Supporting Information
For
A near-infrared fluorescent probe for hydrogen sulfide and its
application in living cells imaging
Tao Chena,b†, Yi Zhengb,c†, Zhaochao Xua,b,*, Miao Zhaob, Yongnan
Xuc,* and Jingnan Cuia,*
a
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012,
China, E-mail: jncui@dlut.edu.cn
b
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China,
E-mail: zcxu@dicp.ac.cn
c
The School Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang
110016, China, E-mail: ynxucn@yahoo.com.cn
† equally contributed to this work.
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Synthesis
(4-azidophenyl)methanol (3). To a solution of 4-aminobenzyl alcohol (300 mg, 2.44 mmol) in
5 mL of 10% HCl aqueous solution was added NaNO2 (201 mg, 2.92 mmol) in 3 mL aqueous
solution at 0 ºC and stirred for 30 min. Then NaN3 (190 mg, 2.92 mmol) in 3 mL aqueous
solution was added at 0 ºC and stirred for another hour. The reaction mixture was warmed to 25
ºC, diluted with ethyl acetate, washed with water and brine, dried over Na2SO4, concentrated in
vacuo and subjected to silica gel chromatography. A yellow oil 3 (315 mg, 86% yield) was
obtained by silica gel column chromatography using Petroleum ether/EtOAc (5:1, v:v) as eluent.
1H
NMR (500 MHz, CDCl3)：δ 7.23 (d, J = 8 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 4.51 (s, 2H),
2.04 (s, br, 1H).
4-azidobenzaldehyde (4). Compound 3 (306 mg, 2.06 mmol) was dissolved in 15 mL dry CH2Cl2.
Dess-Martin reagent (1.3 g, 3.08 mmol) was added and the mixture was stirred for 2 h at room
temperature, at which point oxidation was completed. The mixture was diluted with EtOAc (60
mL), washed with saturated Na2S2O3 (10 M), saturated aqueous NaHCO3 (10 mL), and brine.
Then organic layer was dried with Na2SO4 and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography using Petroleum ether/EtOAc (50:1,
v:v) as eluent to afford 4 as a yellow oil. Yield: 283 mg (93.5%). 1H NMR (CDCl3, 500 MHz): δ
9.85 (s, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H).
3-cyano-2-dicyanomethylene-4,5,5-trimethyl-2,5-dihydrofuran (7). A mixture of 3-hydroxy-3methyl-2-butonone (5) (1.5 g, 14.7 mmol), malonontrile (6) (2.91 g, 44 mmol), magnesium
ethoxide (2.02 g, 17.6 mmol) and absolute ethanol (20 mL) was stirred at 60 oC for 10 h. Then the
S2
solvent was evaporated, and the residue was collected and washed with 20 mL water. Then, the
residue was recrystallized from ethanol to give product 7 as a yellow solid. Yield: 930 mg (31.8%).
1H
NMR (CDCl3, 500 MHz): δ 2.37 (s, 3H), 1.64 (s, 6H).
2-{4-(4’-azidophenylethenyl)-3-cyano-5,5-dimethyl-5H-furan-2-ylidene}-malononitrile (1). To
a 25-ml round-bottom flask with stir bar were added 4-nitrobenzaldehyde (4) (236 mg, 1.6 mmol),
3-cyano-2-dicyanomethylene-4,5,5-trimethyl-2,5-dihydrofuran (7) (319 mg, 1.7 mmol), pyridine
(5 mL), and acetic acid (several drops). The reaction mixture was stirred at room temperature for
24 h and then warmed to 40 oC for another 15 h. Then the reaction was stopped and cooled to
room temperature. The reaction mixture was poured into ice water (500 mL) and stirred for 7 h.
