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Supporting Information

A Near-Infrared Multifunctional Fluorescent Probe with Inherent Tomor-targeting Property for Bioimaging

Xu Zhaoa,b, Yang Lia,b, Di Jina, Yuzhi Xing, Xilong Yana,b * and Ligong Chena,b *

a School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China

b Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, P.R. China

Corresponding authors: X. L. Yan and L. G. ChenTel.: +86 22 27406314, Fax: +86 22 27406314.E-mail address: [email protected] (L. G. Chen), [email protected] (X. L. Yan)

Contents

Materials and equipments ……………………………………………………………∙1

Synthesis and characterization of compounds ………………………………………∙1

Measurement of absorbance and fluorescence spectroscopy ………………………∙∙7

Measurement of absolute quantum yield ……………………………………………∙8

In vitro cytotoxicity experiments ……………………………………………………∙8

pH-dependent cell imaging and subcellular localization experiments………………∙∙9

In vivo imaging experiments ………………………………………………………∙∙10

Fig.S1 UV absorption and fluorescence emission spectra of compound 7…………∙∙10

Fig.S2 UV absorption and fluorescence emission spectra of compound 8…………∙∙11

Fig.S3The decayed curves of compound 10 at different pH values ………………∙∙∙11

Fig.S4 The fluorescent intensity at 814 nm of 10 at pH = 4.01 upon addition of

different interferents ………………………………………………………………∙∙∙11

Fig.S5 Effect of probe 10 on the viability of HeLa cells……………………………∙12

References……………………………………………………………………………121H NMR and 13C NMR spectra………………………………………………………13

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

mailto:[email protected]

mailto:[email protected]

2

Materials and equipments

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-

hydroxysuccinimide (NHS), 2,3,3-trimethyl-5-nitro-3H-indole and ethyl 3-bromo

propionate were purchased from Aladdin Reagent Co. Ltd. (Shanghai, China).

Rhodamine123 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) were purchased from Sigma-Aldrich (Poole, Dorset, England). Fetal bovine

serum and cell culture media were purchased from Invitrogen (Carlsbad, CA, USA).

Diethanolamine was obtained from Yantai Peiyang Fine Chemical Co. Ltd.

(Shandong, China). Other reagents and solvents such as POCl3 and TEA were

purchased from Tianjin chemicals Co. (Tianjin, China) and used without further

purification unless otherwise noted. DMF was purified by refluxing with calcium

hydride for 4 h and then distilled. Deionized water was used throughout this work. 1H and 13C NMR spectra were recorded on a Bruker Avance 600 spectrometer

and Bruker Avance 400 spectrometer, using TMS as an internal standard. High-

resolution mass spectra were measured with a Bruker Daltonics micrOTOF-Q II

instrument (ESI). All pH measurements were measured with a Sartorius basic pH-

meter PB-10. UV-Vis spectra were obtained on an Evolution 300 UV-Visible

spectrophotometer. Fluorescence spectra were recorded on a Jobin Yvon-Spex

Fluorolog3 spectrofluorometer and Hitachi F-4500 spectrofluorometer. The

photophysical properties of compound 10, including fluorescence quantum yield and

lifetime were performed on a PTI QM/TM/NIR system (Photon Technology

International, Birmingham, NJ, USA). The decayed curves of compound 10 emission

at 810 nm excited by the high resolution laser (N2 laser at 337nm tuned by laser dye

PLD665) at 665 nm. Confocal fluorescence images of live cells were taken on a Leica

TCS SP8. The in vivo images of mice were performed with a Bethold NightOWL LB

983 in vivo Imaging System equipped with a CCD camera.

Synthesis and characterization of compounds

N

OO

Cl

OHON

NO2

+ +CH3CH2OH

NN

Cl

OO

NO2

1 2 43

N2

3

CH3CH2OH

SnCl2NN

Cl

OO

NH2

5

ClCl

O

CH2Cl2TEA

NN

Cl

OO

HNO

Cl

6

CH2Cl2

NN

Cl

OO

HNO

NHN

OH

OHOH

OH

7

CH2Cl2

NNN

Cl

OO

HNO

N

EtOH

NaOH / H2O HNN

Cl

OHO

HNO

N

EDC HCl / NHS

DMFN2保护

NN

Cl

OO

HNO

N

NO O

O

OH

OHNH2

HOHO

DIPEA

HCl

DMF

NN

Cl

HNO

N

OOH

HO

HN OHOH

8 9

10O

Scheme S1 Synthetic routes of probes

3-(3,3-dimethyl-2-methyeneiodolinl-yl)-ethyl propionate (1) and 2-chloro-1-

formyl-3-(hydroxymethylene)-cyclohex-1-ene (2) were prepared according to our

previously reported method[1].

