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Supporting Information
A highly selective and sensitive fluorescent probe for simultaneously distinguishing and sequentially detecting hydrogen multiple thiol species in solution and in live cells
Dan Qiaoa, Tangliang Shena, Mengyuan Zhub, Xiao Lianga, Lu Zhanga, Zheng Yina, Binghe Wangb*, Luqing Shanga*
a. College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University Tianjin 300071 (P.R. China) E-
mail: [email protected]
b. Department of Chemistry Georgia State University Atlanta, Georgia 30303 USA E-mail: [email protected]
1
Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2018
Experimental Section
1. Apparatus 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Bruker
AVANCE-400 (400 MHz) (Bruker, Karlsruhe, Germany) NMR spectrometer and TMS
as an internal standard. Electrospray ionization mass spectroscopy (ESI-MS) was
performed on a Shimadzu LC/MS-2020 (Shimadzu, Kyoto, Japan). HRMS were
recorded on a high-resolution ESI-FTICR mass spectrometer (Varian 7.0 T, Varian,
USA). UV-vis spectra were measured by a UH-5300 spectrophotometer (Hitachi).
Fluorescence measurements were recorded on a F-7000 spectrophotometer (Hitachi).
Fluorescence images of cells were taken by a laser scanning confocal microscopy
(Leica TCS SP8).
2. Materials
Unless otherwise stated, all reagents used in this study were commercial products
without further purification. Twice-distilled water was used throughout all experiments.
TLC analysis was performed on silica gel plates and column chromatography was
conducted over silica gel (mesh 200 - 300), both of which were obtained from the
Qingdao Ocean Chemicals. Mito-Tracker Green was obtained from Solarbio.
3. Detection limit
The limit of detection defined by 3 where is the standard deviation of the blank 𝜎⁄𝑘, 𝜎
sample and is the slope of the linear regression equation. 𝑘
4. Theoretical study on the fluorescence mechanism
The structure of NCR was constructed using Gaussian1, which was initially optimized
using the capabilities within these aforementioned programs. Calculations were then
conducted using density functional theory (DFT) with the B3LYP2 exchange functional
employing 6-31G(d)3 level implemented via the Gaussian 09 suite of programs for the
full geometry optimization and frequency calculations of the probe. Imaginary
frequencies were not obtained in any of the frequency calculations.
5. Cells culture
HeLa cells were cultured in DMEM (Dulbecco’s modified Eagle's medium) and
supplemented with 10% FBS (fetal bovine serum), penicillin (100 μg/mL) and
streptomycin (100 μg/mL) in an atmosphere of 5% CO2 and 95% air at 37 °C.
6. MTT assay
2
Cell cytotoxicity was evaluated by MTT assay. Cells were cultivated in a 96-well
plate until 50-70% confluence, and then incubated with different concentrations of
NCR (0-20 μM) for 24 h. 20 μL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT, 5 mg/ mL) was added for 4 h at 37 °C. After removing the MTT, 100
μL DMSO was added. The absorbance intensities at 570 nm were measured using a
plate reader. All experiments were repeated five times, and the data were presented as
the percentage of control cells.
7. Discriminative fluorescence imaging of biothiols in live cells
The cell experiment was divided into six groups. In the first group, HeLa cells were
incubated with NCR (5 μM) for 30 min and imaged after washing by phosphate-
buffered saline (PBS) for three times. For the second group, HeLa cells were pretreated
with NEM (0.5 mM) for 30 min, subsequently incubated with NCR (5 μM) for 30 min,
and then washed by PBS buffer for three times before imaging. In the third to sixth
group, HeLa cells were pretreated with NEM (0.5 mM) for 30 min, subsequently
incubated with NaHS/Cys/Hcy/GSH (500 μM, 30 min) and probe NCR (5 μM, 30 min),
and then washed with PBS buffer for three times. Cells were imaged by a laser scanning
confocal microscopy (Leica TCS SP8; λex = 405 nm, λem = 440-490 nm for the blue
channel; λex = 561 nm, λem = 575-620 nm for the red channel).
8. Sequential fluorescence imaging of biothiols in live cells
The cell experiment was divided into four groups. In the first group, HeLa cells were
pretreated with NEM (0.5 mM) for 30 min, subsequently incubated with NCR (5 μM)
for 30 min, and then imaged after washing three times by PBS buffer, as the control
group. In the second to fourth group, HeLa cells were pretreated with NEM (0.5 mM)
for 30 min, subsequently incubated with Cys /Hcy/GSH (500 μM) for 30 min, washed
and continued to be incubated with NCR (5 μM) for 30 min, and then added 500 μM
NaHS to the culture dish, after 30 min, imaged after washing three times by PBS buffer.
Cells were imaged by a laser scanning confocal microscopy (Leica TCS SP8; λex =
405 nm, λem = 440-490 nm for the blue channel; λex = 561 nm, λem = 575-620 nm for
the red channel).
