Supporting Information

Construction of a ratiometric probe with dual quenching mechanisms for selectively imaging intracellular sulfur dioxide overcoming the interference from cysteine

Qilong Zhang, Zhongqian Cui, Qiufen Wang, Gengxiu Zheng*

School of Chemistry and Chemical Engineering, University of Jinan, No. 336 West Road of NanXinzhuang, Jinan 250022, P. R. China.

Email:chm_zhenggx@ujn.edu.cn

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ContentsMaterialsAnalysisSpectroscopic measurementsCell culture and staining methodsFluorescent imaging methodsCalculation detailsFigure S1.Figure S2Figure S3.Figure S4.Figure S5.Synthesis and Characterization of C-1.Scheme S1Figure S6Figure S7Figure S8Figure S9Figure S10Figure S11

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Materials

All chemicals used are of analytical grade, 2-hydroxybenzoleactone was purchased

from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 4-

(Diethylamino)salicylaldehyde etc. was purchased from J&K Chemical (Beijing,

China). The solvents used in the spectral measurement are of chromatographic grade.

Analysis1H- and 13C- NMR were recorded on a Bruker 400MHz spectrometer using CDCl3 as

solvent. Chemical shift values are reported in ppm with the solvent as the internal

standard (CDCl3: δ 7.26 for 1H, δ 77.16 for 13C). Data are reported as follows:

chemical shifts (δ), multiplicity (s = singlet, d = doublet, dd = double of doublet, t =

triplet, q = quartet, m = multiplet) etc., coupling constants J (Hz), and integration.

High Resolution Mass measurements were performed using Agilent Technologies

Mass spectrometer.

Spectroscopic measurements

The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a

U2910 spectrophotometer using a quartz cuvette having 1 cm path length. One-photon

fluorescence spectra of dilute solutions were obtained on a HITACH F-2700

spectrofluorimeter equipped with a 450-W Xe lamp. PBS buffer solution: 10 mM,

NaCl, NaHPO4·12H2O, NaH2PO4·2H2O, pH = 7.40.

Cell culture and staining methods

HeLa cells were grown in H-DMEM (Dulbecco’s Modified Eagle’s Medium, High

Glucose) supplemented with 10% FBS (Fetal Bovine Serum) in a 5% CO2 incubator

at 37 °C. For living cells imaging experiment of the probes, the culture medium

surrounding the cells were firstly removed, and the cells were washed with PBS

twice. Then the cells were incubated in 1 mL of PBS. On the other hand, 0.1 mM

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stock solutions of the probe in DMSO were prepared. After that, 2 μL of stock

solutions were mixed evenly with 1 mL PBS (pH 7.4) in a tube. The cells were

incubated with the above mixed solutions at 37 °C. After incubation, the cells were

imaged immediately without further washing procedure.

Fluorescent imaging methods

Confocal fluorescence images were obtained with a Nikon A1R confocal laser

scanning microscope. The differential interference contrast (DIC) images were taken

with 561 nm sapphire laser.

Calculation details

The initial structures of the NCCA, NCCA-Cys, and NCCA-SO2 were obtained with

Gaussian View software. Afterwards, the geometrical structures of these probes were

optimized sequentially using the basic set of pm3, b3lyp/3-21g, b3lyp/6-31g, cam-

b3lyp/tzvp, with Gaussian 09 software package. The frontier orbitals were obtained

with the final structure.

430 480 530 580 630 680 7300

900

1800

2700

3600

4500

Fluo

resc

ence

Inte

nsity

Wavelength [nm]

NCCA-Cys

NCCA-SO2

pH = 5

pH = 8

Figure S1. The fluorescence spectra of NCCA in the presence of 80 μM NaHSO3 and

Cys in buffer solutions with different pH values (5, 6, 7, 8). Excitation wavelength:

405 nm.

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430 480 530 580 630 6800

500

1000

1500

2000

2500

3000

3500

+Asp +Glu +Gly +His +Ile +Ser +Thr +VC +KNO3

+Na2SO4

+NaCl +NaHCO3

Fluo

resc

ence

Inte

nsity

Wavelength [nm]

Probe SO2

+Cys +GSH +Hcy +Na2S +H2O2

+NaClO +NO +NaNO2

+Asn

0 5 10 15 20

2

4

6

8

10

12

I 485/I

565

H2O2 NaClO(a) (b)

