Supporting Information - rsc.org of Emission intensity of L 1 ... with Hg2+ in MCF7 cells 26 ......

Saha et. al., ChemComm. DOI: 10.1039/c2cc34490d [Supporting Information]

1

Supporting Information

Sukdeb Saha,a Prasenjit Mahato,a Mithu Baidya,b Sudip K. Ghosh,b* and Amitava Dasa*

aCSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat-364002, India.

bDepartment of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal-721302, India.

Contents Page No.

1. Synthetic methodology of L1 2

2. 1H NMR spectra of L1 3

3. 13C NMR spectra of L1 4

4. ESI-Ms spectra of L1 5

5. FTIR spectra of L1 6

6. Synthetic methodology for the receptor L2 7

7. 1H NMR spectra of L2 8

8. 13C NMR spectra of L2 9

9. ESI-Ms spectra of L2 10

10. 13C NMR of L1 in absence and in presence of Hg2+ 11

11. Beneshi-Hildebrand plot of absorbance titration between L1 and Hg2+ 12

12. Beneshi-Hildebrand plot of Emission titration between L1 and Hg2+ 13

13. Equation used for energy transfer efficiency calculation 14

14. ESI-Ms spectra of L1 in presence of Hg(ClO4)2 showing 1:1 binding 15

15. 1H NMR titration of L1 as a function of Hg(ClO4)2 16

16. Interference study of L1 with Hg2+ in presence of various metal ions 17

17. Uv-Vis study for establishing the reversible binding of Hg2+ to L1 18

18. Change of Emission intensity of L1 as a function of pH 19

19. FTIR Spectra of L1 in presence of 5 equivalent of Hg(ClO4)2 20

20. Bar diagram for changes in the absorbance intensity for L1 with Metal ions 21

21. Bar diagram for changes in the Emission intensity for L1 with Metal ions 22

22. Job’s plot for L1 with Hg2+ showing 1:1 stoichiometry 23

23. Absorption spectra of L1 in presence of Hg2+ at different pH 24

24. Emission spectra of L1 in presence of Hg2+ at different pH 25

25. Confocal microscopic images of L1 with Hg2+ in MCF7 cells 26

26. Fluorescence microscopic images of L1 with Hg2+ 27

27. Reversibility studies for binding of the L1.Hg2+with KI with in the cells 28

28. MTT assay studies for evolution of cytotoxicity of the reagent L1 29

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2012


2

Synthetic methodology for L1:

Amino ethyl rhodamine (L) (200 mg, 0.413 mmole) was dissolved in 20 ml of dry

Tetrahedrofuran (THF). To this, Et3N (62µl, 0.454 mmole) was added and the resulting

solution was kept under N2 for 15 minutes. Then 4-bromomethyl-7-methoxy coumarin (111

mg, 0.413 mmole) was taken in 10 ml of dry THF and added into the stirring solution in drop

wise fashion. It was kept under reflux condition with stirring for 10h until all the starting

materials become consumed. After that, solvent was removed with rotary evaporator. Then it

was dissolved in 20 ml of CHCl3 and washed with 10 ml of water. The organic layer was

collected and dried over anhydrous Na2SO4 before concentration. It was finally purified by

column chromatography using silica gel as stationary phase and CHCl3 as solvent to isolate

an off-white solid L1 in pure form with 87% yield (yield was calculated based on the starting

reagents). 1H NMR (500 MHz, CD3CN, SiMe4, J (Hz), ppm): 7.811 (1H, m, H18), 7.512-

7.483 (3H, m, H17, H19, H20), 6.991 (1H, t, J= 3.5Hz, H7), 6.857 (1H, s, H9), 6.837(1H, d, J =

1.5 Hz, H6), 6.398 (1H, s, H27), 6.380 (1H, s, H33), 6.329 (1H, d, J = 2.5 Hz, H30,24), 6.306

(1H, s, H31), 6.286 (1H, s, H25), 6.091 (1H, s, H2) , 3.849 (3H, s, H11), 3.55 (2H, s, H12), 3.288

(8H, q, J= 7 Hz,H31, 40, 34, 36), 3.208 (2H, t, J= 6Hz, H14), 2.451 (2H, t, J=7 Hz, H13), 1.069

(12H, t, J=7 Hz, H35,36,39,41). 13C NMR (500 MHz, CD3CN, SiMe4, ppm) : 167.637, 162.041,

160.458, 154.917, 154.139,153.417, 152.791, 148.437, 132.118, 130.710, 128.160,

128.011, 127.792, 123.017, 121.905, 116.900, 111.613, 111.218, 108.984, 107.789,

104.963, 100.355, 96.882, 64.166, 55.180, 47.924, 46.976, 39.247, 28.926, 11.358. ESI-MS

(+ve mode, m/z): 673.36 (M + H+), Calc. for C41H44N4O5 is 672.33.



