Supplementary Material

A ratiometric fluorescent probe with DNBS group for

biothiols in aqueous solution

Xi Daia, Tao Zhangb, Jun-Ying Miaob,*, Bao-Xiang Zhaoa,*

a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering,

Shandong University, Jinan 250100, P.R. China. E-mail: bxzhao@sdu.edu.cn (B.X.

Zhao); Tel.: +86 531 88366425; fax: +86 531 88564464;

b Institute of Developmental Biology, School of Life Science, Shandong University,

Jinan 250100, P.R. China. E-mail: miaojy@sdu.edu.cn (J.Y. Miao)

1. Synthesis of Probe DPCS

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1.1 Synthesis of compound H-DPC

Compound H-DPC was synthesized by reported method [S1].

1.2 Synthesis of 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-2-oxo-2H-chromen-7-

yl 2,4-dinitrobenzenesulfonate (probe DPCS)

A solution of compound H-DPC (1 mmol) in CH2Cl2 (20 mL) and Et3N (0.5 mL)

were added to a round-bottomed flask, and the mixture was stirred in an ice-bath. A

solution of 2,4-dinitrobenzene sulfonyl chloride (1.2 mmol) in CH2Cl2 (10 mL) was

added to the mixture over 0.5 h and the mixture was stirred at 0 oC for 1 h. Then the

mixture was stirred at room temperature for 2 h until TLC indicated the end of

reaction. The organic phase was washed with water (3 × 50 mL), and then dried over

Mg2SO4. The solvent was concentrated under reduced pressure. The residue was

purified by column chromatography on silica gel to afford compound DPCS in 80%

yield. Red solid, m.p. 237-238 oC. IR (KBr, cm-1): 3097 (Ar-H), 2921 (-CH2-), 1735,

1601 (C=O), 1545, 1491 (C=C), 1348 (-NO2). 1H NMR (400 MHz, CDCl3): δ = 3.36

(dd, J = 18.3 and 7.1 Hz, 1H, pyrazoline, 4-Ha), 4.06 (dd, J = 18.3 and 12.8 Hz, 1H,

pyrazoline, 4-Hb), 5.36 (dd, J = 12.8 and 7.1 Hz, 1H, pyrazoline, 5-H), 6.84 (t, J = 7.3

Hz, 1H), 7.08 (dd, J = 8.7 and 0.9 Hz, 2H), 7.19-7.32 (m, 9H), 7.60 (d, J = 8.6 Hz,

1H), 8.27 (d, J = 8.6 Hz, 1H), 8.35 (s, 1H, coumarin, C4-H), 8.53 (dd, J = 8.6 and 2.2

Hz, 1H), 8.69 (d, J = 2.2 Hz, 1H). 13C NMR (100 MHz, CDCl3): 44.92, 65.03, 110.37,

113.80, 118.73, 119.35, 120.25, 120.51, 121.75, 125.67, 126.52, 127.74, 129.02,

129.18, 129.44, 133.83, 135.44, 141.77, 142.02, 143.65, 149.76, 151.22, 153.76,

158.57. HRMS: m/z [M+H]+ calcd for [C30H20N4O9S + H]+: 613.1029, found:

613.0906.

2. Supplement figures

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Figure S1. Fluorescence spectra of H-DPC (1 × 10-5 M) at different excitation

wavelengths in PBS buffer solution (CTAB 10 mM, pH 7.4, slit: 10.0 nm/4 nm).

Figure S2. UV-vis absorption spectra of probe DPCS (1 × 10-5 M) with or without

biothiols (1 × 10-4 M) in the PBS buffer solution (CTAB 10 mM, pH 7.4). Inset

photograph is DPCS with or without biothiols in visible light.

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Figure S3. UV-Vis absorption spectra of DPCS (1 × 10-5 M) with a) Cys, b) GSH and

c) Hcy at various equivalents (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.4, 1.6, 1.8, 2.0, 3.0, 4.0 and

5.0 equiv.) in PBS buffer solution (CTAB 1 mM, pH 7.4); d) The plot of absorbance at

458 nm as a function of biothiols equivalent. Data are meanSE (bars) (n = 3).

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Figure S4. (a-e) Fluorescence spectra; (f) Left: Ratiometric responses (△I = I540/I470),

Right: The enhancement ratio (■) of DPCS (1 × 10-5 M) with (red) or without (black)

GSH (1 × 10-4 M) at different excitation wavelengths in PBS buffer solution (CTAB 1

mM, pH 7.4, slit: 10.0 nm/4.0 nm).

Figure S5. Fluorescence intensity (I540) of DPCS (1 × 10-5 M) without or with thiols

(1 × 10-4 M) in the presence of various analytes (1 × 10-4 M) in PBS buffer solution

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(CTAB 1 mM, pH 7.4, λex = 450 nm, slit: 10.0 nm/4.0 nm).

Figure S6. Fluorescence titration spectra of DPCS (1 × 10-5 M) vs Cys (a), GSH (b)

and Hcy (c) at various equivalents (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2,

1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 4.0 and 5.0 equiv.) in PBS buffer solution (CTAB 1 mM,

pH 7.4, λex = 450 nm, slit: 10.0 nm/4.0 nm). (d) The plot of fluorescence intensity at

540 nm as a function of biothiols equivalent. Data are meanSE (bars) (n = 3).

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Figure S7. Fluorescence intensity (I540) of probe DPCS (1 × 10-5 M) toward different

equiv. of thiols in the PBS buffer solution (CTAB 1 mM, pH 7.4, λex = 450 nm, slit:

10.0 nm/4.0 nm). Data are meanSE (bars) (n = 3).