Synthesis of compound 4: To a mixture of compound 1 (7.75 g, 29.88 mmol) and compound 2（5.14 g, 29.78 mmol）in ethanol was added compound 3 (6.13 g, 30.01

mmol), then the resulting solution was stirred at 50 ℃ for 5 h under N2 atmosphere. After cooling to room temperature, the reaction mixture was poured into brine (100 mL) and extracted with dichloromethane (3×30 mL). The organic phases were combined, dried over MgSO4, filtered and concentrated to afford a red solid. The crude product was purified by flash chromatography with gradient elution (petroleum ether / ethyl acetate of 10/1 to 2/1, V/V) to obtain compound 4 as a red solid (7.10 g, 39.78 %).

1H NMR (600MHz, CDCl3) δ(ppm): 8.30(1H, d, J =16.2 Hz, -CH=CH-),

8.26(1H, d, J =9.0 Hz, ArH), 8.17(1H, d, J =2.4 Hz, ArH), 7.63(1H, d, J = 8.4 Hz,

4

ArH), 7.60(1H, d, J =12.6 Hz, -CH=CH-), 7.21-7.18(2H, m, ArH), 6.92(1H, t, J = 7.2

Hz, ArH), 6.74(1H, d, J = 7.8 Hz, ArH), 6.59(1H, d, J = 16.2 Hz, -CH=CH-), 5.55(1H,

d, J = 12.6 Hz, -CH=CH-), 4.13(2H, q, J = 7.2 Hz, CH2), 4.02(2H, t, J = 7.2 Hz, CH2),

2.69(2H, t, J = 7.2 Hz, CH2), 2.62-2.59(4H, m, CH2), 1.93-1.89(2H, m, CH2), 1.66(6H,

s, CH3), 1.53(6H, s, CH3), 1.21(3H, t, J = 7.2 Hz, CH3).13C NMR (150MHz, CDCl3) δ(ppm): 189.15, 189.10, 171.45, 159.93, 159.05,

147.73 145.08, 143.65, 139.67, 139.05, 128.44, 127.86, 125.24, 124.78, 122.49,

121.85, 120.67, 120.06, 117.16, 116.91, 106.66, 93.64, 61.02, 53.00, 46.18, 38.29,

31.23, 29.69, 28.23, 26.88, 26.32, 24.31, 14.10.

HRMS (ESI Positive): calc. for C35H38ClN3O4, [M+H]+ 600.2629, found

600.2623.

Synthesis of compound 5: To an ethanol (50 mL) solution of compound 4(3.60 g,

6.00 mmol), tin(II) chloride dihydrate (4.06 g, 15.00 mmol) was added, and then the

resulting mixture was stirred at room temperature for 12 h. After this, the solvent was

removed, then the residue was dispersed in ice and water mixture (150 mL) and

alkalized to pH=8 with 2 mol/L NaOH solution. The suspension was extracted with

dichloromethane (4×30 mL) and the combined organic phases were washed with

brine, dried over MgSO4, filtered and concentrated to obtain the crude product as a

red solid. Finally, the crude product was purified by flash chromatography with

gradient elution (petroleum ether / ethyl acetate of 5/1 to 1/1, V/V) to offer the title

compound as a red solid (2.67 g, 78.17%).1H NMR (400MHz, CDCl3) δ(ppm): 7.97(1H, d, J =16.4 Hz, -CH=CH-),

7.50(1H, d, J =12.4 Hz, -CH=CH-), 7.41(1H, d, J =7.6 Hz, ArH), 7.19(1H, t, J =8.4

Hz, ArH, 1H, d, J =16.8 Hz, -CH=CH-), 6.90(1H, t, J =7.6 Hz, ArH), 6.71(1H, d, J

=7.6 Hz, ArH), 6.66(1H, s, ArH), 6.63(1H, d, J =8.8 Hz, ArH), 5.53(1H, d, J =12.4

Hz, -CH=CH-), 4.14(2H, q, J = 7.2 Hz, CH2), 4.01(2H, t, J = 7.2 Hz, CH2), 3.77(12H,

s, NH2), 2.69(2H, t, J = 7.2 Hz, CH2), 2.61-2.58(4H, m, CH2), 1.91-1.88(2H, m, CH2),

1.67(6H, s, CH3), 1.48(6H, s, CH3), 1.23(3H, t, J = 7.2 Hz, CH3).13C NMR (150MHz, CDCl3) δ(ppm): 180.48, 171.59, 157.41, 149.33, 146.66,

144,93, 143.90, 139.00, 136.33, 134.90, 126.98, 127.76, 125.34, 125.57, 121.80,

121.15, 120.98, 120.20, 114.26, 108.25, 106.33, 93.41, 60.97, 52.21, 45.90, 38.21,

31.21, 28.17, 26.87, 26.43, 25.00, 21.53, 14.10


5

570.2886.

