S1

Electronic Supplementary Information

Development of a red fluorescent light-up probe for highly selective and sensitive detection of vicinal dithiol-containing

proteins in living cells

Yuanyuan Wang, Xiao-Feng Yang,* Yaogang Zhong, Xueyun Gong, Zheng Li, Hua Li*

a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of

Ministry of Education, College of Chemistry & Materials Science, Northwest

University, Xi’an 710069, P.R. China. xfyang@nwu.edu.cn (X. F. Yang). b College of Life Sciences, Northwest University, Xi’an 710069, P.R. China. c College of Chemistry and Chemical Engineering, Xi'an Shiyou University, Xi'an

710065, P.R. China.

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2015

S2

CONTENTS: Materials and instruments

General procedure for the spectral measurements

SDS-PAGE and fluorescence imaging of gels

Cell Cultures and fluorescence imaging

MTT assay

Colocalization of FAsH and rhodamine 123

Determination of the fluorescence quantum yield of FAsH and FAsH-rBSA complex

Synthesis of FAsH and F4

References

Figure S1. Absorption spectra of F1 in water/1, 4-dioxane solvent mixtures.

Figure S2. Fluorescence spectra of F1 in the absence and presence of BSA.

Figure S3. Absorption and fluorescence spectra of F1, F4 and FAsH.

Figure S4. Vicinal cysteine pairs in rBSA.

Figure S5. Fluorescence time profile of FAsH upon binding to and dissociating from rBSA.

Figure S6. The linear relationship between the fluorescence intensity and rBSA concentration.

Figure S7. Fluorescence response of FAsH to different proteins and their reduced forms.

Figure S8. Fluorescence response of FAsH to rBSA in the presence of different competing

analytes.

Figure S9. Absorption spectra of FAsH in the presence of different concentrations of rBSA.

Figure S10. DLS analysis of the particle size distribution of FAsH in the presence of rBSA.

Figure S11. Fluorescence spectra of FAsH with the addition of rBSA and subsequently treating

with GndHCl.

Figure S12. Time course of the fluorescence intensity of FAsH in the presence of FBS, DTT or

the mixture of FBS and DTT.

Figure S13. Cell viability estimated by MTT assay.

Figure S14. Confocal fluorescence images of intracellular VDPs in SMMC-7721 cells by FAsH.

Figure S15. Semiquantitative determination of endogenous VDPs in SMMC-7721 cells.

Figure S16 – S28. NMR and MS data for compound PAO-EDT, F1, F2, F3, FAsH, compound 2

and F4.

S3

Materials and instruments

Unless stated otherwise, all the chemicals were obtained from commercial suppliers

and used without further purification. Rhodamine 123 and DAPI were obtained from

Sigma-Aldrich. Solvents were dried by standard procedures before use. Other

chemical reagents were of analytical grade and commercially available. Double

distilled water was used throughout the experiments. FAsH and F4 were dissolved in

acetone as stock solution. rBSA (0.15 mM) was prepared and stored according to the

literature [S1]. The fluorescence spectra and relative fluorescence intensity were measured with a

Perkin Elmer LS-55 fluorescence spectrometer equipped with a xenon lamp excitation

source and a Hamamatsu (Japan) 928 PMT. Absorption spectra were recorded using a

Shimadzu UV-2550 spectrophotometer. High-resolution mass spectra were collected

using a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp., USA) in

electrospray ionization (ESI) mode. 1H NMR and 13C NMR spectra were recorded at

400 and 100 MHz on an INOVA-400 spectrometer (Varian Unity) respectively, using

tetramethylsilane (TMS) as the internal standard. Fluorescence imaging was

performed by an Olympus FV1000 confocal laser scanning microscope (Japan).

Dynamic light scattering (DLS) measurements were performed using

Particle Size & Zeta Potential Analyzer （ZetaPlus, Brookhaven）. The scattering angle

was 90o, and the laser wavelength was 660 nm. The pH measurements were carried

out on a Sartorius PB-10 pH meter. All the measurements were operated at room

temperature (25 °C).

