A selective and sensitive phthalimide-based fluorescent probe for hydrogen sulfide with a large Stokes shift

Article abstract of DOI:10.1039/c5ra19081a

A phthalimide-based fluorescent probe for hydrogen sulfide has been developed with high sensitivity and excellent selectivity. Upon treatment with hydrogen sulfide, this probe displays a strong fluorescence enhancement (196-fold) with a large Stokes shift (105 nm). Moreover, the potential application of using this probe in biological systems has been demonstrated by imaging hydrogen sulfide in living cells.

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A selective and sensitive phthalimide-based fluorescent probe for
hydrogen sulfide with a large Stokes shift
Liu Yang,‡a Xingjiang Liu,‡a Li Gao,a Fengpei Qi,a Huihui Tian,a and Xiangzhi Songab*
Received 00th January 20xx,
Accepted 00th January 20xx
A phthalimide-based fluorescent probe for hydrogen sulfide has been developed with high sensitivity and excellent
selectivity. Upon the treatment with hydrogen sulfide, this probe displays a strong fluorescence enhancement (196-fold)
with a large stokes shift (105 nm). Moreover, the potential application using this probe in biological systerm has been
demesnstrated by imaging hydrogen sulfide in living cells.
DOI: 10.1039/x0xx00000x
www.rsc.org/
minimize self-absorption and reduce the interference from
auto-fluorescence.17 So far there are only a few H2S fluorecent
probes with large Stokes shifts reported.17,18 3-
Introduction
Hydrogen sulfide (H2S) with rotten-egg odor is generally
considered as a toxic gas to induce the toxication of central
nervous system and the inhibition of the respiratory system.1
H2S can be endogenously produced through enzymatic actions
by cystathionine-β-synthase,2 cystathionine-γ-lyase3 and 3-
mercaptopyruvate sulfurtransferase4 in mammalian system.
Recently, it has been found that endogenous H2S exerts vital
functions in many physiological activities such as vasodilation,5
neuromodulation,6 anti-inflammation,7 angiogenesis,8 anti-
apoptosis,5 and inhibition of insulin signaling9 and is therefore
regarded as the third most important endogenous
gasotransmitter alongside nitric oxide (NO) and carbon
monoxide (CO). In addition, H2S servers as an antioxidant or
scavenger for endogenous reactive oxygen species (ROS)
involving in the regulation of the cellular redox state.10 Due to
its important roles in physiological processes, abnormal levels
of H2S can cause cardiovascular dysfunction,11 Alzheimer's
Disease,12 Down syndrome,13 diabetes,14 and liver cirrhosis.15
As a result, it is extremely meaningful to explore an efficient
method to detect H2S in environmental and biological systems.
Owning to its high sensitivity, high spatiotemporal
resolution and real-time imaging ability, fluorescent probes to
detect and image H2S in vitro/vivo have drawn considerable
attention in the past decades.16 Fluorescent probes with a
large Stokes shift are more desirable because the large
spectral separation between absorption and emission can
Aminophthalimide derivatives, with an excited-state
intramolecular proton transfer (ESIPT) characteristic, have an
emission in green spectral region with a large Stokes shift and
a relatively high fluorescent quantum yield.19 In addition, 3-
aminophthalimide derivatives can be easily prepared.
Therefore, 3-aminophthalimide might be an ideal scaffold to
construct fluorescent probes.
In this report, we designed and synthesized a derivative of
3-azidophthalimide as a turn-on fluorescent probe for the
detection of H2S with a large Stokes shift. We speculated that
the azido group in this probe would efficiently quench the
fluorescence and the treatment with H2S would reduce the
probe into 3-aminophthalimide which exhibits strong green
fluorescence.16a, 16d, 18b, 20
Results and discussion
Synthesis
Scheme 1 Synthetic route of Probe 1. (a) Butylamine, acetic acid, 120 °C, 2.5 h,
yield 85.7%. (b) Palladium/C, H2, CH3OH, 65 °C, 12 h, 73.5%. (c) (1) NaNO2, HCl,
0 °C, 30 min; (2) NaN3, 0 °C, 40 min, yield 81.7%.
Probe
Scheme 1. First, 3-nitrophthalic anhydride reacted with
butylamine to give compound as a white solid in 85.7% yield.
