S1

Electronic Supplementary Information (ESI)

for

A colorimetric and near-infrared fluorescent probe for

biothiols and its application in living cells

Dehuan Yu, Qiong Zhang, ShuangShuang Ding and Guoqiang Feng*

Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry,

Central China Normal University,152 Luoyu Road, Wuhan 430079, P. R. China.

E-mail: gf256@mail.ccnu.edu.cn

Contents

1. Structure characterizations of compound 2 and probe 1…………….…Page S2-S5

2. Additional spectra………………………….……………………………….………….………Page S5-S11

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2014

S2

1. Structure characterizations of compound 2 and probe 1

0.01.02.03.04.05.06.07.08.09.010.52.0

7.27.88.4

1H-NMR spectrum of compound 2 in d6-DMSO

13C-NMR spectrum of compound 2 in d6-DMSO

O

NC CN

OH2

O

NC CN

OH2

S3

MS (EI) spectrum of compound 2

4x10

0.25

0.5

0.75

1

1.25+ESI Scan (15.539 min) Frag=160.0V WorklistData8.d

313.0956

647.1601 921.9993

Counts vs. Mass-to-Charge (m/ z)100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400

HR-MS spectrum of compound 2

0.01.02.03.04.05.06.07.08.09.010.52.0

6.16.46.77.07.37.67.9

1H-NMR spectrum of probe 1 in CDCl3

O

NC CN

OO

probe 1

O

NC CN

OH2Chemical Formula: C20H12N2O2

Molecular Weight: 312.32

CDCl3

H2O

O

NC CN

OH2

H+

(2 + H+): C20H13N2O2+

Exact Mass: 313.0972

S4

020406080100120140160180200220

0.0

63

.1

76

.77

7.0

77

.31

07

.01

15

.61

16

.71

18

.91

22

.41

27

.61

29

.11

32

.41

33

.21

37

.7

15

2.2

15

2.3

15

2.8

15

7.2

16

4.2

13C-NMR spectrum of probe 1 in d6-DMSO

MS (EI) spectrum of probe 1

O

NC CN

OO

probe 1

Chemical Formula: C23H14N2O3Molecular Weight: 366.37

O

NC CN

OO

probe 1

M+

(M - CH2=CHCO)+

S5

HR-MS spectrum of probe 1

2. Additional spectra

300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

Ab

s

Wavelength(nm)

(a)

probe 1 (0-50 M)

0 10 20 30 40 50 60

0.0

0.2

0.4

0.6

0.8

1.0

[1] M)

Ab

s (

38

0 n

m)

Equation y = a + b*x

Weight No Weightin

Residual Sum of Squares

0.00251

Pearson's r 0.99835

Adj. R-Squar 0.99634

Value Standard Err

?$OP:A=1 Intercept 0.0231 0.00942

?$OP:A=1 Slope 0.0166 3.18421E-4

(b)

300 400 500 600 700 800

0.0

0.3

0.6

0.9

1.2

1.5

Ab

s

Wavelength(nm)

(c)Compound 2 (0-50 M)

0 10 20 30 40 50 60

0.0

0.3

0.6

0.9

1.2

1.5

[2] (M)

Ab

s (

56

0 n

m)

(d)

Equation y = a + b*x

Weight No Weighting

Residual Sum of Squares

3.35918E-4

Pearson's r 0.99992

Adj. R-Square 0.99983

Value Standard Error

?$OP:A=1 Intercept -0.00535 0.00345

?$OP:A=1 Slope 0.02795 1.16501E-4

600 650 700 750 800 850 9000

30

60

90

120

150

180(e)

Flu

ore

scen

t In

ten

sity

Wavelength(nm)

Compound 2 (0-50 M)

0 10 20 30 40 500

30

60

90

120

150

180(f)

[2] (M)

Inte

nsi

ty (

706

nm

)

Fig. S1 (a) Absorption spectra of probe 1 at different concentrations. (b) Absorbance intensity

changes at 380 nm of probe 1 as a function of its concentration from 0-50 M. (c) Absorption

O

NC CN

OO

probe 1

H+

(M + H+): C23H15N2O3+

Exact Mass: 367.10772

S6

spectra of compound 2 at different concentrations. (d) Absorbance intensity changes at 560 nm of

compound 2 as a function of its concentration from 0-50 M. (e) Fluorescence spectra of

compound 2 at different concentrations. (f) Fluorescence intensity changes of compound 2 at 706

nm as a function of its concentration from 0-50 M. All spectra were collected in PBS buffer (10

mM, pH 7.4) with 50% DMSO at 37 °C. For fluorescence measurement, λex = 560 nm with slit

width: (10, 10).