Figure S2. The fluorescence spectra (a) and intensity ratio of 485 nm to 565 nm (b) of

the probe NCCA in the presence of different reagents. 1-blank, 2-NaHSO3, 3-

NaHSO3+Cys, 4- NaHSO3+GSH, 5- NaHSO3+Hcy, 6- NaHSO3+Na2S, 7-

NaHSO3+H2O2, 8- NaHSO3+NaClO, 9- NaHSO3+NO, 10- NaHSO3+NaNO2, 11-

NaHSO3+Asn, 12- NaHSO3+Asp, 13- NaHSO3+Glu, 14- NaHSO3+Gly, 15-

NaHSO3+His, 16- NaHSO3+Ile, 17- NaHSO3+Ser, 18- NaHSO3+Thr, 19-

NaHSO3+VC, 20- NaHSO3+KNO3, 21- NaHSO3+Na2SO4, 22- NaHSO3+NaCl, 23-

NaHSO3+NaHCO3. λex = 405 nm.

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O ON

NH+S

COOH

Figure S3. The HRMS spectra of NCCA-Cys.

O O

Cl

SO3Na

OH

H+

N

Figure S4. The HRMS spectra of NCCA-SO2.

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0.0

0.2

0.4

0.6

0.8

1.0

Cel

l Via

bilit

y

2 h 12 h 24 h

Figure S5. The cell viability of HeLa cells incubated with 5 μM NCCA or different time.

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Synthesis and Characterization of C-1.

Scheme S1. The synthetic routine of NCCA.

To a solution of 4-(diethylamino)salicylaldehyd (3.86 g, 20 mmol) and ethyl

acetoacetate (4.8 g, 40 mmol) in anhydrous ethanol(60 mL) was added piperazine (0.7

g, 0.4 mmol) at room temperature, and then the reaction mixture was heated to reflux

for 12 h. The mixture was cooled down to 0-5°C and stirred for 2 h, The precipitated

solids were filtered, washed with ethanol (30 mL), and dried at 50 °C to afford AC-1

as a bright yellow solid (4.1 g, yield of 80%). 1H NMR (400 MHz, CDCl3) δ 8.44 (s,

1H), 7.41 (d, J = 8.9 Hz, 1H), 6.64 (dd, J = 8.9, 2.2 Hz, 1H), 6.49 (d, J = 2.0 Hz, 1H),

3.46 (q, J = 7.1 Hz, 4H), 2.68 (s, 3H), 1.25 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz,

CDCl3) δ 195.66, 160.83, 158.72, 152.92, 147.81, 131.89, 110.07, 109.96, 108.26,

96.71, 45.23, 30.57, 12.42. HRMS (ESI): m/z, Calc., 260.1281; found, 260.1284.

Synthesis and Characterization of NCCA.

POCl3 (1 mL, 10.8 mmol) was slowly added to a stirred mixture of anhydrous DMF

(10 mL) at 0-5°C, and then the mixture was stirred for 3 h at the same temperature.

Compound AC-1 (2.0 g, 7.7 mmol) was dissolved in anhydrous DMF (10 mL) and

added dropwise through a dropping funnel over 30 min at 0°C, and then the reaction

mixture was heated to 80°C and stirred for 5 h. The mixture was cooled down to room

temperature, and poured into ice-water under stirring. NaOH was added to the mixture

and the pH was adjusted to 7 under 5°C. 100 mL of dichloromethane was used to

extract it twice, and then dried with anhydrous MgSO4, filtered, and concentrated in

reduced pressure to obtain the crude product. Pure product AC-2 was finally obtained

by column chromatography (ethyl acetate/ Petroleum ether, 1:5), presented as pale red

powder (1.7 g, yield of 72%). 1H NMR (400 MHz, CDCl3) δ 10.27 (d, J = 6.9 Hz,

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1H), 8.38 (s, 1H), 7.68 (d, J = 6.9 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 6.70 (dd, J = 8.8,

1.5 Hz, 1H), 6.52 (s, 1H), 3.47 (q, J = 7.0 Hz, 4H), 1.26 (t, J = 7.0 Hz, 6H). 13C NMR

(100 MHz, CDCl3) δ 192.43, 158.36, 156.83, 152.57, 145.28, 144.85, 131.03, 126.17,

113.13, 110.27, 108.62, 96.79, 45.39, 12.42. HRMS (ESI): m/z, Calc., 306.0891;

found, 306.0897.

Figure S6. 1H NMR spectra of C-1 in CDCl3.

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Figure S7. 13C NMR spectra of C-1 in CDCl3.

Figure S8. HRMS of C-1 in methanol.

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Figure S9. 1H NMR spectra of NCCA in CDCl3.

Figure S10. 13C NMR spectra of NCCA in CDCl3.

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Figure S11. The HRMS spectra of NCCA.

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