3
Table S1. Summary of the optical properties of representative fluorescent probes for
distinguishing biothiols with multiple sets of fluorescence signals.
4
Multisite probe Distingui
shing
targets
for
detection
Number
of
emission
bands
Emission
wavelength/
nm
Emission
distance/
nm
Solubility Targeting
mitochon
dria
References
ON
S
O
CF3
CF3O
N
O
Cys/Hcy 2 474,694 220 Ethanol/
PBS
(40:60
v/v)
NO 4 .Anal. Chem.,
2014,86,1800-
1807
O
Cl
N OCN
CN GSH,
H2S
2 517,564 47 DMF/P
BS (3:7
v/v)
NO 5
.Chem.Commun.
(Camb.),2016,
52, 4628-4631.
O
S
O
N N
Cys/Hcy
, GSH
2 546,622 76 PBS NO 6 . Chem. Sci.,
2014, 5, 3183.
O
Cl
N
O
O
Cys/Hcy
, GSH
2 407/503,
546/503
33/43 PBS NO7. Anal. Chim.
Acta,2015, 900,
103-110.
O O
CN
NCN
ClCys,
GSH
2 500,560 60 CH3CN/
PBS
(3:7 v/v)
NO8. Biosens.
Bioelectron.,201
7, 90,117-124.
5
F
F
F
F
CN
CN Cys,
Hcy,
GSH
2 450, 500 50 PBS NO9 .Chem. Sci.,
2016, 7,256-260.
O
N
OCl
NS
Cys,
GSH
2 420,512 92 PBS
(1m M
CTAB)
NO10. J. Am. Chem.
Soc.,2014, 136,
574-577.
NO2
O2NS OONH
SN
NC
Cys,
GSH
2 522,490 32 PBS NO11. Anal. Chem.,
2015,87,3460-34
66.
O
N
OO
O Cys,
SO2
2 514,576 62 DMSO/
PBS
(1:1 v/v)
NO 12. J. Am. Chem.
Soc., 2017, 139,
3181-3185.
O
N
O
Cl
CN
CNGSH 1 505 - DMSO/
PBS
(1:1 v/v)
NO13. Sensors
Actuators B:
Chem., 2017,
253,42-49.
N
SO N O
N
NO2 H2S,
Cys/Hcy
, GSHp
2 465,550 85 DMSO/
PBS
(1:4 v/v)
NO14. Sensors
Actuators B:
Chem., 2016,
235,691-697.
ON O
N
S
S
O
Cys 2 494,644 150 DMF/P
BS (1:9
v/v)
YES 15. RSC Adv.,
2014,4,64542-
64550.
6
NB N
F F ClNNN
OHO
3
Cys/Hcy
, GSH
2 564,588 24 CH3CN/
HEPES
(5:95
v/v)
NO16. J. Am. Chem.
Soc.,2012,134,1
8928-18931.
ON
HN
H2N
N
Cys,
GSH
2 536,618 82 CH3CN/
PBS
(3:7 v/v)
YES17. J. Am. Chem.
Soc.,2014,136,1
2520-12523.
N
CO2nBu
CO2nBu
ONN
NO2 Cys/Hcy
, GSH
2 764,810 46 HEPES YES18. J. Am. Chem.
Soc.,2014, 136,
7018-7025.
O
OO
N
NON
NO2
Cys/Hcy
, GSH
2 545,621 76 CH3CN/
HEPES
(3:7 v/v)
NO19. Anal. Chem.,
2016,88,3638-36
46.
O
N
O
NON NO2
Cys/Hcy
, GSH
2 550,716 166 DMSO/
PBS
(5:95
v/v)
NO20.Biosens.Bioel
ectron.,2016,81,
341-348.
SN
OH
N
O
NO
N
NO2H2S,
Cys/Hcy
, GSH
3 485,546,6
09
61/63 CH3CN/
PBS
(2:8 v/v)
YES21 .Chem. Sci.,
2017,8,6257-626
5.
O
Cl
O
CHO
N
Cys,
Hcy
2 480,542 62 DMSO/
PBS
(2:8 v/v)
NO 22. Chem. Asian
J., 2017, 12,
2098-2103.
O
O
CN
N
CNCl Cys,
Hcy,
GSH
3 457,559,5
29
72/30 DMSO/
PBS
(6:4 v/v)
NO 23.Angew.Chem.
Int.Ed.,2018,57,4
991-4994.