Synthesis of compound 6: To a chilled dichloromethane solution (25 mL) of

compound 5 (2.63 g, 4.61 mmol) and TEA (1.8 mL, 13.0 mmol) was dropwise added

the solution of chloroacethyl chloride (450 μL, 5.98 mmol) in dichloromethane (10

mL), and the resulting mixture was stirred under an ice bath for 5 h. Then the mixture

was poured into water (100 mL) and extracted with dichloromethane (3×30 mL). The

organic phases were combined and dried over MgSO4, concentrated to give the title

compound without further purification (2.68 g, 89.93%). 1H NMR (600MHz, CDCl3) δ(ppm): 8.25(1H, s, -NHCO-), 8.02(1H, d, J =16.2

Hz, -CH=CH-), 7.66(1H, d, J =1.8 Hz, ArH), 7.50(1H, d, J =8.4 Hz, ArH), 7.45(1H, d,

J =13.2 Hz, -CH=CH-), 7.25(1H, dd, J1= 1.8 Hz, J2= 7.8 Hz, ArH), 7.12-7.09(2H, m,

ArH), 6.82(1H, t, J =7.2 Hz, ArH), 6.64(1H, d, J =7.8 Hz, ArH), 6.55(1H, d, J =16.2

Hz, -CH=CH-), 5.45(1H, d, J =12.6 Hz, -CH=CH-), 4.15(2H, s, CH2), 4.05(2H, q, J

=7.2 Hz, CH2), 3.93(2H, t, J = 7.2 Hz, CH2), 2.60(2H, t, J = 7.2 Hz, CH2), 2.53-

2.51(4H, m, CH2), 1.84-1.79(2H, m, CH2), 1.58(6H, s, CH3), 1.43(6H, s, CH3),

1.14(3H, t, J = 7.2 Hz, CH3).

HRMS (ESI Positive): calc. for C37H41Cl2N3O3, [M+H]+ 646.2603, found

646.2605.

Synthesis of compound 8: To a solution of compound 6 (1.04 g, 1.61 mmol) in

dichloromethane (25 mL) was added TEA (1.0 mL, 7.2 mmol）) at room temperature

under N2 atmosphere. After stirring for 12 h, the mixture was poured into water (100

mL) and then extracted with dichloromethane (30 mL×3). The organic phases were

combined and dried over MgSO4, concentrated to give a purple red solid. The crude

product was purified by flash chromatography (dichloromethane / methanol of 5/1,

V/V) to obtain 1.01 g compound 8 as a purple red solid (88.60 % yield).1H NMR (400MHz, CDCl3) δ(ppm): 11.60(1H, s, NH-C=O), 8.11(1H, d, J =

16.4 Hz, -CH=CH-), 7.82(2H, d, J =6.4 Hz, ArH), 7.57-7.53(2H, m, ArH, -CH=CH-),

7.21(2H, t, J =8.0 Hz, ArH), 6.92(1H, t, J =7.2 Hz, ArH), 6.74(1H, d, J =8.0 Hz, ArH),

6.64(1H, d, J = 16.4 Hz, -CH=CH-), 5.55(1H, d, J = 12.4 Hz, -CH=CH-), 4.78(2H, s,

CH2), 4.16(2H, q, J = 7.2 Hz, CH2), 4.03(2H, t, J = 7.2 Hz, CH2), 3.70(6H, q, J = 7.2

Hz, CH2), 2.71(2H, t, J = 6.8 Hz, CH2), 2.63-2.61(4H, m, CH2), 1.95-1.90(2H, m,

CH2), 1.68(6H, s, CH3), 1.54-1.51(15H, s, CH3), 1.25(3H, t, J = 6.8 Hz, CH3).

6

13C NMR (150MHz, CDCl3) δ(ppm): 183.71, 171.49, 160.93, 158.14, 150.77,

147.71, 143.78, 139.03, 137.87, 137.01, 135.33, 128.67, 127.78, 127.01, 125.47,

121.83, 120.42, 120.23, 120.12, 119.54, 113.60, 106.48, 93.51, 60.98, 57.65, 55.21,

52.73, 46.15, 46.04, 31.22, 28.19, 26.86, 26.37, 24.70, 21.45, 14.09, 8.43.

HRMS (ESI Positive): calc. for C43H56ClN4O3+, [M]+ (m/z) 711.4041, [M+H]2+

(m/z) 356.2060, found 711.4037, 356.2058.