0 300 600 900 1200 1500 18000

40

80

120

= max + (0 - max) e(-kobsT)

kobs = 0.167 min-1

R2 = 0.99622

Inte

nsi

ty (

706

nm

)

Time (s)

1 + 1 eq Cys

Fig. S2 Time-dependent of fluorescence kinetics spectra of probe 1 (20 M) upon addition of Cys

(20 M) in PBS buffer (10 mM, pH 7.4) with 50% DMSO at 37˚C. The reaction is monitored at

706 nm. λex = 560 nm, slit width: (10, 10).

300 400 500 600 700 8000.0

0.2

0.4

0.6

Ab

s

Wavelength (nm)

(a)

[Cys]

0 40 80 120 160 2000.0

0.2

0.4

0.6

[Cys] (M)

Ab

s

(b)

Fig. S3 (a) Absorption spectra changes of probe 1 (20 µM) upon addition of different

concentrations of Cys in PBS buffer (10 mM, pH 7.4) with 50% DMSO at 37 °C. Final

concentration of Cys: 0, 6, 10, 30, 40, 50, 60, 70, 100, 120, 140, 160 and 180 M, respectively. (b)

Absorbance changes at 560 nm of probe 1 (20 μM) against concentration of Cys. Each spectrum

was obtained 30 min after Cys addition.

S7

600 650 700 750 800 850 9000

40

80

120

160

Flu

ore

scen

t In

ten

sity

Wavelength (nm)

(a)

[Cys]

0 50 100 150 2000

40

80

120

160

[Cys] (M)

Inte

nsi

ty

(b)

Fig. S4 (a) Fluorescence spectra changes of probe 1 (20 µM) upon addition of different

concentrations of Cys in PBS buffer (10 mM, pH 7.4) with 50% DMSO at 37 °C. Final

concentration of Cys: 0, 2, 6, 10, 20, 40, 60, 80, 100, 120, 140, 160 and 180 M, respectively. (b)

Fluorescence intensity changes at 706 nm of probe 1 (20 μM) against concentration of Cys. Each

spectrum was obtained 30 min after Cys addition. λex = 560 nm, slit width: (10, 10).

5 6 7 8 9 100.0

0.2

0.4

0.6

0.8

1.0

Ab

s

pH

Cys

none

(a)

5 6 7 8 9 100

40

80

120

160

200

Flu

ore

scen

t In

ten

sity

pH

Cys

none

(b)

Fig. S5 (a) Absorbance responses of probe 1 (20 M) at 560 nm in the presence and absence of Cys (5

eq) under different pHs. (b) Fluorescence intensity responses of probe 1 (20 M) at 706 nm in the

presence and absence of Cys (5 eq) under different pHs. All experiment was performed in PBS buffer

(10 mM) with 50% DMSO at 37 °C and each data was obtained 30 min after addition of Cys. For

fluorescence measurements, λex = 560 nm, slit width: (10, 10).

Data for investigation of the sensing mechanism

This experiment was performed according to a reported procedure (see Ref: X. Yang, Y. Guo and

R. Strongin, Angew. Chem., Int. Ed., 2011, 50, 10690–10693). To a 100 mL flask, probe 1 (50 mg,

0.14 mmol) and Cys (21 mg, 1.25 eq) were combined in 30 mL of MeOH: H2O (90: 10, v/v)

solution, and the mixture stirred at room temperature for 1 h. Then, Et3N (30 μL) was added and

the solution stirred overnight. The solvents was removed under reduced pressure and the crude

S8

product was subjected to column chromatography (eluted with CH2Cl2: MeOH, 5:1, v/v) to afford

a red solid (23 mg), which was proved to be compound 2 (see below).

600 650 700 750 800 850 9000

50

100

150

Flu

ore

scen

t In

ten

sity

Wavelength (nm)

probe 1 the isolated product the reference sample of 2

(b)

Fig. S6 (a) The thin layer chromatography (TLC) plate under different light to compare probe 1,

the reference sample of 2 and the isolated product from the reaction mixture of probe 1 with Cys.

Spots on the TLC plate are: a. probe 1; b. the isolated reaction product of probe 1 and Cys; c.

mixture of b and 2; d. the reference sample of 2. The eluent for TLC: hexane:ethyl acetate = 3:1

(v/v). (b) Fluorescent spectra of probe 1, the isolated product and the reference sample of 2 in PBS

buffer (10 mM, pH 7.4) with 50% DMSO at 37 °C. λex = 560 nm, slit width: (10,10).

Fig. S7 1H NMR spectrum of the isolated product from the reaction of probe 1 and Cys, which is

identical to that of the reference sample of 2 (see above).

S9

YUDH010-1 #622 RT: 2.90 AV: 1 SB: 837 0.04-3.07 , 3.45-4.27 NL: 1.47E4T: + c Full ms [40.00-500.00]

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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

312.30

148.08

113.14 193.29 283.29233.07 330.1593.0563.12

Fig. S8 MS spectrum of the isolated product from the reaction of probe 1 and Cys, which showed

the expected mass of 2.