7
OS
O
ON
O
O
OO
NN
NON
O2N
S
H2S,
Cys/
Hcy,
GSH
2 467/588 121 DMSO/
PBS
(1:99
v/v)
YES This work
COOH
HSLAH
HS
OH + N SS N
N SS
OH
PPh3,CBr4
N SS
Br
+O
N
OHO
K2CO3
DMF
O
N
OO
SS
N
+HS OH
O
O
N
OO
SSHO
O
NO
N
NO2
Cl
+HN
NBoc CsCO3
CH3CN
NO
N
NO2
N
NBoc
CF3COOHN
ON
NO2
N
NH
+O O
Cl
HO
TEA
CH3CN
NO
N
NO2
N
N
O
O
OH
HO OH
+
O
OO
ClZnCl2
Ethanol O O
Cl
HO
6
6
NC,DPTS,DIC
DCM
O
N
OO
SS
O
NON
NO2
NN
OO O
1
2 3
4 NCR
5
NC
DCM
DCM
Scheme S1. The synthetic route of probe NCR.
Synthesis of 2. Compound 1 was prepared via previous method24. Triphenylphosphine
(632 mg, 2.4 mmol) and carbon tetrabromide (800 mg, 2.4 mmol) were dissolved
absolutely in dichloromethane. Compound 1 (400 mg, 1.6 mmol) was slowly added.
TLC was applied to monitor the reaction. After complete reaction, the solvent was
evaporated under reduced pressure. The resulted residue was purified by silica gel
column chromatography with PE/EA (v/v 100:1) to give a yellow oil as compound 2
(200 mg, 40%).
Synthesis of 3. Resorufin (136.6 mg, 0.64 mmol) and potassium carbonate (132.7 mg,
0.96 mmol) were dissolved absolutely in dry DMF. Under the protection of nitrogen,
compound 2 (200 mg, 0.64 mmol) was slowly added. The mixture was stirred for
overnight at 55 oC. After complete reaction, 50 mL water was added into the flask and
the reaction mixture was extracted with EA (100 mL × 3). The organic phase was
8
collected and dried using MgSO4. Then the solvent was removed under reduced
pressure affording the crude product, which was purified by flash chromatography
column using CH2Cl2/MeOH (v/v = 250:1) to afford red solid as compound 3 (210 mg,
73%).1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.66 – 7.54 (m,
4H), 7.39 (t, J = 9.6 Hz, 3H), 7.11 (s, 1H), 6.98 (d, J = 8.9 Hz, 1H), 6.83 (d, J = 10.6
Hz, 2H), 6.31 (s, 1H), 5.13 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 186.3, 162.3, 159.3,
149.8, 149.7, 145.8, 145.6, 137.3, 136.8, 134.7, 134.6, 134.3, 131.6, 128.6, 128.3,
127.6, 121.1, 119.7, 114.2, 106.8, 101.1, 70.3. HRMS: m/z [M+Na]+ calculated for C24H16N2NaO3S2: 467.0495; measured 467.0497.
Synthesis of 4. Compound 3 (210 mg, 0.47 mmol) was dissolved absolutely in
dichloromethane. 3-Mercaptopropionic acid (60.4 mg, 0.57 mmol) was slowly added.
The reaction was monitored by TLC. After complete reaction, the solvent was
evaporated under reduced pressure. The resulted residue was purified by silica gel
column chromatography with CH2Cl2/MeOH (v/v 100:1) to afford dark red solid as
compound 4 (102 mg, 49%).1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 7.77 (t, J = 9.6 Hz, 1H), 7.54 (dt, J =
19.2, 9.9 Hz, 5H), 7.17 (dd, J = 33.5, 9.6 Hz, 2H), 6.79 (d, J = 9.9 Hz, 1H), 6.27 (s,
1H), 5.28 (s, 2H), 2.95 (t, J = 6.8 Hz, 2H), 2.62 (t, J = 6.8 Hz, 2H). 13C NMR (101
MHz, DMSO-d6) δ 185.8, 173.0, 162.7, 150.2, 145.8, 145.7, 136.9, 135.6, 135.4, 134.2,
131.8, 129.4, 128.5, 127.8, 114.8, 106.2, 101.7, 70.2, 34.0, 33.9. HRMS: m/z [M-H+]
calculated for C22H16NO5S2: 438.0475; measured 438.0473.
Synthesis of 6. Resorcinol (10 g, 90.8 mmol), Ethyl 4-Chloroacetoacetate (14.9 g, 90.8
mmol) was dissolved absolutely in ethanol. Zinc chloride(18.5 g, 136 mmol)was
added. The mixture was heated and reflux for 12 hours. After complete reaction, cooled
to room temperature. The cooled reaction mixture was poured into 100 mL of 0.1 N
aqueous hydrochloride acid. Then, the solvent was evaporated under reduced pressure.
The resulted residue was purified by silica gel column chromatography with
CH2Cl2/MeOH (v/v 100:1) to afford white solid as compound 6 (11 g, 86%). 1H NMR (400 MHz, DMSO-d6) δ 7.65 (d, J = 8.8 Hz, 1H), 6.83 (d, J = 6.6 Hz, 1H),
6.75 (s, 1H), 6.40 (s, 1H), 4.92 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.9, 160.6,
155.7, 151.3, 126.9, 113.5, 111.5, 109.8, 102.9, 41.8. HRMS: m/z [M+Na]+ calculated for C10H7ClNaO3: 232.9976; measured 232.9978.