General procedure for the spectral measurements

In a set of test tubes containing 0.5 mL phosphate buffer (0.2 M, pH 7.4) and 50

μL of FAsH (0.1 mM), different concentrations of analytes were added and the

reaction mixture was diluted to 5.0 mL with H2O. The resulting solution was

well-mixed and kept at room temperature for 5 min, and then the absorption or

fluorescence spectra were recorded.

SDS-PAGE and fluorescence imaging of gels

S4

The selectivity of FAsH was proved by SDS-PAGE. Samples were labeled in PBS

buffer with a final concentration of protein at 3.75 μM, FAsH (or F4) at 50 μM for 30

min. After labeling, the samples were mixed with 5 × loading buffer, and run on a

10% polyacrylamide resolving gel and a 3% stacking gel. Molecular mass standards

(Thermo Scientific, Waltham, USA) were run with all gels. For fluorescence assay,

the gel was scanned on the red fluorescence channel (635 nm excitation/650 nm LP

emission) using a phosphorimager (Storm 840, Molecular DynamicsInc. USA). After

that, the same gel was stained with alkaline silver for comparison.

Cell Cultures and fluorescence imaging

SMMC-7721 cells were seeded in 6-well culture plates containing sterile coverslips

and were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine

serum (FBS), penicillin (100 U mL-1) and streptomycin (100 μg mL-1) at 37 oC in a

humidity atmosphere under 5% CO2 for 24 h. The medium was removed and the

adherent cells were washed with PBS buffer (pH 7.4) three times. After the cells were

incubated with 5.0 μM FAsH (or 5.0 μM F4) in serum-free RPMI medium at 37 °C

for 20 min, the medium was removed. The cells were further incubated with nucleus

staining dye DAPI (1.0 μg mL-1) in the same medium for 10 min. After that, the

staining solution was replaced with fresh PBS to remove the remaining free dye. Cell

imaging was then performed by an Olympus FV1000 confocal laser scanning

microscope immediately. Excitation wavelength for FAsH: 635 nm; Emission

collection: 655-755 nm; Excitation wavelength for DAPI: 405 nm; Emission

collection: 425-475 nm.

MTT assay

The cytotoxicity of FAsH was performed by a standard MTT assay. SMMC-7721

cells were seeded in 96-well flat bottom microtiter plates for 24 h before detection

with about 80% intensity. The cells were then incubated with various concentrations

of FAsH (0 – 20 μM) (n = 4) in FBS-free RPMI 1640 medium in a humidified

environment containing 5% CO2 at 37 oC for 12 h. 20 μL of MTT solution (5.0 mg

S5

mL-1) was then added to each well. After 4 h, 100 μL of the supernatant was removed,

and 150 μL of DMSO was added to each well to dissolve the formazan crystals. After

that, the plate was shaken for 10 min and then the absorbance was measured at 490

nm with a microplate reader (WD-2102A Microplate Reader, Beijing Liuyi

Instruments Inc.).

Colocalization of FAsH and rhodamine 123

For colocalization experiments, SMMC-7721 cells were incubated with 5.0 μM

FAsH, 2.5 μg mL-1 rhodamine 123 in FBS-free RPMI 1640 medium for 20 min. After

the above medium was removed, the cells were further incubated with nucleus

staining dye DAPI (1.0 μg mL-1) in RPMI 1640 medium for 10 min. Coverslips were

then transferred to fresh PBS buffer for imaging. Excitation wavelength for FAsH:

635 nm; Emission collection: 655-755 nm; Excitation wavelength for rhodamine 123:

488 nm; Emission collection: 500-600 nm. ImageJ software was used for analysis of

the images.