300 400 500 600 700 8000.0

0.2

0.4

0.6(a)

Cys

other anions

Ab

s

Wavelength(nm)

0.0

0.2

0.4

0.6

(b)

Ab

s (5

60 n

m)

OH

H2 O

2

ClO -

O2 -

S 2-

HS -

Cys

CN -

SO3 2-

SO4 2-

CO

3 2-

SC

N -

AcO -

NO

2 -

NO

3 -

S2 O

3 2-

I -Br -

Cl -

F -blank

Fig. S9 (a) Absorption spectral responses of probe 1 (20 μM) upon addition of 100 μM various

anions and Cys. Anions are F−, Cl−, Br−, I−, NO3−, NO2

−, AcO−, SCN−, CO32−, SO4

2−, SO32−,

S2O32−, HS−, S2−, CN−, O2•

−, ClO−, H2O2 and OH•. (b) A comparison of absorbance intensity

changes of probe 1 (20 μM) at 560 nm upon addition of 100 μM various anions and Cys. All

experiment was performed in PBS buffer (10 mM, pH 7.4) with 50% DMSO at 37 °C and each

spectrum was obtained 30 min after addition of various anions.

600 650 700 750 800 850 9000

40

80

120

160(a)

Cys

other anions

F

luo

resc

ent

Inte

nsi

ty

Wavelength(nm)

0

40

80

120

160(b)

Inte

nsi

ty (

706

nm

)

OH

H2 O

2

ClO -

O2 -

S 2-

HS -

Cys

CN -

SO3 2-

SO4 2-

CO

3 2-

SCN -

AcO -

NO

2 -

NO

3 -

S2 O

3 2-

I -Br -

Cl -

F -blank

Fig. S10 (a) Fluorescent spectral responses of probe 1 (20 μM) upon addition of 100 μM various

anions and Cys. Anions are F−, Cl−, Br−, I−, NO3−, NO2

−, AcO−, SCN−, CO32−, SO4

2−, SO32−,

O

NC CN

OH2Chemical Formula: C20H12N2O2

Molecular Weight: 312.32

S10

S2O32−, HS−, S2−, CN−, O2•

−, ClO−, H2O2 and OH•. (b) A comparison of fluorescence intensity

changes of probe 1 (20 μM) at 706 nm upon addition of 100 μM various anions and Cys. All

experiment was performed in PBS buffer (10 mM, pH 7.4) with 50% DMSO at 37 °C and each

spectrum was obtained 30 min after addition of various anions. λex = 560 nm , slit width: (10, 10).

300 400 500 600 700 8000.0

0.2

0.4

0.6

Ab

s

Wavelength (nm)

0-30 min

Cys

(a)

300 400 500 600 700 8000.0

0.2

0.4

0.6

0-30 min

Ab

s

Wavelength (nm)

Hcy

(b)

 

300 400 500 600 700 8000.0

0.2

0.4

0.6

0-30 min

Ab

s

Wavelength (nm)

(c)

GSH

0 5 10 15 20 25 300.0

0.2

0.4

0.6

A

bs

at 5

60

nm

Time (min)

Cys

Hcy

GSH

none

(d)

Fig. S11 (a)-(c): Scanning kinetics of the UV-vis spectra changes of probe 1 (20 µM) against time

in the presence of 5 equiv of Cys (a), Hcy (b) and GSH (c), respectively. All spectra were

measured in PBS buffer (10 mM, pH 7.4) with 50% DMSO at 37 °C. (d) The corresponding

absorbance changes of probe 1 at 560 nm against time for Cys, Hcy and GSH.

0 300 600 900 1200 1500 18000

40

80

120

160

Inte

nsi

ty (

706

nm

)

Time (s)

Cys

Hcy

GSH

none

Fig. S12 Fluorescence kinetics of probe 1 (20 µM) at 706 nm in the absence and presence of

biothiols (Cys, Hcy and GSH, 100 µM each). All spectra were measured in PBS buffer (10 mM,

pH 7.4) with 50% DMSO at 37 °C. λex= 560 nm, slit width (10,10).

S11

Generation of reactive oxygen species (ROS)

ClO−

The NaOCl solution was added to the probe 1 testing solution at 25 °C.

H2O2

H2O2 was added and the mixtures were stirred for 1 hour at 25 °C.

OH•

Hydroxyl radicals were generated by the addition of Fe2+ and H2O2 at room temperature in PBS

buffer (pH 7.4) and the mixture was then stirred for 30 min. (Ref. Y. Zhou, J.-Y. Li, K.-H. Chu, K.

Liu, C. Yao, J.-Y. Li, Chem Commun. 2012, 48, 4677–4679.)

O2•−

O2•− was generated by an improved pyrogallol autoxidation method reported recently (Ref. X. Li,

J. Agric. Food Chem., 2012, 60, 6418−6424).