Next, compound was hydrogenated with palladium/carbon
catalyst in 73.5% yield. Finally, Probe was obtained in 81.7%
1 was readily prepared in three steps, as outlined in
a.College of Chemistry & Chemical Engineering, Central South University, Changsha,
Hunan Province, P. R. China, 410083.
b.State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian,
Liaoning Province, P. R. China, 116024.
2
2
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See DOI:
10.1039/x0xx00000x
1
yield by the classical diazotization-azidation reaction from
‡
These authors contribute equal to this work.
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compound 3. All the reactions were neat with good yields and 0.99988) was obtained between the fluorescence intensity at
did not need column chromatography. The detailed synthetic 492 nm and the concentration of H2S in the range of 0.0-40.0
procedures were described in experimental section and the µM. The detection limit was calculated to be 13.6 nM based on
characterization data for the investigated compounds was the signal-to-noise (S/N = 3), which was sufficiently low for the
presented in supporting information.
detection of H2S in environmental and biological samples.
These results indicated that Probe 1 was highly sensitive to H2S.
Sensing property of Probe 1 to H2S
Mechanism studies
The sensing property of Probe
1 was studied in PBS buffer (pH
7.4) with 1.0 mM CTAB (cetyltrimethylammonium bromide) at The optical changes of Probe
25 °C. The solution of Probe was colorless (λabsmax = 344 nm) that H2S reduced the azido group into amino group in Probe
and essentially non-fluorescent. Upon the addition of H2S to generate dye , which displayed a strong green fluorescence
(Na2S, a standard H2S source), the solution of Probe with a large Stokes shift due to the ESIPT process. The
1 in response to H2S suggested
1
1
3
1
exhibited a significant fluorescence enhancement with λemmax proposed mechanism was illustrated in Scheme 2. In order to
= 492 nm and a pale yellow color with λabsmax = 387 nm, which
were coincident with the hallmark of dye
3
(Fig. 1, Fig. S1 and
with H2S
S2). As expected, the reaction product of Probe
1
Scheme 2 The proposed sensing mechanism of Probe
1 with H2S.
1
further confirm this sensing mechanism, we performed the H
NMR and mass spectral analysis on the reaction product of
Probe
the reaction product was in good agreement with that of
reference dye . The mass spectrum of the reaction product
exhibited a peak at m/z = 219.1131 (Fig. S12) which is nearly
identical to the exact molecular weight of dye ([M+1] =
219.1134). Furthermore, we conducted HPLC analysis on Probe
, dye and the reaction solution of Probe with H2S (shown
in Fig. 3). Probe showed a single peak with a retention time
at 9.7 min (Fig. 3(a)) and dye produced a single peak at 5.7
min (Fig. 3(d)). When treated the solution of Probe with 1.0
equiv. of H2S, the peak assigned to Probe decreased with the
1
with H2S. As shown in Fig. S4, the 1H NMR spectrum of
3
3
1
3
1
Fig. 1 Fluorescence spectra of Probe 1 (10.0 µM) upon the addition of H2S (0.0-12.0
equiv.) in PBS buffer (10.0 mM, pH = 7.4, containing 1.0 mM CTAB). Inset: photographs
of Probe 1 before (left) and after (right) the addition of H2S. Spectra were recorded
after incubation with H2S for 40 min. λex = 380 nm. Excitation and emission slits: 5.0
nm/2.5 nm.
1
3
1
1
appearance of a new peak at 5.7 min. When the solution of
exhibited a 105 nm Stokes shift. The fluorescence intensity of
Probe
H2S. The fluorescence enhancement was up to 196-fold when
the solution of Probe (10.0 µM) was treated with 12.0 equiv.
of H2S. As depicted in Fig. 2, a linear calibration curve (R =
1 was increased with increasing the added amount of
1
Fig. 3 The HPLC chromatograms: (a) Probe 1 (100.0 μM); (b) Probe 1 (100.0 μM) with
1.0 equiv. of H2S incubated for 1 h in CH3CN/H2O (v/v, 2/1); (c) Probe 1 (100.0 μM) with
10.0 equiv. of H2S incubated for 1 h in CH3CN/H2O (v/v, 2/1); (d) reference dye 3 (100.0
μM). Condition: eluent, H2O/CH3CN (v/v, 3/7), flow rate, 1.0 mL/min; temperature,
25 °C; detection wavelength, 360 nm; injection volume, 20.0 μL.
Fig. 2 Fluorescence intensity of Probe
1 (10.0 µM) at 492 nm as a function of H2S
concentration in PBS buffer (10.0 mM, pH = 7.4, containing 1.0 mM CTAB). Inset:
the linear relationship between the fluorescence intensity at 492 nm and the
concentration of H2S (0.0 - 40.0 µM).
