Host–guest interaction enhanced aggregation-induced emission and its application in

cell imaging

Jiong Zhou, Bin Hua, Li Shao, Hao Feng and Guocan Yu*

Department of Chemistry, Zhejiang University, Hangzhou 310027, P. R. China.

Fax and Tel: +86-571-8795-3189 Email address: guocanyu@zju.edu.cn

Electronic Supplementary Information (12 pages)

1. Materials and methods S2

2. Syntheses of compounds H, G and G′ S4

3. Investigation of the interactions between model compound G′ and H S10

4. Fluorescence spectra of G in THF/water mixtures with different fractions of water S11

5. Tyndall effect of a solution containing HG S12

References S12

S1

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2016

1. Materials and methods

All reagents were commercially available and used as supplied without further purification. Solvents

were either employed as purchased or dried according to procedures described in the literature.

Compounds 1S1 and 2S2 were synthesized according to published literature procedures. NMR spectra were

recorded with a Bruker Avance DMX 400 spectrophotometer, a Bruker Avance DMX 500

spectrophotometer, or a Bruker Avance DMX 600 spectrophotometer with the deuterated solvent as the

lock and the residual solvent or TMS as the internal reference. Low-resolution electrospray ionization

mass spectra (LRESI-MS) were obtained on a Bruker Esquire 3000 Plus spectrometer (Bruker-Franzen

Analytik GmbH Bremen, Germany) equipped with an ESI interface and an ion trap analyzer. High-

resolution electrospray ionization mass spectra (HRESI-MS) were obtained on a Bruker 7-Tesla FT-ICR

mass spectrometer equipped with an electrospray source (Billerica, MA, USA). The melting points were

collected on a SHPSIC WRS-2 automatic melting point apparatus. The fluorescence experiments were

conducted on a RF-5301 spectrofluorophotometer (Shimadzu Corporation, Japan).

Fabrications of NPs. The NPs were obtained through reprecipitation method. Briefly, H (8.16 mg) and 1

equiv. of G (5.18 mg) were dissolved in acetone (10 mL). A 50 μL acetone solution of the host−guest

system was injected into a certain amount of water with controlled stirring for 12 h to afford the

corresponding NPs.

Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) Studies. The

nanostructures of NPs were revealed using TEM. TEM samples were prepared by drop-coating the

solution onto a carbon-coated copper grid. TEM experiments were performed on an HT-7700

instrument. The corresponding solution was left to stand overnight and the insoluble precipitate was

eliminated by using a microporous membrane before DLS tests. Dynamic light scattering (DLS)

measurements were carried out using a 200 mW polarized laser source Nd:YAG (λ = 532 nm). The

polarized scattered light was collected at 90° in a self-beating mode with a Hamamatsu R942/02

photomultiplier. The signals were sent to a Malvern 4700 submicrometer particle analyzer system.

Cell Culture. HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing

10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells grew as a monolayer and were

detached upon confluence using trypsin (0.5% w/v in PBS). The cells were harvested from cell

culture medium by incubating in trypsin solution for 5 min. The cells were centrifuged, and the

supernatant was discarded. A 3 mL portion of serum-supplemented DMEM was added to neutralize

any residual trypsin. The cells were resuspended in serum-supplemented DMEM at a concentration of

S2

1 × 104 cells/mL. Cells were cultured at 37 °C and 5% CO2.

Evaluation of Cytotoxicity. The cytotoxicity of the NPs against HeLa cells was determined by 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays in a 96-well cell culture plate. All

solutions were sterilized by filtration with a 0.22 μm filter before tests. HeLa cells were seeded at a

density of 1 × 104 cells/well in a 96-well plate, and incubated for 24 h for attachment. Cells were then

incubated with NP at various concentrations for 4 h and 24 h. After washing the cells with PBS buffer, 20

μL of a MTT solution (5 mg/mL) was added to each well. After 4 h of incubation at 37 °C, the MTT

solution was removed, and the insoluble formazan crystals that formed were dissolved in 100 μL of

dimethylsulfoxide (DMSO). The absorbance of the formazan product was measured at 570 nm using a

spectrophotometer (Bio-Rad Model 680). Untreated cells in media were used as a control. All

experiments were carried out with five replicates.