Compound 7 was obtained by the similar procedure in 79.86 % yield.1H NMR (400MHz, CDCl3) δ(ppm): 9.80(1H, s, -NHCO-), 8.10(1H, d, J = 16.4

Hz, -CH=CH-),7.97(1H, s, ArH), 7.56-7.51(2H, m, ArH, -CH=CH-), 7.35(1H, d, J

=8.0 Hz, ArH), 7.21(2H, t, J =8.0 Hz, ArH), 6.92(1H, t, J =7.2 Hz, ArH), 7.43(1H, d,

J =7.6 Hz, ArH), 6.64(1H, d, J =16.4 Hz, -CH=CH-), 5.54(1H, d, J =12.4 Hz, -

CH=CH-), 4.15(2H, q, J = 7.2 Hz, CH2), 4.02(2H, t, J = 7.2 Hz, CH2), 3.79(4H, t, J =

4.8 Hz, CH2), 3.44(2H, s, CH2), 2.89(4H, t, J = 4.8 Hz, CH2), 2.70(2H, t, J = 7.2 Hz,

CH2), 2.62-2.60(4H, m, CH2), 1.93-1.87(2H, m, CH2), 1.68(6H, s, CH3), 1.51(6H, s,

CH3), 1.24(3H, t, J = 7.2 Hz, CH3).13C NMR (150MHz, CDCl3) δ(ppm): 183.40, 171.55, 169.68, 157.98, 149.98,

147.97, 143.83, 139.05, 136.67, 136.18, 136.13, 128.67, 127.78, 126.71, 125.47,

121.83, 120.36, 120.28, 119.70, 119.13, 112.91, 106.42, 93.46, 60.99, 59.86, 58.46,

56.89, 52.72, 46.01, 31.22, 29.70, 28.20, 26.88, 26.39, 24.68, 18.44, 14.10.


715.3925.

Synthesis of compound 9: To an ethanol solution (25 mL) of intermediate 8 (1.82

g, 2.55 mmol), 2 mol/L NaOH solution (17.5 mL) was added, and then the resulting

mixture was stirred at 50 ℃ for 3 h under N2 atmosphere. After cooling to room

temperature, the solvent was removed, then the residue was dissolved in water (50 mL)

and acidified to pH=6 with 1 mol/L HCl solution. The precipitation was collected by

filtration and washed with water (30 mL×3), then purified by flash chromatography

with gradient elution (petroleum ether/ethyl acetate of 3:1 to methanol) to afford

compound 9 as a blue solid (1.46 g, 83.91 %).1H NMR (400MHz, MeOD) δ(ppm): 8.14(1H, d, J = 16.0 Hz, -CH=CH-),

7.78(1H, s, ArH), 7.56(1H, d, J = 12.4 Hz, -CH=CH-), 7.45(2H, s, ArH), 7.20-

7.15(2H, m, ArH), 6.90-6.85(2H, m, ArH), 6.55(1H, d, J = 16.0 Hz, -CH=CH-),

7

5.67(1H, d, J = 14.8 Hz, -CH=CH-), 4.94(2H, s, CH2), 4.00(2H, t, J = 7.2 Hz, CH2),

3.63(6H, q, J = 7.2 Hz, CH2), 2.55-2.51(6H, m, CH2), 1.82(2H, t, J = 7.2 Hz, CH2),

1.62(6H, s, CH3), 1.45(6H, s, CH3), 1.38(9H, t, J = 7.2 Hz, CH3).

HRMS (ESI Positive): calc. for C41H52ClN4O3+, [M]+ (m/z) 683.3728, [M+H]2+

(m/z) 342.1903, found 683.3780, 342.1918 .

Synthesis of compound 10 (TM): The mixture of EDC·HCl (0.29 g, 1.62 mmol)

and NHS (0.21 g, 1.82 mmol) was added to a solution of compound 9 (1.03 g, 1.51

mmol) in anhydrous DMF (15 mL) on an ice bath under N2 atmosphere. After stirring

for 1 h, the mixture was allowed to warm up to room temperature and continually

stirred for 12 h in the dark. After this, N,N-diisopropylethylamine (DIPEA) (820 μL,

4.70 mmol) and D-glucosamine hydrochloride (0.39 g, 1.81 mmol) were added. The

resulting mixture was stirred at room temperature for an additional 5 h under N2

atmosphere, and then poured into acetone (50 mL). The precipitation was collected by

filtration and purified by flash chromatography (CH2Cl2/Methanol of 10:1) to obtain

the title compound as a red solid (0.73 g, 57.03 %).