3

1H NMR spectra of L1 in CD3CN:

SI Figure 1: 1H NMR of L1 in CD3CN.



4

13C NMR spectra of L1 in CD3CN:

SI Figure 2: 13C NMR spectra of L1 in CD3CN.



5

ESI-Ms spectra of L1:

SI Figure 3: ESI-Ms spectra of L1.

100 200 300 400 500 600 700 800 900 1000 1100m/z0

100

%

SS B2 25HG 13 (0.130) 1: TOF MS ES+ 2.30e3239.11

237.10

97.09

88.09

84.09

235.08

128.10141.57

673.36

282.10

261.10

310.10468.28

424.22353.17

427.25477.21

629.29515.16

601.22

674.37

675.37

711.31753.26 859.23889.34 949.25 998.97 1049.53

L1+ H+



6

FTIR Spectra of L1:

SI Figure 4: FTIR spectra of L1.

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

8.7

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24.3

cm-1

%T

SSB2-25

3364

3079

2964

2924

2856

2666

2363

1876

1714

1683

1614

1543

1513

1461

1431

1388

1351

1307

1270

1218

1151

1118

1022

977

888

817

785

760

699

632

578

534

485

450

424



7

Synthetic methodology for the receptor 4-((benzylamino)methyl)-7-methoxy-2H-

chromen-2-one L2:

Benzyl amine (0.26 mmole) was dissolved in 5 ml of dry Tetrahedrofuran (THF). To this, Et3N (28µl)

was added and the resulting solution was kept under N2 for 15 minutes. Then 4-bromomethyl-7-

methoxy coumarine (66.2 mg, 0.26 mmole) was taken in another 5 ml of dry THF and added into the

stirring solution in drop wise fashion. It was kept at reflux temperature under N2 atmosphere with

stirring for 10h until all the starting materials become consumed. After that, solvent was removed

with rotary evaporator. Then it was dissolved in 10 ml of CHCl3 and washed with 5ml of water. The

organic layer was collected and dried over anhydrous Na2SO4 before concentration. It was finally

purified by column chromatography using silica gel as stationary phase and MeOH: CHCl3 (1:49) as

solvent to isolate yellow solid L2 in pure form with 91% yield (yield was calculated based on the

starting reagents). 1H NMR (500 MHz,CD3CN: CDCl3,1:1, SiMe4, J (Hz), ppm): 7.456 (1H, dd, J=

2.5 Hz), 7.380-7.320 (4H, m), 7.257 (1H, t, J= 7.5 Hz), 6.889-6.869 (3H), 6.337 (1H, s), 3.91 (2H),

3.861 (3H, s), 3.843 (2H, s). 13C NMR(500 MHz, CDCl3, ppm): 50.605, 55.361, 57.579, 102.850,

112.425, 114.164, 126.950, 129.226, 130.006, 130.433, 141.244, 155.474, 157.470, 163.333,

164.423. ESI-MS (+ve mode, m/z): 296.42 (M + H+, 100%), Calc. for C18H17NO3 is 295.33.



8

1H NMR spectra of L2 in CD3CN/CDCl3:

SI Figure 5: 1H NMR of L2 in CD3CN/CDCl3 (1:1, v/v).



9

13C NMR spectra of L3 in CD3CN/CDCl3:

SI Figure 6: 13C NMR of L2 in CD3CN/CDCl3 (1:1, v/v).



10

ESI-Ms spectra of L2.

SI Figure 7: ESI-Ms spectra of L2.

L3 + H+



11

160 140 120 100 80 60 40 20ppm

L1

L1+ 1eqv Hg

2+

13C NMR of L1 in absence and in presence of Hg2+ in CDCl3/CD3CN:

SI Figure 8: 13C NMR of L1 in absence and in presence of Hg2+ in CDCl3/CD3CN.



12

Beneshi-Hildebrand (B-H) plot obtained from the spectrophotometric titration of L1

with Hg2+:

SI Figure 9: Beneshi-Hildebrand (B-H) plot obtained from the spectrophotometric titration of

L1 with Hg2+ supported 1:1 binding stoichiometry (R2=0.99723).

