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: shanglq@nankai.edu.cn

b. Department of Chemistry Georgia State University Atlanta, Georgia 30303 USA E-mail: wang@gsu.edu

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

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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