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of Probe
min, assigned to Probe
only the peak at 5.7 min, assigned to dye
1
was treated with 10.0 equiv. of H2S, the peak at 9.7 Probe
, completely disappeared whereas be concluded that Probe
, was left. These toward H2S.
1
in the detection of H2S (shown in Fig. 5). Thus, it could
1
1
exhibited an excellent selectivity
3
results strongly supported the proposed sensing mechanism in
Scheme 2.
Kinetic studies
Time-dependent fluorescence experiments were performed on
Probe (10.0 μm) with 12.0 equiv. of H2S by monitoring the
for H2S, we fluorescence intensity at 492 nm. After the addition of H2S to
in response to the solution of Probe , the fluorescence enhancement was
Selectivity and competition studies
1
In order to evaluate the selectivity of Probe
investigated the fluorescence change of Probe
various relevant species. As shown in Fig. 4, no fluorescence
1
1
1
21-fold within 1 min and reached a plateau (196-fold) within
40 min (shown in Fig. 6). In contrast, no fluorescence
enhancement occurred when the solution of Probe
1 was in
the absence of H2S within 40 min. The observed first order rate
constant, kobs, was determined to be 0.064 min−1 (shown in Fig.
S4).
Fig. 4 Fluorescence spectra of Probe 1 (10.0 µM) upon the addition of various
species in PBS buffer (10.0 mM, pH = 7.4, 1.0 mM CTAB). These species included
3-
H2S, F-, Cl-, Br-, I-, NO3-, NO2-, CH3COO-, N3-, SO42-, S2O32-, S2O52-, CN-, SO32-, PO4
CO32-, H2O2, ClO-, Cys, Hcy and GSH.
,
Fig. 6 Time-dependent fluorescence behavior of Probe 1 (10.0 µM) (492 nm) in the
presence of H2S (12.0 equiv.) in PBS buffer (10.0 mM, pH = 7.4, containing 1.0 mM
CTAB).
response was observed when the solution of Probe
1 was
-
-
treated with the common anions (F-, Cl-, Br-, I-, NO3 , NO2 ,
-
3-
2-
,
CH3COO-, N3 , SO42-, S2O32-, S2O52-, CN-, SO32-, PO4 and CO3
0.4 mM for each) and reactive oxygen species (H2O2 and ClO-,
0.4 mM for each). The counter ion for all the anions was Na+
except (n-Bu)4N+CN- was used for CN-. To our delight, none of
the biothiols (Cys and Hcy, 0.5 mM for each; 1.0 mM GSH), the
main competitive species in biological systems, induced
obvious fluorescence. Moreover, the co-existence of all these
above mentioned species had little impact on the ability of
pH effect studies
Moreover, we sought to study pH influence on the
fluorescence behavior of Probe
shown in Fig. 7. In the pH range of 2.0 to 12.0, the
fluorescence spectra of Probe remained unchanged,
1 in the detection of H2S. As
1
indicating that this probe was stable in wide pH range. When
Fig. 5 The fluorescence responses of Probe 1 (10 μM) to H2S (12.0 equiv.) in the
Fig. 7 Fluorescence intensity of Probe 1 (10.0 µM) at 492 nm under different pH values
presence of various relevant species in PBS buffer (10.0 mM, pH = 7.4, 1 mM CTAB). (1)
in the absence/presence of H2S (12.0 equiv.).
-
H2S (120.0 μM); (2-18) F-, Cl-, Br-, I-, NO3 , NO2-, CH3COO-, N3-, SO42-, S2O32-, S2O52-, CN-,
SO32-, PO43-, CO32-, H2O2 and ClO- (0.4 mM for each); (19, 20) Cys and Hcy (0.5 mΜ for
Probe
1 (10.0 µM) was incubated with H2S (12.0 equiv.),
each); (21) GSH (1.0 mM).
negligible fluorescence was observed at pH 2.0 and 3.0, and
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week fluorescence signal was obtained at pH 4.0. It suggested Chemicals, China. Unless otherwise noted, all reagents and
that H2S could not reduce N3 group to NH2 in strong acid starting materials were obtained from commercial suppliers
solution (shown in Scheme S1). However,
fluorescence signal was observed in the solution of Probe
with H2S in the pH range (5.0 to 12.0). The wide pH range for lines (HNE-2 cells) were provided by Xiangya Third People’s
Probe in the detection of H2S implied its potential application Hospital of Central South University (China).
a
strong and used as received. Double distilled water was used
1
throughout all the experiments. Nasopharygeal carcinoma cell
1
in environmental and biological studies (pH = 7.4 under
physiological condition).