Determination of the fluorescence quantum yield of FAsH and FAsH-rBSA

complex

Fluorescence quantum yields of FAsH and FAsH-rBSA complex were determined

by using F1 (φF = 0.76 in CH2Cl2) as fluorescence standard [S2]. The fluorescence

quantum yields of F1 and FAsH-rBSA were determined in phosphate buffer (pH 7.4,

20 mM, containing 1% acetone as cosolvent). Spectral data of FAsH-rBSA complex

was measured 10 min after the mixture of 2.4 μM of rBSA with FAsH in phosphate

buffer. The quantum yield was calculated using the following equation:

φF(X) =φF(S)(ASFX/ AXFS) (nX/nS)2

Where φF is the fluorescence quantum yield, A is the absorbance at the excitation

wavelength, F is the area under the corrected emission curve, and n is the refractive

index of the solvent used. Subscript “S” and “X” refer to the standard and the

unknown compound, respectively.

Synthesis of FAsH and F4

S6

N OH

O COOH N

O

ON

COOH

N1

ON

N

O

N

F2

ON

N

O

N

HN

NHO

O

AsS

S

+

F1

ΗΝ O

O

ON

N

O

N

NH2

F3

NH2

AsSS

AsS

SNH

COOH

O

NH2

AsOHHO O

NH2

AsOHHO

PAO PAO-EDT PAO-EDT-C

FAsH

NH

COOH

OO

O

O ON

N

O

N

HN

NHO

O

F4

+

NH2

h) i)

2

a) b) c)

d) e)

f) g)

Scheme S1. Synthesis of FAsH and F4. Reagents and conditions: (a) phenylhydrazine,

methanol, reflux, 60 min; (b) ethanedithiol, ethanol, reflux, 10 min, 79%; (c) succinic

anhydride, toluene, reflux, 3 h, 97%; (d) concentrated H2SO4, 90 °C, 1.5 h, 67%; (e)

4-(N-Boc-amino)piperidine, EDC, HOBt, CH2Cl2, RT, 6 h, 51%. (f) CF3COOH,

CH2Cl2, RT, overnight, 73%; (g) PAO-EDT-C, EDC, HOBt, CH2Cl2, RT, 4 h, 54%;

(h) succinic anhydride, toluene, reflux, 3 h, 96%; (i) F3, EDC, HOBt, CH2Cl2, RT, 6 h,

51%. Counterions are omitted for clarity. EDC = 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride, HOBt = 1-hydroxybenzotriazole.

S7

Sythesis of 2- p-aminophenyl-1, 3, 2-Dithiarsenolane (PAO-EDT) [S3]

4-Aminophenylarsenoxide (PAO) was prepared according to the literature

procedure [S3]. PAO (0.96 g, 4.78 mmol) was dissolved in anhydrous ethanol (10 mL)

and heated to reflux. Then, ethanedithiol (0.48 mL, 6.30 mmol) was added dropwise

to the above solution, and the mixture was refluxed with stirring for 10 min. The

reaction mixture was then cooled with ice-cold water, and the resulting precipitate

was filtered off to give the crude product, which was further recrystallized from

ethanol to afford PAO-EDT as a white solid (0.98 g, 79% yield). 1H NMR (400 MHz,

DMSO-d6), δ (ppm): 7.26 (d, J = 7.6 Hz, 2H), 6.55 (d, J = 7.2 Hz, 2H), 5.42 (s, 2H),

3.32-3.27 (m, 2H), 3.26-3.19 (m, 2H). HRMS (ESI): m/z calcd for C9H10AsNS2 [M +

H]+ 259.9549; found: 259.9547.

Sythesis of PAO-EDT-C

PAO-EDT-C was prepared according to the literature procedure [S4]. To a solution

of PAO-EDT (0.10 g, 0.39 mmol) in toluene (8 mL) was added succinic anhydride

(43 mg, 0.42 mmol). The above suspension was reflux for 3 h. After cooled to room

temperature, the resulting precipitate was filtered off and dried under vacuum to give

PAO-EDT as a white solid (0.14 g, 97% yield), which was then used for next step

reaction without further purification.