0.0 6.0x104

1.2x105

1.8x105

15

30

45

1/(

A-A

0)

1/[Hg2+

]



13

Beneshi-Hildebrand (B-H) plot obtained from the emission titration of L1 with Hg2+:

SI Figure 10: Beneshi-Hildebrand (B-H) plot obtained from the emission titration of L1 with

Hg2+ supported 1:1 binding stoichiometry (R2=0.994).

2.0x104

4.0x104

6.0x104

4.0x10-4

8.0x10-4

1/(

I-I 0

)

1/[Hg2+

]



14

Equation used for calculation of Energy transfer efficiency (ETE %):

Reference:

C. Thivierge, J. Han, R. M. Jenkins and K. Burgess, J. Org. Chem., 2011, 76, 5219.



15

ESI-Ms spectral evidence for the 1:1 adducts formation of L1 with Hg2+:

SI Figure 11: ESI-Ms spectral evidence for 1:1 adducts formation of L1 with Hg2+.

Lig+Hg2++2H2O



16

Partial 1H NMR titration of L1 as a function of Hg(ClO4)2:

SI Figure 12: A plot of change in 1H NMR spectral pattern for the receptor (i) L1; (ii) L1 with

0.5 eqv Hg2+; (iii) L1 with 0.75 eqv Hg2+ and (iv) L1 with 1 eqv. Hg2+ in CD3CN medium.



17

Spectrophotometric interference study of L1 with Hg2+ in presence of various metal ions:

Hg

2+/

Li+

Hg

2+/

Na

+

Hg

2+/

K+

Hg

2+/

Mg

+2

Hg

2+/

Ca

+2

Hg

2+/

Ba

+2

Hg

2+/

Sr+

2

Hg

2+/

Cr+

3

Hg

2+/

Cu

+2

Hg

2+/

Ni+

2

Hg

2+/

Pb

+2

Hg

2+/

Zn

+2

Hg

2+

Hg

2+/

Co

+2

Hg

2+/

Fe

+2

Hg

2+/

Cd

+2

Li Na K Mg Ca Ba Sr Cr Cu Ni Pb Zn Hg Co Fe Cd

0.00

0.04

0.08

Ab

so

rba

nce

SI Figure 13: Spectrophotometric interference study of L1(6.7 x 10

-6 M) with Hg2+ (1.2 x 10

-3 M)

in presence of various metal ions (6.0 x 10-4

M) in MeOH/ HEPES buffer(1:1, v/v).



18

Uv-Vis spectral studies for establishing the reversible binding of Hg2+ to the L1:

SI Figure 14: Uv-Vis studies for establishing the reversible binding of Hg2+ (1.53 x 10-4 M)

to L1(6.9 x 10-5 M) in presence of EDTA2-(2 x 10-3 M) in MeOH-Water(1:1, v/v).

450 5400.0

2.0x10-2

4.0x10-2

6.0x10-2

L1+Hg

2+

L1

L1+Hg

2++EDTA

2-

Absorb

ance

Wavelength(nm)



19

Change of Emission intensity of L1 at 585 nm as a function of the solution pH:

SI Figure 15: Change in emission intensity at 585 nm with variation in pH of the MeOH-

water (1:1, v/v) solution for L1 (6.7 x 10-6 M).



20

FTIR Spectra of L1 in presence of 5 equivalent of Hg(ClO4)2:

SI Figure 16: FTIR spectra of L1 in presence of 5 equivalent of Hg(ClO4)2.

FTIR spectra have been recorded to understand the binding mode of Hg2+ with L1 in presence of 5

mole equivalent of Hg(ClO4)2. In L1, two carbonyl stretching were observed which are belong to

coumarin C=O bond and amide carbonyl moiety from rhodamine unit. The peak appeared at the

stretching frequency 1714 cm-1 could be appeared from coumarine moiety while peak at 1682 cm-1

could be appeared from amide carbonyl of rhodamine as amide stretching frequency should be

lowered compared to the ester carbonyl frequency. The lowering of the stretching frequency of the

amide carbonyl from 1682 cm-1 to 1661 cm-1 indicated definite coordination of Hg2+ to the amide

C=O bond. However, a slight increment in the stretching frequency of the ester C=O was due to

lowering of overall electron density on the coumarin moiety after coordination to metal ions thus no

participation of ester C=O in Hg2+ coordination.

