford AcHS-1 as a pale yellow
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for financial support from the National Natural
Science Foundation of China (Grant No. 21272224) and the
Science Foundation of Anhui Province (128085MB19), facility
support from Prof. Jun Wang’s laboratory at USTC for cell
culture, and Prof. Hongping Zhou of Anhui University for
measurements of two-photon excitation spectra of two probes
and their sensing products (3 and 4).
1
solid (15 mg, 6.8%): H NMR (DMSO-d6, 400 MHz) δ 8.56 (d, J =
8.0 Hz, 1H), 8.52 (d, J = 8.8 Hz, 1H), 8.47 (d, J = 8.4 Hz, 1H), 7.83 (t,
J = 8.0 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 1H),
7.21 (d, J = 8.4 Hz, 2H), 5.48 (s, 2H), 4.04 (t, J = 7.4 Hz, 2H), 1.61
(m, 2H), 1.35 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H) ppm; 13C NMR
(DMSO-d6, 75 MHz) δ 13.7, 19.8, 29.6, 40.0, 66.0, 117.1, 118.1, 119.2,
122.2, 123.8, 126.3, 128.3, 129.2, 130.2, 130.9, 131.6, 133.1, 139.3,
REFERENCES
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140.6, 153.9, 162.9, 163.4; IR (Nujol) ν 3294 (s), 2959 (s), 2115 (m),
̅
1692 (s), 1650 (s), 1538 (s), 1248 (s), 772 (s) cm−1; UV/vis (pH 7.0
PBS buffer) λmax (ε) 370 nm (13400 mol−1 dm3 cm−1); FTMS (ESI)
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I
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