In Vitro Cell Accumulation of the NPs Determined by Confocal Laser Scanning Microscopy

(CLSM). HeLa cells were treated with the NPs (the concentration of the NPs was kept at 5 μM) in the

culture medium at 37 °C for 4 h. The cells were washed three times with PBS and fixed with fresh 4.0%

formaldehyde at room temperature for 15 min. After washing with PBS, the cells were stained with DAPI

(1 μg/mL) for 15 min. The images were taken using a LSM-510 confocal laser scanning microscope

(CLSM, ZEISS LSM780).

S3

2. Syntheses of compounds H, G and G′

2.1. Synthesis of compound H

NaOH

1 H

H

H

O

O 4 O

O

O

H

H

H

H

O

O 4 O

O

HOO

H

H

O Na

Synthesis of H: 1 (300 mg, 0.380 mmol) and NaOH (152 mg, 3.80 mmol) were added to H2O (25

mL) and stirred at room temperature for 12 h. Water was removed under reduced pressure to give H as a

white solid (310 mg, 100%). Mp 165.2−166.5 °C. The proton NMR spectrum of H is shown in Fig. S1. 1H NMR (600 MHz, acetone-d6/D2O = 5:3, 293 K) δ (ppm): 7.17 (s, 1H), 6.87–6.85 (m, 9H), 4.25 (s, 2H),

3.87 (s, 2H), 3.76–3.71 (m, 35H). The 13C NMR spectrum of H is shown in Fig. S2. 13C NMR (100 MHz,

chloroform-d, 293 K) δ (ppm): 152.16, 151.08, 150.90, 150.75, 150.47, 148.68, 128.66, 128.38, 128.18,

127.67, 114.59, 114.04, 113.78, 55.84, 55.25, 52.99, 29.63, 29.37. LRESIMS is shown in Fig. S3: m/z

793.3 [M − Na]−. HRESIMS: m/z calcd for [M − Na]− C46H49O12−, 793.3224, found 793.3236, error −2

ppm.

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)

35.0

72.

00

2.01

9.07

1.00

2.18

3.87

4.25

4.45

5.56

6.85

6.87

7.17

Fig. S1 1H NMR spectrum (600 MHz, acetone-d6/D2O = 5:3, 293K) of H.

S4

0102030405060708090100110120130140150160f1 (ppm)

29.3

729

.63

52.9

955

.25

55.8

4

76.7

277

.04

77.3

5

113.

7811

4.04

114.

59

127.

6712

8.18

128.

3812

8.66

148.

6815

0.47

150.

7515

0.90

151.

0815

2.16

Fig. S2 13C NMR spectrum (100 MHz, chloroform-d, 293K) of H.

Fig. S3 Electrospray ionization mass spectrum of H. Assignment of the main peak: m/z 793.3 [M Na] .

S5

2.2. Synthesis of compound G

N

+ BrToluene

Reflux

NBr

2 GSynthesis of G: Bromoethane (5.24 g, 49.0 mmol) was added to a solution of 2 (0.200 g, 0.490

mmol) in toluene (30 mL). The mixture was heated under nitrogen at reflux for 24 h. The cooled reaction

mixture was evaporated under vacuum to yield G as a yellow solid (0.250 g, 100%). Mp 181.7−183.7 °C.

The proton NMR spectrum of G is shown in Fig. S4. 1H NMR (400 MHz, chloroform-d, 293K) δ (ppm):

9.33 (d, J = 8 Hz, 2H), 8.12 (d, J = 8 Hz, 2H), 7.53 (d, J = 8 Hz, 2H), 7.23–7.03 (m, 17H), 5.01–4.95 (m,

2H), 1.73 (t, J = 8 Hz, 3H). The 13C NMR spectrum of G is shown in Fig. S5. 13C NMR (100 MHz,

chloroform-d, 293K) δ (ppm): 155.64, 148.81, 142.86, 139.24, 132.87, 131.03, 127.76, 127.18, 126.97,

124.44, 56.30, 17.16. LRESIMS is shown in Fig. S6: m/z 438.3 [M − Br]+. HRESIMS: m/z calcd for [M −

Br]+ C33H28N+, 438.2222, found 438.2207, error 3 ppm.

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)

3.04

2.06

17.0

6

2.08

2.00

1.99

1.61

1.72

1.73

1.75

4.96

4.97

4.99

5.01

5.30

7.02

7.03

7.04

7.05

7.06

7.26

7.52

7.54

8.11

8.13

9.32

9.34

Fig. S4 1H NMR spectrum (400 MHz, chloroform-d, 293K) of G.