Sythesis of 2-(4-dimethylaminophenyl)-4-(2-carboxyphenyl)-7-diethylamino-1-

benzopyrylium (F1) [S2]

Compound F1 was prepared according to the literature procedure [S3]. A mixture

of 1-(4-(dimethylamino) phenyl)ethanone (82 mg, 0.5 mmol) and

2-(4-diethylamino-2-hydroxybenzoyl) benzoic acid (1) [S5] (0.16 g, 0.5 mmol) in

concentrated H2SO4 (10 mL) was stirred at 90 °C for 1.5 h under argon atmosphere.

After cooling to room temperature, the mixture was then poured in ice-cold water

solution (60 mL), and then perchloric acid (70%, 0.5 mL) was added. The resulting

precipitate was filtered, washed with cold water, and then dried under vacuum to give

F1 as a purple-red solid (0.18 g, 67% yield), which can be used in the next step

S8

reaction without further purification. HRMS (ESI): m/z calcd for C28H29N2O3+[M ]+

441.2178; found: 441.2211.

Synthesis of F2 To a mixture of F1 (90 mg, 0.17 mmol), 4-(N-Boc-amino)-piperidine (50 mg, 0.25

mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 58

mg, 0.3 mmol), and HOBt (48 mg, 0.3mmol) was added dry CH2Cl2 (5 mL). The

resulting solution was allowed to stir at room temperature (RT) for 6 h. The reaction

solution was then washed with water, dried over anhydrous Na2SO4, filtered, and

evaporated under reduced pressure. The crude reside was purified by silica gel flash

column chromatography (CH2Cl2/MeOH = 20:1, v/v) to give compound F2 (63 mg,

51% yield). 1HNMR (400 MHz, DMSO-d6), δ (ppm): 8.24 (d, J = 9.2 Hz, 2H),

7.71-7.69 (m, 3H), 7.60 (br, 1H), 7.56 (d, J = 4 Hz, 1H ), 7.33-7.30 (m, 2H ), 7.21 (d,

J = 9.2 Hz, 1H ), 6.95 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 6.4 Hz, 1H ), 4.05-3.97 (m, 1H),

3.40 (2H), 3.64 (q, J = 7.2 Hz, 4H), 3.19 (s, 6H), 1.65 (br, 2H), 1.35(s, 9H), 1.22 (t, J

= 6.8 Hz, 6H), 1.09 (m, 2H). HRMS (ESI): m/z calcd for C38H47N4O4+ [M]+ 623.3597;

found: 623.3605.

Synthesis of compound F3

Compound F2 (0.16 g, 0.22 mmol) was dissolved in CH2Cl2 (8 mL), and then

CF3COOH (8 mL) was added. The mixture solution was stirred at room temperature

overnight. The solvent was then evaporated under reduced pressure, and the resulting

reside was purified by silica gel flash column chromatography (CHCl3/MeOH = 10:1,

v/v) to give F3 in 73% yield (0.10 g). 1 HNMR (400 MHz, DMSO-d6), δ (ppm): 8.24

(d, J = 8.8 Hz, 2H), 8.02 (br, 2H, -NH2), 7.75-7.71 (m, 3H), 7.60-7.53 (m, 2H ),

7.35-7.31 (m, 2H ), 7.21 (d, J = 8.8 Hz, 1H ), 6.95 (d, J = 9.2 Hz, 2H), 4.19 (br, 1H)

3.63 (q, J = 6.0 Hz, 4H), 3.19 (s, 6H), 1.85 (br, 2H), 1.38 (m, 2H), 1.22(t, J = 7.2 Hz,

6H). HRMS (ESI): m/z calcd for C33H39N4O2+ [M]+ 523.3073; found: 523.3063.

(Some peaks of piperidine ring are overlapped with DMSO and H2O in solvent, so it

can not be precisely integrated.)