Synthesis of compound 2
To a solution of 3-nitrophthalic anhydride (1.9361 g, 10.0
mmol) in acetic acid (20.0 mL) was slowly added butylamine
Fluorescence imaging of H2S in living cells
Encouraged by the above merits of Probe
1 in aqueous (1.0951 g, 15.0 mmol) within 5 min. After stirring at room
solution, we sought to evaluate its ability to visualize temperature for 10 min, the resulting mixture was refluxed at
intracellular H2S in living cells. When HNE-2 cells, 120 °C for 2.5 h. Next, the reaction mixture was cooled to
nasopharygeal carcinoma cell lines, were treated with H2S room temperature and then poured into 50.0 mL cold water to
(120.0 μM) for 30 min at 37 °C, washed with PBS buffer three afford a precipitate. The solid was collected and washed with
times and then incubated with Probe
30 min, a strong green fluorescence was observed inside the (2.1252 g, 85.7% yield). H NMR (500 MHz, DMSO-d6, TMS) δH
cells (Fig. 8b). However, the cells only incubated with Probe 8.27 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 7.5 Hz, 1H), 8.05 (t, J = 7.8
1 (10.0 μM) for another water (3.0 mL × 3) to give pure compound 2 as a white solid
1
1
(10.0 μM) for 30 min at 37 °C (Fig. 8d) exhibited negligible Hz, 1H), 3.57 (t, 2H), 1.63-1.51 (m, 2H), 1.38-1.24 (m, 2H), 0.90
fluorescence. These fluorescence imaging experiments (t, 3H). 13C NMR (125 MHz, DMSO-d6) δC 166.5, 163.8, 144.6,
demonstrated that Probe
living cells.
1
had a potential for imaging H2S in 136.5, 134.1, 128.6, 127.2, 123.5, 38.1, 30.2, 19.9, 13.9.
Synthesis of compound 3
Compound
2 (0.4964 g, 2.0 mmol) was hydrogenated in
methanol (15.0 mL) under reflux at 65 °C for 12 h with 10%
Pd/C (0.0496 g) as a catalyst. Then, the reaction mixture was
filtered through celite to remove the catalyst. Next, the filtrate
was concentrated in vacuum to give a solid. The solid was
collected and washed with cold ethanol to give pure
1
(0.3210 g, 73.5% yield) as a pale yellow solid. H
compound
3
NMR (500 MHz, CDCl3, TMS) δH 7.42 (dd, J = 8.3, 7.1 Hz, 1H),
7.16 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 5.13 (s, 2H),
3.65 (t, 2H), 1.63-1.70 (m, 2H), 1.35-1.42 (m , 2H). 0.95 (t, 3H).
13C NMR (125 MHz, CDCl3) δC 170.4, 168.8, 145.2, 135.02,
132.9, 121, 112.6, 111.4, 37.4, 30.8, 20.1, 13.7.
Fig. 8 (a, c) Bright-field and (b, d) fluorescence images of living HNE-2 cells. Top row:
cells pretreated with H2S (120.0 µM) for 30 min at 37 °C, washed with PBS buffer and
then incubated with Probe 1 (10.0 μM) for 30 min at 37 °C (a, b). Bottom row: cells
incubated with Probe 1 (10.0 μM) for 30 min at 37 °C. The cells were magnified 20
times.
Synthesis of Probe 1
The mixture of compound
3 (0.2183 g, 1.0 mmol) and
concentrated hydrochloric acid (4.0 mL) was stirred at 0 °C for
10 min. Then, the solution of sodium nitrite (0.1383g, 2.0
mmol in 1.0 mL water) was added dropwise and the reaction
mixture was stirred for 30 min to afford a clear solution. Next,
the solution of sodium azide (0.1301 g, 2.0 mmol in 1.0 mL
water) was added dropwise to the above reaction mixture.