9
Synthesis of NC. Compound 5 was prepared via previous method25. Compound 5 (237
mg, 0.95 mmol), Triethylamine (144.7 mg, 1.43 mmol) was dissolved absolutely
acetonitrile, compound 6 (281 mg, 0.95 mmol) was added. The mixture was heated and
reflux for 24 hours. After complete reaction, cooled to room temperature, the solvent
was evaporated under reduced pressure. The resulted residue was purified by silica gel
column chromatography with CH2Cl2/MeOH (v/v 200:1) to afford red solid as
compound NC (360 mg, 75%). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.49 (d, J = 9.1 Hz, 1H), 7.79 (d, J =
8.7 Hz, 1H), 6.97 – 6.47 (m, 3H), 6.32 (s, 1H), 4.17 (s, 4H), 3.74 (s, 2H), 2.76 (s, 4H),13C NMR (101 MHz, DMSO-d6) δ 161.6, 160.9, 155.7, 152.7, 145.8, 145.3, 145.3,
136.8, 127.3, 121.7, 113.3, 111.3, 110.7, 104.1, 102.7, 57.6, 52.9, 49.8. HRMS: m/z
[M+Na]+ calculated for C20H17N5NaO6: 446.1071; measured 446.1075.Synthesis of NCR. Compound 4 (50 mg, 0.11 mmol) was dissolved in anhydrous
dichloromethane (5 mL) in a round-bottom flask. NC (48.2 mg, 0.11 mmol) was added,
followed by the addition of 4-(dimethylamino)-pyridinium-4-toluene sulfonate(DPTS)
(33.5 mg, 0.11 mmol) and N, N-Diisopropylcarbodiimide (14.4 mg, 0.11 mmol). The
reaction mixture was stirred room temperature. The reaction was monitored by TLC,
after complete reaction, diluted with dichloromethane, and washed with brine. The
organic layer was dried over anhydrous Na2SO4, then the solvent was evaporated under
reduced pressure. The resulted residue was purified by silica gel column
chromatography with CH2Cl2/MeOH (v/v 150:1) to afford red solid as compound NCR
(60 mg, 62.5%). 1H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.58
(dd, J = 34.3, 8.4 Hz, 3H), 7.42 – 7.26 (m, 3H), 7.07 – 6.88 (m, 3H), 6.82 – 6.68 (m,
2H), 6.47 (s, 1H), 6.33 – 6.14 (m, 2H), 5.10 (s, 2H), 4.07 (t, J = 4.9 Hz, 4H), 3.67 (s,
2H), 3.06 – 2.91 (m, 4H), 2.76 (t, J = 5.0 Hz, 4H).
13C NMR (101 MHz, CDCl3) δ 186.2, 169.5, 162.4, 160.2, 154.5, 152.9, 150.4, 149.7,
145.7, 145.5, 145.0, 144.8, 144.7, 137.3, 135.0, 134.7, 134.6, 134.2, 131.6, 128.5,
128.3, 127.9, 125.6, 118.0, 116.5, 114.8, 114.2, 110.5, 106.7, 102.9, 101.1, 70.3, 58.6,
52.9, 49.3, 33.9, 32.9.
HRMS: m/z [M+Na] + calculated for C42H32N6NaO10S2: 867.1514; measured 867.1518.
10
References
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11
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12
9. Proposed responding mechanism
H 2S
O
SS
O
ON
O
O
O
ON
NN
ON
NO2
H 2S
H 2S
SH
O
ON
O
S
O
N
OHO
OHOO
NHN
O
S
O
O
ON
NH
SH
S SO
NO
N
O2N SH
Cys/HcyO
SS
O
ON
O
O
O
ON
NN
ON
NO2
Cys/H
cy
SH
O
ON
O
O
N
OHO
O
S
O
ON
NNON
O2N
S
NH2
COOHn
n=1 for Cysn=2 for Hcy
OH
OO
NN
NO
N
O2N
SS
NH
COOH
O
n
C n=1 for CysH n=2 for Hcy
GSH
O
SS
O
ON
O
O
O
ON
NN
ON
NO2
GSHSH
O
ON
O
O
N
OHO
O S
O
O
O
NN
NON
O2N
SNH
OCOOH
NH2
HN O
HOOC
R
NCR
N
R
R
NC
NC-GSH
S
D
O
SS
O
ON
O
O
O
ON
NN
ON
NO2
S
O
Scheme S2. Proposed responding mechanism of probe NCR for simultaneously
distinguishing H2S and biothiols.