1H NMR (600MHz, MeOD) δ(ppm)：8.14(1H, d, J =15.6 Hz, -CH=CH-),

7.77(1H, d, J =28.8 Hz, -CH=CH-), 7.60-7.55(2H, m, ArH), 7.45(1H, dd, J1= 2.4 Hz,

J2= 8.4 Hz, ArH), 7.19-7.14(2H, m, ArH), 6.87(1H, t, J =7.2 Hz, ArH), 6.77(1H, d, J

=8.4 Hz, ArH), 6.56(1H, d, J =15.0 Hz, -CH=CH-), 5.59(1H, d, J =10.8 Hz, -

CH=CH-), 5.32(1H, d, J =3.0 Hz, -NH-), 4.05(6H, q, J = 7.2 Hz, CH2), 3.84-3.80(2H,

m, CH2), 3.79-3.76(2H, m, CH2), 3.54(1H, d, J = 16.8 Hz, CH), 3.44(1H, t, J = 9.6 Hz,

CH), 3.33-3.32(1H, m, CH), 2.66(2H, t, J = 6.6 Hz, CH2), 2.62-2.58(4H, m, CH2),

1.85(2H, t, J = 5.4 Hz, CH2), 1.60(6H, s, CH3), 1.45(6H, s, CH3), 1.15(9H, t, J = 7.2

Hz, CH3).

HRMS (ESI Positive): calc. for C47H63ClN5O7+, [M+H]+ 845.4494, found

845.4487.

Measurement of absorbance and fluorescence spectroscopy

Absorption spectra were recorded in a quartz cuvette (1 cm×1 cm) under ambient

atmosphere at room temperature. Before the measurement, a stock solution of probe

(1×10-3 M) was prepared by dissolving the required amount of 10 (0.0085 g) in

ethanol and diluting it to the mark in a 10 mL volumetric flask. Further dilution

yielded the working solution (1×10-5 M) by diluting 2.5 mL of stock solution to 250

8

mL with ethanol-water (2:8, V/V). For investigating the optical response of this probe

towards pH, the accurate pH of the working solution was adjusted by 0.1 M NaOH

and 0.1 M HCl (from 2.53 to 11.70), and then the absorption spectra at different pH

value were recorded in the wavelength range of 300~1000 nm. Fluorescence spectra

were collected in a quartz cuvette (1 cm×1 cm) at a 90-degree angle relative to the

excitation light path. Excitation and emission slits were both 8 nm and the excitation

wavelength was 760 nm. The Absorption spectra and fluorescence spectra of control

molecule (compounds 7 and 8, 1×10-5 M) upon pH were evaluated in the similar

method.

The biologically relevant analytes, including metal ions (Na+, K+, Mg2+, Zn2+,

Ca2+) made from their chloride salts, thiols (GSH, Cys, Hcy) and H2O2 were dissolved

in deionized water to obtain the stock solutions of the interferents (1×10-3 M). In

selectivity experiments, working solution (9 mL) was added to a 10 mL volumetric

flask, to which stock solutions of interferents (100 μL) were respectively added using

a micropipettor, and then it was diluted to the mark with deionized water.

Measurement of absolute quantum yield

The absolute fluorescence quantum yield (ɸf) of solution of compound 10

(excitation at 760 nm) at room temperature was determined on a PTI QM/TM/NIR

system (Photon Technology International, Birmingham, NJ, USA) with an integration

sphere attachment (Labsphere, North Sutton, NH, USA). A circular quartz cuvette for

the working solution of sample is set in the integrating sphere. The absolute

photoluminescence QY is given by:

ɸ𝑓=𝑃𝑁(𝑒𝑚)𝑃𝑁(𝑎𝑏𝑠)

=∫𝐿𝑠𝑎𝑚𝑝𝑙𝑒

∫𝐸𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 -∫𝐸𝑠𝑎𝑚𝑝𝑙𝑒

Where PN(abs)and PN(em) are the number of photons absorbed by the sample and

photons emitted from the sample, respectively. Lsample is the fluorescence intensity of

the working solution of sample, Ereference and Esample are the excitation light intensities

of the reference and the sample, respectively. Ethanol-water (2/8, V/V) solution was

employed as reference.

In vitro cytotoxicity experiments[2]

HeLa cells were used for in vitro cytotoxicity evaluation of the probe (10) based

on the MTT assay. HeLa cells were seeded in a 96-well plate at a density of 5×104

cells/well in 100 μL DMEM with 10% FBS at 37 ℃ under 5% CO2 humidified

9

atmosphere. After 24 h incubation, the original medium was replaced with fresh

culture medium containing different concentrations of 10 (0.5, 1, 5 and 10 equiv. of

testing concentration (1×10-5M)) and then incubation for another 24 h (each

concentration was done in triplicate). Afterwards, the medium in each well was then

removed and washed with PBS buffer three times. Then solution of MTT (30 μL, 5

mg/mL in PBS buffer) and PBS buffer (1 mL) were added to each well followed by

incubation for additional 4 h under the same condition. Subsequently, the medium in

each well was removed and washed twice with PBS buffer, then 2 mL DMSO was

added to dissolve the internalized purple formazan crystals. The plate was subjected

to gently agitation for 30 min in order to dissolve all the crystals. The absorbance at

wavelength of 570 nm was recorded on a microplate reader (Thermo Fisher). The

average absorbance of the blank wells without cells was subtracted from the readings

of the other wells. And then the cell viability was determined by the following

equation: viability (%) = Ax/Acontrol×100, (Ax is the average absorbance of the date

with a certain concentration of 10, Acontrol is the average absorbance of the control

wells in which the probe was absent.).