The suspension was filtered .The brown precipitate was purified by silica gel column
chromatography (CH2Cl2 : PE = 2 : 1) to afford compound 1 as a yellow solid. Yield: 196 mg
(37.4%). 1H NMR (500 MHz, DMSO-d6): δ 7.95 (d, J = 8.5 Hz, 2H), 7.89 (d, J =16.5 Hz, 2H),
7.24 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 16.5 Hz, 1H), 1.77 (s, 6H); 13C NMR (500 MHz, DMSO-d6):
δ 177.5, 175.6, 146.8, 143.8, 131.8, 131.7, 120.5, 115.2, 113.1, 112.3, 111.3, 99.8, 99.6, 54.8, 25.6.
HRMS (API-ES) calcd for C18H12N6O [M+] 328.1073, found 328.1079.
DCDHF-NH2. To a 25 mL round bottomed flask equipped with a magnetic stirrer, compound 8
(700 mg, 2.1 mmol) was added to a suspension of 37.4% HCl (4 mL) and stannous chloride
dehydrate (3.6 g, 15.75 mmol) at room temperature. Stirring was continued for 12 h at refluxing
temperature. Then the solution was poured onto 20 g of ice and made alkaline using sodium
hydroxide solution (5 M). The resulting suspension was then extracted with EA (3×100 mL). The
organic layer was washed with water (50 mL) and then dried over anhydrous sodium sulfate. The
solvent was removed to obtain pasty residue which was purified by silica gel column
chromatography (CH2Cl2:PE = 1:1) to afford the compound as a purplish red solid in 81% yield.
1H
NMR (500 MHz, DMSO-d6): δ 7.88 (d, 1H, J = 15.5 Hz, trans), 7.67 (d, 2H, J = 9 Hz, Ar-H),
6.82 (d, 3H, J = 16 Hz, NH2, trans), 6.66 (d, 2H, J = 9 Hz, Ar-H), 1.74 (s, 6H, 2×CH3).
Imaging of HUVEC
HUVEC were cultured in DMEM/F12 medium (Hyclone, China) under 95% humidified
atmosphere with 5% CO2 at 37° C. Besides DMEM/F12, HUVEC culture medium contained 1%
penicillin-streptomycin and 10% fetal bovine serum which were purchase from Invitrogen, USA.
Cells passage was conducted every two days with 0.025% trypsin solution digest.
The staining experiment was conduct when HUVEC were confluence around 70%-80%. 50
M 1 in the culture media was added to the cells and the cells were incubated for 1 h at 37 oC.
After washing twice to remove the remaining sensor, the cells were treated with 50 μM NaHS for
60 min. The cell sample was observed by IX 73 Olympus microscope.
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400
200
0
600
700
Figure S1. Fluorescent emission spectra of 100 M compound 1 with 1 eq H2S and 100 M DCDHF-NH2 in
aqueous solution (CH3CN:50 mM HEPES = 1:2, pH = 7.4). Excitation at 574 nm.
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Figure S2. MS spectra of 100 M compound 1 in the presence of 1 equiv of H2S in aqueous solution (CH3CN:50
mM HEPES = 1:2, pH = 7.4). MS: calcd for C18H13N4O [M-H]+ 301.1, found 301.0.
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300
200
100
0
600
650
700
Figure S3. Fluorescence emission spectra of 100 M compound 1 in the presence of different concentration of
H2S in aqueous solution (CH3CN:50 mM HEPES = 1:2, pH = 7.4). Excitation at 574 nm.
400
200
0
600
700
Figure S4. Time-dependent fluorescence intensity of 100 M compound 1 in the presence of 1 equiv of H2S in
aqueous solution (CH3CN:50 mM HEPES = 1:2, pH = 7.4). Excitation at 574 nm. Time points represent 0, 10, 20,
30, 40, 50, and 60 min after the addition of H2S.
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Figure S5. 1H-NMR spectra of compound 3 in CDCl3.
Figure S6. 1H-NMR spectra of compound 4 in CDCl3.
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Figure S7. 1H-NMR spectra of compound 7 in CDCl3.
Figure S8. 1H-NMR spectra of compound 1 in DMSO-d6.
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Figure S9. 13C-NMR spectra of compound 1 in DMSO-d6.
Figure S10. 1H-NMR spectra of compound DCDHF-NH2 in DMSO-d6.
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