13
OH
OO
NN
NO
N
O2N
NCCys/Hcy
GSH
O
S
O
O
ON
NNON
O2N
S
NH
O
COOHH2N
HN
O
COOH
NC-GSH
H2S
NO
N
O2N SH
N
+
D
H2S
NO
N
O2N SH
N O
S
O
O
ON
HN
S
NH
O
COOHH2N
HN
O
COOH
D-GSH
+
O
S S
O
ON
O
O
O
ON
N
NO
NNO2
NCR
OHOO
NNH
Scheme S3. Proposed reaction mechanism of NCR pre-treated with Cys, Hcy and GSH
upon continued addition of H2S.
10. Supplementary Spectra
Fig. S1. The fluorescence intensity of NCR (10 μM) at (A) 467 nm, (B) 588 nm, upon
addition of H2S (0–100 equiv.), Cys (0–100 equiv.), Hcy (0–100 equiv.) and GSH (0–
100 equiv.) and (C) 467 nm pretreated with Cys (100 equiv.), Hcy (100 equiv.), and
GSH (100 equiv.) upon continued addition of H2S (0-100 equiv.) in DMSO/PBS buffer
(25 mM, pH 7.4, 1:99, v/v). ex = 380 nm, em =467 nm for the first and third column.
ex=560 nm, em =588 nm for the second column. Data are expressed as mean ± SD of
five parallel experiments.
14
Fig. S2. The fluorescence spectra of NCR (10 μM) pre-treated with Cys (100 equiv.),
Hcy (100 equiv.), and GSH (100 equiv.) upon continued addition of H2S (0-100 equiv.)
in DMSO/PBS buffer (25 mM, pH 7.4, 1:99, v/v). ex = 380 nm, em =467nm. Upon
continuing addition of increasing amount of H2S to the mixture of NCR (10 μM) and
Cys (100 equiv.), a significant emission centered at 467 nm (50-fold) was generated. A
similar phenomenon also occurred in the mixture of NCR and Hcy (60-fold). However,
the mixture of NCR and GSH has no obvious emission band at 467 nm (4-fold).
Fig. S3. Linear correlation between the fluorescence emission intensity of NCR (10
μM) and (a) H2S (0 - 40 equiv.), (b) Cys (0 – 40 equiv.), (c) Hcy (0 – 50 equiv.) and (d)
GSH (0 - 60 equiv.) in DMSO/PBS buffer (25 mM, pH 7.4, 1:99, v/v).
15
400 450 500 550 600 650 7000
1000
2000
3000
4000
5000
6000
7000
8000Fl
.inte
nsity
(a.u
.)
Wavelength(nm)
400 450 500 550 600 650 7000
1000
2000
3000
4000
5000
6000
7000
8000
Fl.in
tens
ity(a
.u.)
Wavelength(nm)
400 450 500 550 600 650 7000
1000
2000
3000
4000
5000
6000
7000
8000
Fl.in
tens
ity(a
.u.)
Wavelength(nm)
(A1) (A2) (A3)
0 3 6 9 12 15 18 21 24 27 300
1000
2000
3000
4000
5000
6000
7000
8000 Excited at 380 nm Blank H2S Cys Hcy GSH
F 467(a
.u.)
Time(min)
0 3 6 9 12 15 18 21 24 27 300
1000
2000
3000
4000
5000
6000
7000
8000
9000 Excited at 560 nm Blank H2S Cys Hcy GSH
F 588(a
.u.)
Time(min)
0 3 6 9 12 15 18 21 24 27 300
1000
2000
3000
4000
5000
6000
7000 Excited at 380 nm Blank Cys+H2S Hcy+H2S GSH+H2S
F 467(a
.u.)
Time(min)
(a) (b) (c)
Fig. S4. Time-dependent fluorescence spectra of NCR (10 μM) in the presence of Cys
(100 equiv.), Hcy (100 equiv.), GSH (100 equiv.), H2S (100 equiv.) within 30 min in
DMSO/PBS buffer (25 mM, pH 7.4, 1:99, v/v). (a) Fluorescence intensity at 467 nm,
excitation at 380 nm; (b) Fluorescence intensity at 588 nm, excitation at 560 nm; (c)
The fluorescence spectra of NCR (10 μM) pre-treated with 100 equiv. Cys /Hcy and
GSH upon continued addition of H2S (100 equiv.) within 30 min in DMSO/PBS buffer
(25 mM, pH 7.4, 1:99, v/v). Fluorescence intensity at 467 nm, excitation at 380 nm.