S9

Synthesis of FAsH

Compound PAO-EDT-C (54mg, 0.15 mmol), F3 (61mg, 0.10mmol), EDC (50 mg,

0.26 mmol) and HOBt (42 mg, 0.26 mmol) were dissolved in dry CH2Cl2 (10 mL),

and the reaction mixture was allowed to stir at room temperature for 4 h. The reaction

solution was then washed with water, dried over anhydrous Na2SO4, filtered, and

evaporated under reduced pressure. The resulting residue was purified by silica gel

flash chromatography (CH2Cl2/MeOH = 20: 1, v/v) to afford FAsH as a dark blue

solid (52 mg, 54% yield). 1HNMR (400 MHz, DMSO-d6), δ (ppm):10.03 (s, 1H), 8.25

(d, J = 8.4 Hz, 2H), 7.84 (d, J = 7.2 Hz, 1H), 7.73-7.70 (m, 3H), 7.60-7.53 (m, 5H),

7.34-7.31 (m, 2H), 7.24-7.19 (m, 1H), 7.06 (d, J = 9.6 Hz, 1H), 6.96 (d, J = 8.4 Hz,

2H), 4.04-3.92 (m, 1H), 3.64 (q, J = 7.2, 4H), 3.38-3.35 (m, 2H), 3.19 (s, 6H),

3.17-3.11 (m, 2H), 2.35 (t, J = 7.0 Hz, 2H), 1.68 (br, 2H), 1.22(t, J = 7.2 Hz, 6H), 1.12

(2H). 13C NMR (100 MHz, DMSO-d6), δ(ppm): 170.69, 170.58, 166.74, 156.11,

154.93, 154.14, 140.24, 136.87, 131.33, 130.23, 129.57, 129.17, 127.20, 119.64,

118.62, 116.69, 114.74, 112.68, 109.03, 96.53, 46.59, 45.05, 41.36, 31.60, 30.85,

30.16, 12.48. HRMS (ESI): m/z calcd for C45H51AsN5O4S2+ [M]+ 864.2598; found:

864.2617.

Synthesis of compound 2

Compound 2 was prepared by the reaction of phenylamine with succinic anhydride

using the same procedure described for the synthesis of PAO-EDT-C. Yield, 96%. 1H

NMR (400 MHz, DMSO-d6), δ (ppm): 12.13 (s, 1H), 9.94 (s, 1H), 7.57 (d, J = 7.6 Hz,

2H), 7.27 (t, J = 7.8 Hz, 2H), 7.00 (t, J = 7.2 Hz, 1H), 2.55-2.51 (m, 4H).

Synthesis of compound F4

Compound F4 was prepared via the reaction of F3 with F2 using the procedure

described for the synthesis of FAsH. Yield: 51% 1HNMR (400 MHz, DMSO-d6), δ

(ppm):9.92 (s, 1H), 8.25 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 7.2 Hz, 1H), 7.73-7.68 (m,

3H ), 7.61-7.55 (m, 4H ), 7.34-7.22 (m, 5H), 7.07-6.95 (m, 3H ), 4.05-3.94 (m, 1H),

3.64 (q, J = 6.8 Hz, 4H), 3.19 (s, 6H), 2.37 (t, J = 6.8 Hz, 2H), 1.68 (br, 2H), 1.23 (t, J

S10

= 6.8 Hz, 6H). HRMS (ESI): m/z calcd for C43H48N5O4+ [M]+ 698.3706; found:

698.3727.

References

[S1] X. Pan, Z. Liang, J. Li, S. Wang, F. Kong, K. Xu and B. Tang, Chem. Eur. J.,

2015, 21, 2117–2122.

[S2] P. Czerney, G. Graneβ, E. Birckner, F. Vollmer and W. Rettig, J. Photochem.

Photobiol., A, 1995, 89, 31-36.

[S3] N. Wimmer, J. A. Robinson, N. Gopisetty-Venkatta, S. J. Roberts-Thomson, G. R.

Monteith and I. Toth, Med. Chem., 2006, 2, 79-87.

[S4] C. Huang, Q. Yin, J. Meng, W. Zhu, Y. Yang, X. Qian and Y. Xu, Chem. Eur. J.,

2013, 19, 7739–7747.

[S5] E. Nakata, Y. Yukimachi, H. Kariyazono, S. Im, C. Abe, Y. Uto, H. Maezawa, T.

Hashimoto, Y. Okamoto and H. Hori, Bioorg. Med. Chem., 2009, 17, 6952-6958.