pH-dependent cell imaging and subcellular localization experiments[3]

HeLa cells were seeded in a 6-well plate at a density of 5×105 cells/well and

incubated for 24 h for cell attachment. Afterwards, the original medium was removed

and the cells were further incubated with 1×10-5 M of 10 in culture media for

additional 2 h at 37 ℃ under 5% CO2 humidified atmosphere. Then the culture

medium was removed and washed twice with PBS buffer. For the pH-dependent cell

imaging study, the above cells were treated with PBS buffer at various pH values

from 4.0 to 7.0, and then confocal fluorescence imaging was acquired on Leica TCS

SP8 confocal laser-scanning microscope with a 40×oil-immersion object lens.

Fluorescence was excited at 633 nm and emission was collected by a 700~800 nm

band pass filter.

Next, HeLa cells were incubated on another 6-well plate for subcellular

localization study. After incubation for 24 h, 10 (1×10-5 M) was added to one of the

well, and compounds 7 and 8 (1×10-5 M) were respectively added into another well as

control. The culture medium was removed and washed twice with PBS buffer after

incubated for 2 h, and then fresh culture medium containing rhodamine123 (1×10-6 M)

was added to these wells and incubated for another 1 h. Afterwards, the medium was

10

removed and washed 3 times, and then the cells were treated with PBS buffer at pH 5

in order to activate the fluorescence of 10, 8 and 7. Subsequently, fluorescence images

of probe (10, 8 and 7) and rhodamine123 were measured.

In vivo imaging experiments[4]

Male nude mice were purchased from Tianjin Medical University Cancer Institute

and Hospital at 4 weeks of ages. All animal experiments were conducted according to

protocols approved by the Animal Ethics Committee of the Institute of Hematology

＆ Hospital of Blood Diseases, Chinese Academy of Medical Sciences ＆ Peking

Union Medical College. For in vivo imaging, HeLa cells (5×106 cells suspended in

100 μL PBS) were inoculated into the right hind limb of the nude mice after

anesthetization by injection 4% chloral hydrate (150 μL). After inoculation for two

weeks, the mice were chosen for in vivo imaging experiments with the tumor

diameters reached approximately 0.5 cm. With the HeLa tumor-bearing nude mice on

the hand, 10 (1×10-5 M in PBS containing 1% DMSO, 200 μL) was injected in suit

into the tumor-bearing nude mice and subcutaneously injected into the homologous

normal tissus (left hind limb) with the same amount as a control.The fluorescence

images of the tumor-bearing nude mice were performed at the special time points

(before injection, 0.5 h, 1 h, 4 h, 8 h, 12 h and 24 h after injection) with a Bethold

NightOWL LB 983 in vivo Imaging System equipped with the CCD camera. The

excitation filter and emission filter were set as 630 nm and 800 nm respectively, the

constant time was 0.1 s.

300 400 500 600 700 800 900 1000 1100

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Abso

rban

ce

Wavelength (nm)

2.13 2.88 3.08 3.43 3.83 4.26 4.77 5.04 5.54 6.06 6.30 6.50 7.04 7.36 8.85 10.56 11.70

(A)

780 790 800 810 820 830 840 8500

5000000

10000000

15000000

20000000

25000000

30000000

35000000

Fluo

resc

ence

Inten

sity

Wavelength (nm)

2.57 3.09 3.38 3.64 4.17 4.78 5.14 5.41 5.96 6.23 6.50 7.05 7.42 8.95 10.06 11.52

(B)

Fig.S1 UV absorption spectra (A) and fluorescence emission spectra (B) of compound 7 (1×10-5 M) upon pH in ethanol-water (2/8, V/V) solution

11

300 400 500 600 700 800 900 1000 1100

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4Ab

sorb

ance

Wavelength (nm)

2.56 3.22 3.54 4.01 4.56 5.10 5.61 5.82 6.01 6.32 6.63 7.40 8.93 10.31 11.70

(A)

780 790 800 810 820 830 840 850

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

Fluo

resc

ence

Inte

nsity

Wavelength (nm)

2.64 3.01 3.64 3.95 4.34 4.84 5.14 5.36 5.95 6.26 6.63 6.91 7.39 9.01 10.25 11.47

(B)

Fig.S2 UV absorption spectra (A) and fluorescence emission spectra (B) of compound 8 (1×10-5 M) upon pH in ethanol-water (2/8, V/V) solution

135 140 145 150 155 160 165 1700

500

1000

1500

2000

2500

3000

3500

4000

Inten

sity

(a.u

.)