After stirring for 40 min at 0 °C, the precipitated solid was
collected and washed with water (3.0 mL × 3) to give pure
Experimental section
Instruments and Materials
1H NMR and 13C NMR spectra were recorded on a Bruker 500
NMR spectrometer using tetramethylsilane (TMS) as the
internal standard for chemical shifts. Mass spectra were
obtained on a high resolution mass spectrometer (IonSpec4.7
T FTMS-MALDI/DHB). UV-vis absorption spectroscopy was
Probe 1 as a white solid (0.1993 g, 81.7% yield). HRMS (EI) m/z:
calcd for C12H12N4NaO2 [M + Na]+, 267.0858; found, 267.0860.
1H NMR (500 MHz, CDCl3) δH 7.71 (t, J = 7.5, 1H), 7.63 (d, J = 7.0
Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 3.69 (t, J = 7.2 Hz, 2H), 1.75 -
1.58 (m, 2H), 1.48 - 1.27 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C
NMR (125 MHz, CDCl3) δC 167.5, 166.4, 138.1, 135.2, 134.3,
124.9, 120.9, 119.4, 37.9, 30.5, 20.0, 13.6.
measured with
a
UV-2450 spectrophotometer and
fluorescence spectra was recorded on a Hitachi F-7000
spectrometer. Fluorescence imaging experiments were
performed on an Olympus IX83 inverted microscope. pH
measurement was carried out on a Leici PHS-3C meter. TLC
silica gel plates and silica gel (mesh 200-300) for column
chromatography were purchased from Qingdao Ocean
Synthesis of the reaction product of Probe 1 with H2S
To a solution of Probe
1 (0.0244 g, 0.1 mmol) in a mixture of
acetonitrile (15.0 mL) and PBS buffer (10.0 mL) was added a
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solution of Na2S (0.0782 g, 1.0 mmol in water 5.0 mL) at room
temperature. Next, the reaction mixture was stirred at room
temperature for 2 h and then extracted with dichloromethane
(20.0 mL × 3). The organic layer was separated, combined and
dried over anhydrous Na2SO4. After removal of the solvent, the
solid was purified by silica gel flash chromatography using
petroleum ether/dichloromethane (v/v, 1:1) as eluent to
afford the target product (0.0148 g, 67.7% yield). HRMS (EI)
m/z: calcd for C12H15N2O2 [M + 1]+, 219.1134; found, 219.1131.
1H NMR (500 MHz, CDCl3) δ 7.40 (dd, J = 8.2, 7.2 Hz, 1H), 7.14
(d, J = 7.1 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 5.25 (s, 2H), 3.64 (t, J
= 7.3 Hz, 2H), 1.71 - 1.59 (m, 2H), 1.44 - 1.30 (m, 2H), 0.95 (t,
3H).
3
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Imaging of HNE-2 cells
HNE-2 cells were cultured in Dulbecco’s Modified Eagle’s 11
Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS, Hyclone) as well as 1% penicillin, and incubated at 37 °C 12
in 5% CO2 and 95% air. One day before imaging, cells were
passaged and plated on 9-well plate. Before used, the growth 13
medium was removed and the cells were washed three times
with PBS buffer. The cells were incubated with H2S (120.0 μM) 14
for 30 min at 37 °C, washed with PBS buffer and then
Y.-H. Liu, M. Lu, L.-F. Hu, P. T.-H. Wong, G. D. Webb and J.-S.
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incubated with Probe
1
(10.0 μM) for another 30 min. After 15
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washing with PBS buffer, cell imaging experiments were
performed. For a control experiment, HNE-2 cells were only
incubated with Probe
1
(10.0 μM) for 30 min at 37 °C.
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Conclusions
To close, we have developed a phthalimide-based fluorescent
probe for H2S in both aqueous solution and living cells. This
turn-on ESIPT-based probe features a significant fluorescence
enhancements (196-fold) and low detection limit (13.6 nM) in
response to H2S. Importantly, this probe shows a large Stokes
shift (105 nm) which can minimize the self-absorption and
reduce the interference from auto-fluorescence. Preliminary
biological experiments demonstrates that this probe is cell
permeable and capable of detecting H2S in living cells.
Acknowledgements
This research was supported by the Fundamental Research
Funds for the Central South University and State Key
Laboratory of Fine Chemicals (KF1202). We thank Modern
Analysis and Testing Center of Central South University for
NMR spectral data of all the compounds.
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ARTICLE
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Graphical Abstract
A phthalimide-based fluorescent probe for H2S with a large Stokes shift has been
developed. This probe displayed good selectivity and high sensitivity toward H2S.
Imaging intracellular H2S by using this probe was successfully achieved in living
cells.