S11

500 550 600 650 700 750 8000.00

0.05

0.10

0.15

0.20

0.25

0.30

Abs

orba

nce

Wavelength (nm)

fd (vol%)

0 10 20 30 40 50 60 70 80

Δf

high

low

Figure S1. Absorption spectra of F1 (10 μM) in water/1, 4-dioxane solvent mixtures

with different fractions of 1, 4-dioxane (fd).

600 625 650 675 7000

120

240

360

480

600

720

Fluo

resc

ence

inte

nsity

(a.u

.)

Wavelength (nm)

F1 only F1 + BSA

Figure S2. Fluorescence spectra (λex =580 nm) of F1 (1.0 μM) in the absence and

presence of BSA (1.0 mg mL-1) in phosphate buffer (20 mM, pH 7.4, containing 1%

acetone as cosolvent).

400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

Abso

rban

ce

Wavelength (nm)

F1 F4 FAsH

a)

600 630 660 690 720 7500

100

200

300

400

500

600

700

Fluo

resc

ence

Inte

nsity

(a.u

)

Wavelength (nm)

F1 F4 FAsH

b)

Figure S3. (a) Absorption spectra of F1, F4 and FAsH (both 10 μM) in phosphate

buffer (20 mM, pH 7.4, containing 1% acetone as cosolvent). (b) Fluorescence spectra

(λex =580 nm) of F1, F4 and FAsH (both 1.0 μM) in CH2Cl2.

S12

(1) 53-62; (2) 75-91; (3) 90-101; (4) 123-168; (5) 167-176; (6) 199-245; (7) 244-252;

(8) 264-278; (9) 277-288; (10) 315-360; (11) 359-368; (12) 391-437; (13) 436-447;

(14) 460-476; (15) 475-486; (16) 513-558; (17) 557-566.

Figure S4. Vicinal cysteine pairs in rBSA. In BSA the disulfide bonds are located in

the above positions, and the vicinal cysteine pairs are highlighted yellow.

0 5 10 15 20 250

250

500

750

1000

1.0 mM EDT added

0.9 μM rBSA added

Fluo

resc

ence

inte

nsity

(a.u

.)

Time (min)

Figure S5. Fluorescence time profile of FAsH (1.0 μM) upon binding to and

dissociating from rBSA (0.9 μM). The binding was reversed with the addition of EDT

(1.0 mM). λex / λem = 621/651 nm.

0.0 0.2 0.4 0.6 0.8 1.00

80

160

240

320

400

Fluo

resc

ence

inte

nsity

(a.u

.)

rBSA (μM)

Y=334.0537X+12.3236r=0.9933

Figure S6. The linear relationship between the fluorescence intensity and rBSA

concentration. λex / λem = 621 / 651 nm.

S13

lysoz

yme

ovalb

umin

BSAHSA

0

170

340

510

680

850

Fluo

resc

ence

Inte

nsity

(a.u

.) protein reduced protein

Figure S7. Fluorescence response of FAsH (1.0 µM) to different proteins and their

reduced forms (1 equiv of each) in phosphate buffer (20 mM, pH 7.4, containing 1%

acetone as cosolvent) for 5 min. λex / λem = 600/651 nm. The reduced proteins were

prepared by using the same procedure for preparing rBSA.

1 2 3 4 50

200

400

600

800

Fluo

resc

ence

inte

nsity

(a.u

.)

Figure S8. Fluorescence response of FAsH (1.0 µM) to rBSA (0.9 µM) in the

presence of 1.0 mM of different competing analytes. (1) Free; (2) Cys; (3) GSH; (4)

Hcy; (5) ascorbic acid. λex / λem = 600/651 nm.

S14

420 480 540 600 660 720 7800.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Abs

orba

nce

Wavelength (nm)

FAsH only FAsH + 0.3 μM rBSA FAsH + 0.6 μM rBSA FAsH + 0.9 μM rBSA FAsH + 1.2 μM rBSA

Figure S9. Absorption spectra of FAsH (10 μM) in the presence of different

concentrations of rBSA (0 – 1.2 μM) in phosphate buffer (20 mM, pH 7.4, containing

1% acetone as cosolvent).