Time (ns)

pH-3 pH=4 pH=5 pH=6 IRF

Fig.S3 The decayed curves of compound 10 at different pH values

probe Na+ K+ Mg2+ Zn2+ Ca2+ H2O2 GSH Lys Hcy0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

Fluo

resc

ence

inten

sity

Fig.S4 The fluorescent intensity at 814 nm of 10 at pH = 4.01 in ethanol-water (2/8, V/V) solution upon addition of different interferents

12

Fig.S5 Effect of probe 10 on the viability of HeLa cells

References[1] X. Zhao, R. S. Wei, L. G. Chen, D. Jin and X. L. Yan, New J. Chem., 2014, 38,

4791-4798.

[2] X. J. Jiang, P. C. Lo, S. L. Yeung, W. P. Fong and D. K. P. Ng, Chem. Commun.,

2010, 46, 3188-3190; K. H. Xu, F. Wang, X. H. Pan, R. P. Liu, J. Ma, F. P. Kong

and B. Tang, Chem. Commun., 2013, 49, 2554-2556; M. H. Zan, J. J. Li, S. Z.

Luo and Z. S. Ge, Chem. Commun., 2014, 50, 7824-7827.

[3] M. H. Lee, N. Park, C. Yi, J. H. Han, J. H. Hong, K. P. Kim, D. H. Kang, J. L.

Sessler, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2014, 136, 14136-14142, T.

Myochin, K. Kiyose, K. Hanaoka, H. Kojima, T. Terai and T. Nagano, J. Am.

Chem. Soc., 2011, 133, 3401-3409.

[4] Y. Wang and X. P. Yan, Chem. Commun., 2013,49, 3324-3326; A. Abdukayum, J.

T. Chen, Q. Zhao and X. P. Yan, J. Am. Chem. Soc., 2013, 135, 14125-14133.

13

1H NMR and 13C NMR spectra

ppm (t1)0.01.02.03.04.05.06.07.08.09.0

8.31

68.

289

8.27

18.

256

8.17

38.

169

7.64

17.

627

7.61

47.

593

7.20

67.

193

7.18

77.

175

6.92

96.

917

6.90

4

6.74

26.

729

6.60

66.

579

5.55

85.

537

4.14

44.

132

4.12

04.

109

4.03

34.

021

4.00

9

2.69

92.

687

2.67

52.

620

2.60

92.

599

2.58

8

1.93

41.

920

1.91

01.

900

1.89

01.

657

1.52

61.

226

1.21

41.

202

1.001.020.94

1.020.88

1.93

0.970.900.97

0.89

2.611.81

2.064.03

2.275.525.85

3.00

Fig. S6 1H NMR spectrum of compound 4

ppm (t1)0255075100125150175200

189.

147

189.

097

171.

448

159.

934

159.

047

147.

732

145.

084

143.

647

139.

666

139.

053

128.

437

127.

855

125.

242

124.

780

122.

493

121.

851

120.

674

120.

057

117.

156

116.

914

106.

662

93.6

40

61.0

19

52.9

97

46.1

8438

.290

31.2

33

29.6

86

28.2

32

26.8

7926

.322

24.3

08

14.0

96

Fig. S7 13C NMR spectrum of compound 4

NN

Cl

OO

NO2

NN

Cl

OO

NO2

14

ppm (t1)0.01.02.03.04.05.06.07.08.09.0

7.98

87.

947

7.51

27.

481

7.42

17.

401

7.21

27.

190

7.17

0

6.91

96.

901

6.88

2

6.72

36.

704

6.66

26.

642

6.62

05.

540

5.50

94.

169

4.15

14.

133

4.11

5

4.02

54.

007

3.99

03.

769

2.70

82.

690

2.67

2

2.61

02.

596

2.58

21.

908

1.89

41.

879

1.66

8

1.48

31.

250

1.23

21.

214

1.00

1.001.022.05

1.031.032.031.05

0.96

2.251.981.56

2.084.16

2.276.176.243.19

ppm (t1)6.506.757.007.257.507.758.00

7.98

87.

947

7.51

2

7.48

17.

421

7.40

17.

212

7.19

07.

170

6.91

96.

901

6.88

26.

723

6.70

46.

662

6.64

26.

620

1.00

1.001.02

2.05

1.03

1.032.031.05


ppm (t1)050100150200

180.

481

171.

586

157.

413

149.

325

146.

657

144.

928

143.

900

138.

994

136.

333

134.

898

128.

984

127.

759

125.

836

125.

572

121.

797

121.

147

120.

977

120.

204

114.

255

108.

248

106.