Fig. S5. Absorption spectra of (a) NCR (10 μM) in the presence of 100 equiv. of H2S
.(b) NCR (10 μM ) in the presence of 100 equiv. of Cys/Hcy and GSH. (c) NCR (10
μM) was pretreated with 100 equiv. Cys/Hcy/GSH in the presence of 100 equiv. of H2S
(d) NC (20 μM) in the presence of 100 equiv. of H2S in DMSO/PBS buffer (25 mM,
pH 7.4, 1:99, v/v). The UV-vis spectra of free NCR showed a main absorption at 475
16
300 400 500 600 7000.0
0.1
0.2
0.3
0.4
0.5
Abs
orpt
ion
Wavelength (nm)
Blank H2S
300 400 500 600 7000.0
0.1
0.2
0.3
0.4
0.5
Abs
orpt
ion
Wavelength (nm)
Blank Cys Hcy GSH
300 400 500 600 7000.0
0.5
1.0
1.5
2.0
2.5
Abs
orpt
ion
Wavelength (nm)
NC NC+H2S
300 400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
Abs
orpt
ion
Wavelength (nm)
Blank Cys+H2S Hcy+H2S GSH+H2S
(a) (b)
(c) (d)
nm. Upon addition of H2S, an obvious increase was observed at 372 nm and a shoulder
peak simultaneously emerged at 575 nm. These can be attributed to the products R, D
and N. Similarly, only an obvious shoulder peak was observed at 575 nm in the presence
of Cys/Hcy and GSH. After NCR was pre-treated with Cys, Hcy, or GSH (100 equiv.)
individually, addition of H2S resulted in a significant increase in the UV absorption at
372 nm and the simultaneous emergence of a peak at 575 nm in the Cys- and Hcy-
pretreated solutions. However, H2S addition only resulted in a shoulder peak at 575 nm
for the GSH-pretreated solution. To confirm the original attribution of those newly
generated absorption peaks, the absorption spectra of control compounds NC and NC
in the presence of H2S were studied, and the results were consistent with the spectral
performance of NCR.
Extinction coefficient calculations:
0.000002 0.000006 0.000010 0.0000140.0
0.1
0.2
0.3
0.4
Abs
orpt
ion
(OD
)
C (M)
y = 29083 xR2 = 0.9947
Fig. S6. Linear plots of NCR. The experiments were applied in 1 cm cuvette, at the
indicated concentrations in DMSO/PBS buffer (25 mM, pH 7.4, 1:99 v/v). The
measurements were performed at the maximum wavelength of NCR (475 nm).
NCR = 29083[M-1cm-1]
17
Fig S7. 1H-NMR spectra comparison: (a) NCR, (b) NCR-NaHS (c) NCR-Cys, (d)
NCR-Hcy and (e) NCR-GSH. The chemical shifts for hydrogens, Ha linked with
resorufin; and Hc linked with coumarin; and Hb linked with and NBD were observed
during the reaction. In the NCR-NaHS group, it was found that the chemical shifts of
Ha and Hb displayed obvious upfield shifts compared with that of NCR group, which
indicated the release of resorufin and NBD. In NCR-Cys, NCR-Hcy and NCR-GSH
group, only upfield shift of Ha was observed, but the chemical shift of Hb didn’t change,
which indicated that the release of resorufin, and NBD was not removed (Scheme S2
in supporting information).
18
O
SS
O
O
N
O
OOO
NN
NO
N
O2N
Ha
Hb Hc
Hb Hc
NCR
Fig S8. 1H-NMR spectra comparison of (a) NC and (b) NC-NaHS showed that the
chemical shift of Hb in NC-NaHS displayed obvious upfield shifts compared with that
of NC, which indicated that the NBD was removed (Scheme S3 in supporting
information). These results are in good agreement with our proposed reaction
mechanism.
250 500 750 1000 1250 1500 m/z
0
50
100Inten.
212.00
265.05110.95 425.00 524.15 1308.45 1704.80915.40 1439.30745.40 1062.90
220 230 240 250 260 270 280 290 300 310 320 m/z
0
50
100Inten.
261.20
288.20 302.20255.25 263.20 326.35240.15 279.15270.25 318.25228.25 286.20220.25 250.20
180.0 185.0 190.0 195.0 200.0 205.0 210.0 m/z
0
50
100Inten.
195.90
197.90 212.00
Fig. S9. Mass spectra (ESI) of NCR in the presence of H2S in aqueous solution. R
m/z=(M-H+)=212.00, D m/z (M + H+) = 261.20, N m/z (M-H+) =195.90.
19
OH
O
NN
NO
N
O2N
O
Hb Hc
Hb Hc
NC
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z
0
50
100Inten.
212.00
181.15 241.95159.95148.25110.95 283.25130.95 225.00 259.15 269.40199.05
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z
0
50
100Inten.
422.05
327.25790.55 922.95359.10259.00 845.20677.40 1035.75557.00 1347.70 1417.951251.251101.05
Fig. S10. Mass spectra of NCR in the presence of Cys in aqueous solution. R m/z=
(M-H+)=212.00, NC m/z (M-H+) = 422.05, C m/z (M-H+) =206.0024.
140 150 160 170 180 190 200 210 220 230 240 250 260 m/z
0
50
100Inten.
212.00
222.00
253.00188.05 242.00182.00 263.00169.95 176.05 195.95 218.95162.00154.05 204.00 233.05148.00
300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z
0
50
100Inten.