100 1000 10000

0

20

40

60

80

100

Inte

nsity

(a.u

.)

Diameter (nm) Figure S10. DLS analysis of the particle size distribution of FAsH (2.0 μM) in the

presence of 1.5 μM rBSA in phosphate buffer (20 mM, pH 7.4). The solution was kept

at room temperature (25 oC) for 20 min prior to taking the measurements.

S15

620 640 660 680 700 7200

150

300

450

600

750

900

Fluo

resc

ence

inte

nsity

(a.u

.)

Wavelength (nm)

FAsH only FAsH+ rBSA FAsH+ rBSA+GndHCl

Figure S11. Fluorescence spectra (λex = 600 nm) of FAsH (1.0 μM) with the addition

of rBSA (0.9 μM) and subsequently treating with GndHCl (3.0 M) in phosphate buffer

(20 mM, pH 7.4, containing 1% acetone as cosolvent). Each spectrum was recorded 5

min after the addition of the test species.

0 1 2 3 4 50

50

100

150

200

250

Fluo

resc

ence

inte

nsity

(a.u

.)

Time (h)

FAsH + FBS FAsH + DTT FAsH + FBS + DTT

Figure S12. Time course of the fluorescence intensity of FAsH (0.5 μM) in the

presence of FBS (0.3 %), DTT (5 mM) or the mixture of FBS (0.3 %) and DTT (5

mM) in phosphate buffer (20 mM, pH 7.4, containing 0.5% acetone as cosolvent). λex

/ λem =621/651 nm.

S16

0

20

40

60

80

100

120

2015108642

Cel

l Via

bilit

y (%

)

0FAsH concentration (μM)

Figure S13. Cell viability estimated by MTT assay. SMMC-7721cells were incubated

with different concentrations of FAsH (0 - 20 μM) for 12 h.

FAsH+

PAO

FAsH

a b c

d e fFAsH

+ PAO

FAsH

a bb cc

d ee ff

Figure S14. Confocal fluorescence images of intracellular VDPs in SMMC-7721

cells by FAsH. (a) and (d), The cells were stained with DAPI (1.0 μg mL-1); (b) the

cells were stained with FAsH (2.5 μM) for 10 min; (e) the cells were pretreated with

PAO (30 μM) for 30 min, and then incubated with FAsH (2.5 μM) for 10 min; (c),

overlay of (a) and (b); (f), overlay of (d) and (e). Scale bar: 15 μm.

S17

FAsH F40

1

2

3

4

5

6

Inte

nsity

Figure S15. Semiquantitative determination of endogenous VDPs in SMMC-7721

cells according to the averaged fluorescence intensity (650 - 750 nm). The

calculations were conducted by ImageJ software. Error bars represent SD.

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)

1.92

1.97

2.00

2.04

2.00

3.19

13.

212

3.23

53.

302

3.31

53.

323

5.42

0

6.54

36.

561

7.25

37.

272

Figure S16. 1H NMR of PAO-EDT in DMSO-d6

S18

Figure S17. HRMS of PAO-EDT

Figure S18. HRMS of F1.

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

1.97

6.00

8.84

2.27

5.93

1.56

4.20

1.11

0.92

2.01

0.96

2.01

1.01

1.04

3.02

2.00

1.08

61.

206

1.22

31.

239

1.34

8

1.64

8

3.19

13.

400

3.63

13.

649

3.96

84.

015

4.03

24.

045

6.84

36.

859

6.93

66.

958

7.20

3

7.55

07.

601

7.70

3

8.22

68.

249

Figure S19. 1H NMR of F2 in DMSO-d6.

[M]+ Calcd for 441.2178

441.2211

+MS, 0.1-0.4min #(3-22)

0

1

2

3

4

5

65x10

Intens.

100 200 300 400 500 600 700 800 900 m/z

ON

COOH

N

166.8961 226.9514

259.9547

+MS, 0.0-0.3min #(2-19)

0

2

4

6

5x10Intens.