S6

0102030405060708090100110120130140150160170f1 (ppm)

17.1

6

56.3

0

76.7

577

.07

77.3

9

124.

4412

6.97

127.

1812

7.76

131.

0313

2.87

139.

2414

2.86

148.

81

155.

64

Fig. S5 13C NMR spectrum (100 MHz, chloroform-d, 293K) of G.

Fig. S6 Electrospray ionization mass spectrum of G. Assignment of the main peak: m/z 438.3 [M Br] +.

S7

2.3. Synthesis of model compound G′

N

Br+CH3CN

Reflux

N Br

G'Synthesis of G′: Bromoethane (4.28 g, 40.0 mmol) was added to a solution of 4-phenylpyridine

(0.310 g, 2.00 mmol) in acetonitrile (30 mL). The mixture was heated under nitrogen at reflux for 24 h.

The cooled reaction mixture was evaporated under vacuum to yield G′ as a liquid. The proton NMR

spectrum of G′ is shown in Fig. S7. 1H NMR (400 MHz, D2O, 293 K ) δ (ppm): 8.73 (d, J = 8 Hz, 2H),

8.09 (d, J = 8 Hz, 2H), 7.78 (d, J = 8 Hz, 2H), 7.62 (t, J = 8 Hz, 1H), 7.55 (t, J = 8 Hz, 2H), 4.64–4.58 (m,

2H), 1.69 (t, J = 8 Hz, 3H). The 13C NMR spectrum of G′ is shown in Fig. S8. 13C NMR (100 MHz, D2O,

293 K) δ (ppm): 155.34, 143.49, 133.09, 132.10, 129.58, 127.70, 124.47, 56.29, 15.64. LRESIMS is

shown in Fig. S9: m/z 184.1 [M − Br]+. HRESIMS: m/z calcd for [M − Br]+ C33H28N+, 184.1126, found

184.1122, error 2 ppm.

Fig. S7 1H NMR spectrum (400 MHz, D2O, 293K) of G′.

S8

0102030405060708090100110120130140150160f1 (ppm)

15.6

4

56.2

9

124.

4712

7.70

129.

5813

2.10

133.

09

143.

49

155.

34

Fig. S8 13C NMR spectrum (400 MHz, D2O, 293K) of G′.

5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

+ESI Scan (0.3-0.3 min, 2 scans) Frag=100.0V ZJ-36_1.d Subtract

184.1

Counts vs. Mass-to-Charge (m/z)20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

Fig. S9 Electrospray ionization mass spectrum of G′. Assignment of the main peak: m/z 184.1 [M Br] +.

S9

3. Investigation of the interactions between model compound G′ and H

H

H

O

O 4 O

O

O

H

H

O Na

Ha1Ha

Hb Hb1

Hc Hc1 NBr

H1H2H3H4

H5 H6

H7

H G'

Fig. S10 NOESY NMR spectrum (500 MHz, acetone-d6/D2O = 5:3, 293 K) of H (10.0 mM) and G′ (10.0 mM).

S10

Fig. S11 Partial NOESY NMR spectrum (500 MHz, acetone-d6/D2O = 5:3, 293 K) of H (10.0 mM) and G′ (10.0

mM).

4. Fluorescence spectra of G in THF/water mixtures with different water fractions

400 450 500 550 6000

200

400

600

800

Water fraction (%)

FL in

tens

ity

Wavelength/nm

0 1 2 5 7 10 20 30 40 50 60 70 80 90 100

Fig. S12 Fluorescence spectra of G in THF and THF/water mixtures with different water fractions. The

concentration of G was 1.00 × 10–4 M; excitation wavelength = 350 nm.

S11

5. Tyndall effect of a solution containing HG

Fig. S13 Tyndall effect of a solution containing HG

References:S1. B. Xia and M. Xue, Chem. Commun., 2014, 50, 10211023.

S2. (a) M. Banerjee, S. J. Emond, S. V. Lindeman and R. Rathore, J. Org. Chem., 2007, 72, 8054–8061; (b) M.

Wang, Y.-R. Zheng, K. Ghosh and P. J. Stang, J. Am. Chem. Soc., 2010, 132, 62826283.

S12