422.05
485.05359.05 375.05309.25 327.20297.10 395.10 450.15435.15 473.00409.65339.45 384.35
20
Fig. S11. Mass spectra of NCR in the presence of Hcy in aqueous solution. R m/z=
(M-H+)=212.00, NC m/z (M-H+) = 422.05, H m/z (M-H+) =220.0182.
600 700 800 900 1000 1100 1200 1300 m/z
0
50
100Inten.
815.10
903.60 966.60826.40 1017.50 1079.60555.00 713.30 1128.80621.25 1193.20 1262.20 1356.151311.00
200 300 400 500 600 700 800 900 1000 m/z
0
50
100Inten.
212.00
414.00259.05 343.90 494.10 541.25188.70 1036.55599.45 648.95 828.10 938.30893.65744.55
Fig. S12. Mass spectra of NCR in the presence of GSH aqueous solution. R m/z=(M-
H+)=212.00, NC-GSH m/z (M-H+) = 815.10.
100 200 300 400 500 600 700 800 m/z
0
50
100Inten.
261.20
283.15 543.25 647.40413.30167.2587.30 701.35591.40330.20 855.00473.25 786.25
175.0 200.0 225.0 250.0 275.0 300.0 325.0 m/z
0
50
100Inten.
195.95
237.90160.00 179.95 227.85208.20 260.00 276.90 315.45 327.50307.10293.75
Fig. S13. Mass spectra of NCR pre-treated with Cys upon continued addition of H2S.
D m/z (M+H+) = 261.20, N m/z (M-H+) =195.95.
21
200.0 225.0 250.0 275.0 300.0 325.0 350.0 m/z
0
50
100Inten.
261.20
283.15 315.20301.20199.25 273.20 330.20218.20 233.15 243.15 353.25225.25
125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z
0
50
100Inten.
195.90
251.90110.00 149.95 230.95219.90179.95 283.90262.25 301.75135.75 208.30
Fig. S14. Mass spectra of NCR pre-treated with Hcy upon continued addition of H2S.
D m/z (M + H+) = 261.20, N m/z (M-H+) =195.90.
652.5 653.0 653.5 654.0 654.5 655.0 655.5 656.0 m/z
0
50
100Inten.
654.25
655.15
250 500 750 1000 1250 1500 1750 m/z
0
50
100Inten.
195.90
414.90109.95 1030.45536.10 808.35303.65 1401.251277.70614.05 1811.301693.60
Fig. S15. Mass spectra of NCR pre-treated with GSH upon continued addition of H2S.
D-GSH m/z (M + H+) = 654.25, N m/z (M-H+) =195.90.
22
Fig. S16. (A) The fluorescence intensity of NCR (10 μM) at (A1) 467 nm, (A2) 588
nm in the presence of (1) Blank (2) Thr, (3) Phe, (4) Asp, (5) Val, (6) His, (7) Glu, (8)
Pro, (9) Met, (10) Ala, (11) Trp, (12) Ser, (13) NO3-, (14) Br-, (15) ClO-, (16) SO4
2-,
(17) SO32- , (18) S2O3
2- , (19) AcO- , (20) N3-, (21) Ca2+, (22) Cu2+, (23) Mg2+, (24) Fe2+,
(25) Fe3+, (26) Sn2+, (27) Zn2+, (28) Na+, (29) K+, (30) H2O2, (31) Cys, (32) Hcy, (33)
GSH, (34) H2S in DMSO/PBS buffer (25 mM, pH 7.4, 1:99, v/v). Concentrations were
100 equiv. for (2)– (34). (A3) NCR was pre-treated with 100 equiv. (31) Cys (32) Hcy
and (33) GSH, upon continued addition of 100 equiv. H2S. (B). Fluorescence intensity
of NCR (10 μM) at (B1) 467 nm, (B2) 588 nm in the presence or absence of biothiols
at different pH values ranging 4.0–10.0 in DMSO/PBS buffer (25 mM, pH 7.4, 1:99,
v/v). Concentrations were 100 equiv. for Cys/Hcy, GSH, H2S. (B3) NCR was pre-
treated with 100 equiv. Cys/Hcy, GSH, upon continued addition of 100 equiv. H2S.
Excitation at 380 nm for (A1/B1), 560 nm for (A2/B2), and 380 nm for (A3/B3). The
results showed the fluorescence intensity of free NCR at two emission bands was not
influenced by pH in aqueous solutions. However, at pH ranging from 7.0 to 8.0, probe
NCR generated significant enhancement of the fluorescence intensity in the presence
of H2S, Cys/Hcy and GSH. Data are expressed as mean ± SD of five parallel
experiments.