Cys: y = 338.519x + 4.934 R2 = 0.992 LOD = 2.8 × 10-8 M

GSH: y = 587.985x + 7.167 R2 = 0.993 LOD = 1.5 × 10-8 M

Hcy: y = 282.465x + 4.402 R2 = 0.990 LOD = 3.2 × 10-8 M

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Figure S8. Two-photon fluorescence titration spectra of DPCS (1 × 10-5 M) vs Cys

(a), GSH (b) and Hcy (c) (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6,

1.8, 2.0, 2.5, 3.0, 4.0 and 5.0 equiv.) in PBS buffer solution (CTAB 1 mM, pH 7.4, λex

= 900 nm). (d) The plot of fluorescence intensity at 580 nm as a function of biothiols

equivalent. Data are meanSE (bars) (n = 3).

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Figure S9. The plot of fluorescence intensity (I580 nm) of DPCS (1 × 10-5 M) vs thiols

(0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 equiv.) in PBS buffer solution

(CTAB 1 mM, pH 7.4, λex = 900 nm). Data are meanSE (bars) (n = 3).

Cys: y = 731.259x + 188.270 R2 = 0.993 LOD = 5.5 × 10-7 M

GSH: y = 1456.92x + 166.918 R2 = 0.996 LOD = 2.8 × 10-7 M

Hcy: y = 633.231x + 157.3658 R2 = 0.994 LOD = 6.4 × 10-7 M

Figure S10. Fluorescence titration spectra of DPCS (1 × 10-5 M) vs Cys (a), GSH (b)

and Hcy (c) at various equivalents (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2,

1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 4.0 and 5.0 equiv.) in PBS buffer solution (CTAB 1 mM,

pH 7.4, λex = 410 nm, slit: 10.0 nm/4.0 nm). (d) The plot of fluorescence intensity

ratio of I540/I470 as a function of biothiols equivalent. Data are meanSE (bars) (n = 3).

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Figure S11. The plot of fluorescence intensity ratio (I540/I470) of DPCS (1 × 10-5 M) vs

thiols (0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 2.0 equiv.) in PBS buffer

solution (CTAB 1 mM, pH 7.4, λex = 410 nm, slit: 10.0 nm/4.0 nm). Data are

meanSE (bars) (n = 3).

Cys: y = 0.545x + 1.395 R2 = 0.948 LOD = 4.3 × 10-6 M

GSH: y = 0.855x + 1.892 R2 = 0.983 LOD = 2.7 × 10-6 M

Hcy: y = 0.540x + 1.741 R2 = 0.973 LOD = 4.3 × 10-6 M

Table S1 The LOD of biothiols at different excitation wavelengths.

λex / nm LOD / mol L-1

GSH Cys Hcy

410 2.7 × 10-6 4.3 × 10-6 4.3 × 10-6

450 2.8 × 10-8 1.5 × 10-8 3.2 × 10-8

900 5.5 × 10-7 2.8 × 10-7 6.4 × 10-7

[a] The concentration of probe DPCS was 10-5 M with three excitation wavelengths.

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Figure S12. Time-dependent fluorescence intensity at I540 of DPCS (1 × 10-5 M) with

or without thiols (10 equiv.) in the PBS buffer solution (CTAB 1 mM, pH 7.4, λex =

450 nm, slit: 10.0 nm/4.0 nm).

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Figure S13. Fluorescence intensity (I540 nm) of DPCS (1 × 10-5 M) with or without

thiols (1 × 10-4 M) in PBS buffer solution (CTAB 10 mM, pH = 4.99, 5.48, 5.98, 6.49,

6.98, 7.48, 7.99, 8.50, 9.01, 9.52 and 9.99, λex = 450 nm, slit: 10.0 nm/3.5 nm).

Scheme S1. The proposed reaction mechanism of DPCS with thiols.

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Figure S14. High resolution mass spectrum of the conjugate addition adduct of probe

DPCS with Cys.

Figure S15. High resolution mass spectrum of the conjugate addition adduct of probe

DPCS with GSH.

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Figure S16. High resolution mass spectrum of the conjugate addition adduct of probe

DPCS with Hcy.

Figure S17. Fluorescence spectra of DPCS (1 × 10-5 M), compound H-DPC (1 × 10-5

M) and DPCS (1 × 10-5 M) with thiols (1 × 10-4 M) in PBS buffer solution (CTAB 10

mM, pH 7.4, λex = 450 nm, slit: 15.0 nm/5.0 nm).

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Figure S18. Fluorescence spectra of DPCS (1 × 10-5 M) with or without NEM (1

mM) in the presence of thiols (1 × 10-4 M) in the PBS buffer solution (CTAB 10 mM,

pH = 7.4, λex = 450 nm, slit: 10.0 nm/4.0 nm).

Figure S19. Fluorescence images of DPCS (5 μM) with different concentrations of

calf serum. Calf serum was diluted 100 times (1%), 50 times (2%), 20 times (5%), 10

times (10%), 5 times (20%), 2 times (50%) with PBS and not diluted (100%). (n > 3)

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Figure S20. Viability of A549 cells incubated with probe DPCS for 12 h.

Figure S21 (a) Fluorescence images of A549 cells. A549 cells were pre-incubated

with 0, 1 mM, 5 mM N-ethylmaleimide for 1 h, then with 10 µM DPCS for 1 h. (b)

Fluorescence intensity quantitation. Results are presented as means±SE. (n = 3, *, p <

0.05, **, p < 0.01 vs. control).

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Figure S22. 1H NMR spectrum of probe DPCS (CDCl3).

Figure S23. 13C NMR spectrum of probe DPCS (CDCl3).

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References

S1. X. Dai, T. Zhang, Y.Z. Liu, T. Yan, Y. Li, J.Y. Miao and B.X. Zhao, Sensor. Actua. B. 207

(2015) 872-877.

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