327

93.4

09

60.9

74

52.2

0545

.896

38.2

1131

.207

28.1

70

26.8

6526

.425

25.0

03

21.5

31

14.1

02


NN

Cl

OO

NH2

NN

Cl

OO

NH2

15

ppm (t1)-2.0-1.00.01.02.03.04.05.06.07.08.09.0

8.25

08.

036

8.00

97.

665

7.66

27.

504

7.49

07.

463

7.44

1

7.25

77.

254

7.24

47.

240

7.12

17.

108

7.10

17.

096

7.08

96.

835

6.82

36.

811

6.64

16.

628

6.56

56.

538

5.45

55.

434

4.14

6

4.07

04.

058

4.04

64.

034

3.93

73.

925

3.91

32.

614

2.60

22.

590

2.53

42.

525

2.51

52.

505

1.83

51.

825

1.81

51.

805

1.79

41.

579

1.42

6

1.15

41.

142

1.13

0

1.000.94

0.991.041.051.062.12

1.091.011.08

0.97

2.192.042.83

2.094.23

2.28

6.216.22

3.26


ppm (t1)-1.00.01.02.03.04.05.06.07.08.09.010.0

9.79

88.

118

8.07

77.

974

7.55

77.

527

7.50

67.

362

7.34

17.

337

7.22

87.

208

7.18

96.

940

6.92

26.

904

6.74

36.

724

6.65

56.

614

5.55

75.

526

4.18

14.

163

4.14

54.

127

4.04

24.

024

4.00

6

3.80

43.

792

3.78

03.

441

2.89

62.

885

2.87

22.

721

2.70

32.

685

2.62

32.

611

2.59

91.

927

1.91

21.

904

1.89

81.

885

1.88

21.

872

1.68

01.

506

1.26

11.

243

1.22

5

1.000.98

2.041.131.81

1.12

1.07

0.64

2.141.833.89

3.992.213.65

2.09

1.82

3.36

0.850.98

5.695.36


NN

Cl

OO

HNO

Cl

NN

Cl

OO

HNO

N

OH

OH

16

ppm (t1)0102030405060708090100110120130140150160170180190200

183.

403

171.

551

169.

678

157.

977

149.

977

147.

968

143.

825

139.

048

136.

671

136.

183

136.

125

128.

671

127.

776

126.

712

125.

470

121.

826

120.

359

120.

284

119.

697

119.

134

112.

906

106.

422

93.4

58

60.9

92

59.8

61

58.4

61

56.8

9452

.719

46.0

0531

.215

29.6

99

28.1

98

26.8

7726

.392

24.6

8218

.436

14.0

98


ppm (t1)-1.00.01.02.03.04.05.06.07.08.09.010.011.012.0

11.6

02

8.12

88.

087

7.82

37.

807

7.57

07.

562

7.54

87.

529

7.23

07.

210

7.19

16.

941

6.92

36.

905

6.74

56.

725

6.66

06.

619

5.56

05.

529

4.78

44.

184

4.16

64.

148

4.13

0

4.04

54.

027

4.01

03.

721

3.70

43.

686

3.66

82.

723

2.70

62.

689

2.62

72.

615

1.94

81.

945

1.92

81.

915

1.90

1

1.68

31.

544

1.52

71.

511

1.26

41.

246

1.22

9

1.001.041.192.16

2.072.131.13

0.97

0.88

2.00

2.272.05

6.43

2.294.08

2.396.09

3.2515.17


NN

Cl

OO

HNO

N

OH

OH

NN

Cl

OO

HNO

N

17

ppm (t1)0102030405060708090100110120130140150160170180190200

183.

712

171.

493

160.

934

158.

144

150.

769

147.

709

143.

779

139.

033

137.

873

137.

007

135.

332

128.

665

127.

781

127.

008

125.

465

121.

828

120.

423

120.

232

120.

124

119.

543

113.

596

106.

480

93.5

14

60.9

80

57.6

4855

.209

52.7

34

46.1

4546

.036

31.2

22

28.1

89

26.8

6426

.369

24.6

98

21.4

4814

.093

8.42

9


ppm (t1)-1.00.01.02.03.04.05.06.07.08.09.010.0

8.16

48.

124

7.78

37.

572

7.54

1

7.45

1

7.19

77.

179

7.16

67.

147

6.89

76.

886

6.87

86.

868

6.84

9

6.56

96.

529

5.68

25.

650

4.94

44.

022

4.00

43.

985

3.65

23.

635

3.61

73.

599

2.55

32.

535

2.51

41.

817

1.61

5

1.45

11.

392

1.37

51.

357

1.00

1.021.032.03

2.15

2.07

1.00

0.96

2.01

6.19

2.066.166.17

6.97

9.99

2.25


NN

Cl

OO

HNO

N

NN

Cl

OHO

HNO

N

18


NN

Cl

HNO

N

OOH

HO

HN OHOHO

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