Fig. S17. (A) Optimized structures of NCR and the co-regulation of response emission
by FRET and ICT mechanisms. (B) Frontier molecular orbital energy of NCR at the
excited state. Calculations were performed using DFT with the B3LYP exchange
functional employing 6-31G(d) basis sets using Gaussian 09 programs. In the optimized
23
(A) (B)
LUMO -3.05 eV
HOMO -6.01eV
structures of NCR, the calculated distance between coumarin and NBD is 13 Å. Thus,
the FRET donor fluorescence of coumarin can be quenched by the NBD moiety through
the FRET process. In addition, the electrons on both the HOMO and LUMO
efficiently distribute in the respective resorufin and NBD units in NCR, suggesting that
NCR is a typical intramolecular charge transfer (ICT)-based fluorescent probe.
Fig. S18. The cell viability of living HeLa cells treated with 5, 10, or 20 μM NCR for
24 hours measured by standard MTT assay. (Error bars represent the standard
deviations of five trials)
Fig. S19. The images of living HeLa cells pre-treated with NCR (5 μM) for 30 min and
subsequently co-incubated with Mito-Tracker Green (1 μM) for 30 min. (A) Red
channel images of probe NCR (ex= 561 nm, em= 575 - 620 nm); (B) Green channel
images of Mito -Tracker Green (ex = 488 nm, em= 500 - 535 nm); (C) Merged green
and red channel images; (D) The intensity profile of the ROI in merged images. Scale
bar: 10 μm.
24
Fig. S20. 1H NMR of compound 3 in CDCl3.
0102030405060708090100110120130140150160170180190200
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
70.3
101.1
106.8
114.1
119.7
121.1
127.6
128.3
128.6
131.7
134.3
134.6
134.7
136.8
137.3
145.6
145.8
149.7
149.8
159.3
162.4
186.3
Fig. S21. 13C NMR of compound 3 in CDCl3.
25
Fig. S22. HRMS (ESI) of compound 3 m/z [M+Na]+ calculated for C24H16N2NaO3S2 :
467.0495; measured 467.0497.
Fig. S23. 1H NMR of compound 4 in DMSO-d6.
26
0102030405060708090100110120130140150160170180190200
f1 (ppm)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
33.9
34.0
70.2
101.7
106.1
114.8
127.8
128.5
129.4
131.8
134.2
135.4
135.5
136.9
145.7
145.8
150.2
162.7
173.0
185.8
Fig. S24. 13C NMR of compound 4 in DMSO-d6.
Fig. S25. HRMS (ESI) of compound 4 m/z [M-H+] calculated for C22H16NO5S2:
438.0475; measured 438.0473.
27
Fig. S26. 1H NMR of compound 6 in DMSO-d6.
0102030405060708090100110120130140150160170180190200
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
41.8
102.9
109.8
111.5
113.5
126.9
151.3
155.7
160.6
161.7
Fig. S27. 13C NMR of compound 6 in DMSO-d6.
28
Fig. S28. HRMS (ESI) of compound 6 m/z [M+Na]+ calculated for C10H7ClNaO3:
232.9976; measured: 232.9978.
Fig. S29. 1H NMR of compound NC in DMSO-d6.
29
0102030405060708090100110120130140150160170180190200
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
49.8
52.9
57.7
102.7
104.1
110.7
111.3
113.3
121.7
127.2
136.8
145.3
145.3
145.7
152.7
155.7
160.9
161.6
Fig. S30. 13C NMR of compound NC in DMSO-d6.
Fig. S31. HRMS (ESI) of compound NC m/z [M+Na]+ calculated for C20H17N5NaO6:
446.1071; measured: 446.1075.
30
2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
4.13
4.06
2.04
4.02
2.00
2.00
1.01
2.03
3.02
3.03
3.00
1.00
1.00
2.75
2.76
2.77
2.93
2.94
2.95
2.96
2.96
2.97
2.99
3.01
3.02
3.03
3.04
3.04
3.67
4.06
4.07
4.08
5.10
6.20
6.20
6.23
6.25
6.47
6.72
6.72
6.74
6.75
6.78
6.78
6.91
6.92
6.93
6.94
6.98
6.98
7.00
7.00
7.01
7.01
7.31
7.35
7.37
7.52
7.54
7.61
7.63
7.75
7.77
8.30
8.33
Fig. S32. 1H NMR of compound NCR in CDCl3.
0102030405060708090100110120130140150160170180190200
f1 (ppm)
-400
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
32.9
33.9
49.3
52.9
58.6
70.3
101.1
102.9
106.7
110.5
114.2
114.8
116.5
118.0
124.0
125.6
127.9
128.3
128.5
131.6
134.2
134.6
134.7
135.0
137.3
144.7
144.8
145.0
145.5
145.7
149.7
150.4
152.9
154.5
160.2
162.4
169.5
186.2
Fig. S33. 1C NMR of compound NCR in CDCl3.
31
Fig. S34. HRMS (ESI) of compound NCR m/z [M+Na]+ calculated for
C42H32N6NaO10S2: 867.1514; measured 867.1518.
32