50 100 150 200 250 300 350 400 450 500 m/z

[M+H]+ Calcd for 259.9549

NH2

AsSS

S19

Figure S20. HRMS of F2.

Figure S21. 1H NMR of F3 in DMSO-d6.

423.2069

523.3063

579.5341623.2618

+MS, 0.1-0.5min #(3- 28)

0

1

2

3

4x10Intens .

400 500 600 700 800 m/z

[M]+ Calcd for 523.3073 ON

N

O

N

NH2

Figure S22. HRMS of F3

622.5357

623.3605

624.3631

625.3680

627.3004

628.3003631.2142 633.5162

635.5034

636.5075

637.5019 641.5080

+MS, 0.1-0.3min #(3-20)

0

500

1000

1500

2000Intens.

622 624 626 628 630 632 634 636 638 640 m/z

[M]+ Calcd for 623.3597 ON

N

O

N

ΗΝ O

O

S20

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.5f1 (ppm)

1.31

6.00

1.93

1.64

1.73

5.60

1.95

4.41

1.19

1.75

0.52

1.20

1.90

4.80

3.01

0.97

2.03

0.95

1.12

31.

203

1.22

11.

238

1.67

5

2.33

62.

353

2.37

12.

495

2.50

03.

111

3.15

13.

345

3.62

8

3.91

43.

935

4.01

44.

046

6.94

96.

970

7.04

67.

070

7.19

47.

199

7.21

87.

239

7.30

57.

310

7.31

97.

344

7.56

98.

235

8.25

6

10.0

34

Figure S23. 1H NMR of FAsH in DMSO-d6

0102030405060708090100110120130140150160170180f1 (ppm)

12.4

8

30.1

630

.85

31.6

0

41.3

645

.05

45.5

9

96.5

3

109.

0311

2.68

114.

7411

6.69

118.

6211

9.64

127.

2013

0.23

136.

8714

0.23

154.

1415

4.93

156.

11

166.

7617

0.58

170.

69

Figure S24. 13C NMR of FAsH in DMSO-d6

S21

413.2781

619.5284

864.2617

+MS, 0.1-0.2min #(3-13)

0

1

2

3

4x10Intens .

400 500 600 700 800 900 1000 1100 m/z

ON

N

O

N

HN

NHO

O

AsS

S

[M]+ Calcd for 864.2598

413.2781

619.5284

864.2617

+MS, 0.1-0.2min #(3-13)

0

1

2

3

4x10Intens .

400 500 600 700 800 900 1000 1100 m/z

ON

N

O

N

HN

NHO

O

AsS

S

[M]+ Calcd for 864.2598

Figure S25. HRMS of FAsH

0.01.02.03.04.05.06.07.08.09.010.011.012.013.0f1 (ppm)

4.07

1.03

2.11

2.12

1.00

1.03

2.50

92.

522

2.53

62.

548

6.98

67.

004

7.02

27.

252

7.27

27.

291

7.56

07.

579

9.94

4

12.1

30

Figure S26. 1H NMR of 2 in DMSO-d6

S22

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.5f1 (ppm)

6.16

1.90

1.80

6.05

4.29

1.10

2.92

5.19

3.70

2.99

1.09

2.16

1.06

1.20

71.

225

1.24

1

1.67

9

2.34

82.

365

2.38

2

3.18

9

3.63

33.

650

3.93

73.

961

3.96

74.

018

4.05

1

6.95

06.

971

7.00

67.

024

7.05

37.

072

7.22

27.

250

7.27

0

7.55

17.

609

7.72

0

8.23

88.

259

9.91

9

Figure S27. 1H NMR of F4 in DMSO-d6

Figure S28. HRMS of F4.

ON

N

O

N

HN

NHO

O

[M]+ Calcd for 698.3706

430.9270 521.2149

619.5215

698.3728

838.8268

+MS, 0.1-0.2min #(5-13)

0.0

0.5

1.0

1.5

5x10Intens.

300 400 500 600 700 800 900 1000 m/z