2000 1500

L1

1614 cm-1

1714 cm-1

(cm-1)

1682 cm-1

1661 cm-1

1608 cm-1

L1+ Hg

2+

1721 cm-1



21

Hg Ca Cd Cr Cu Fe Lig Ni Sr Zn Co Ba Mg Na K Li0.0

2.0x10-2

4.0x10-2

6.0x10-2

Ab

so

rba

nc

e

Hg

2+

Ca

2+

Cd

2+

Fe

2+

Cu

2+

Zn

2+

Lig

and

Co

2+

Sr2

+

Ni2

+

Cr3

+

Ba

2+

Mg

2+

Na

+

Li2

+

K+

Bar Diagram for changes in absorbance intensity for L1 with metal ions:

SI Figure 17: Bar diagram for the changes in absorbance intensity of L1(6.7 x 10-6 M) with

metal ions(1.0 x 10-4 M).



22

Bar Diagram for changes in emission intensity for L1 with metal ions:

SI Figure 18: Bar diagram for the changes in absorbance intensity of L1(6.7 x 10-6 M) with

metal ions(1.5 x 10-4 M).

Hg2+Ca2+Cd2+Cr3+Cu2+Fe2+ lig Ni2+Sr2+Zn2+Co2+Ba2+Mg2+Na+ K+ Li+0

1x105

2x105

Inte

nsity

Hg

2+

Ca

2+

Cd

2+

Cu

2+

Fe

2+

Cr3

+

liga

nd

Ni2

+

Zn

2+

Sr2

+

Co

2+

Ba

2+

Mg

2+

Na

+

K+ Li+



23

Job’s plot for L1 with Hg2+ showing 1:1 stoichiometry:

SI Figure 19: Job’s plot between L1 and Hg2+ confirmed 1:1 adducts.

0.0 0.2 0.4 0.6 0.8 1.0

0

1

2

3

Absorb

ance

[L1

]/([L1

]+[Hg2+

])



24

Absorption spectra of L1 in presence of Hg2+ at different pH:

SI Figure 20: Absorption spectra for L1(6.9 x 10-6 M) in presence of Hg2+(1.0 x 10-4 M) at

different basic pH.

300 400 500 600

0.1

0.2

0.3

10.5

9.5

8.5

7.5

Abs.

Wavelength(nm)



25

Emission spectra of L1 in presence of Hg2+ at different pH:

SI Figure 21: Emission spectra for L1 (6.9 x 10-5 M) in presence of Hg2+(1.0 x 10-4 M) at

different basic pH (λExt 320 nm).

550 575 6000.0

5.0x104

1.0x105

1.5x105

2.0x105

10.59.5

8.5

7.5

Inte

nsity

Wavelength(nm)



26

Confocal microscopic images of L1 in presence and in absence of Hg2+ in MCF7 cells:

SI Figure 22: Confocal microscopic images of L1 (10 μM) in absence and in presence of

Hg2+ (2-10ppb) in MCF7 cells (λExt 543nm).

L1

+

2 ppb Hg2+

Dark fieldBright field Overlay

Control

L1

+

10ppb Hg2+



27

Fluorescence microscopic images of L1 in presence and in absence of Hg2+ in MCF7

cells:

Bright

Field

Dark

Field

L1

L1

+

2 ppb Hg2+

Overlay

SI Figure 23: Fluorescence microscopic images of L1 (10 μM) in absence and in presence

of Hg2+ (2ppb) in MCF7 cells (λExt 330-385nm).



28

Reversibility studies for binding of the reagent L1 to Hg2+ present in the breast cancer

cell (MCF7 cells) with subsequent treatment with KI:

SI Figure 24: Confocal dark field (A),Bright field (B), and overlay images(C) of MCF7 cells. (1) The cells were supplemented with Hg2+ (10ppb) and then were stained with 10 μM of L1 for 1.0 H at 37°C, (2) with Hg2+(10 ppb), 10 μM L1 and then KI(30 μM) in the growth media for 1.0 h at 37 °C (λext = 543 nm).

Dark

field

Bright

field Overlay

1

2

A B C



29

MTT assay studies for evolution of cytotoxicity of the reagent L1 towards the breast cancer cell (MCF7 cells):

SI Figure 25: Check of viability of L1 on the breast cancer cells (MCF7 cells). Here % of viability was calculated with respect to the growth considering 100% without L1. Cell Cytotoxicity Assay

Cytotoxicity of L1 on MCF7 cells was determined by conventional MTT assay (J. Natl.

Cancer Inst., 1990, 8, 1113-1117). MCF7 cells in their exponential growth phase were

trypsinised and seeded in 96-well flat-bottom culture plates at a density of 3 x 103 cells per

well in 100 μl DMEM complete medium (Himedia, India). The cells were allowed to adhere

and grow for 24 hr at 37 ºC in CO2 incubator (New Brunswick Scientific, U.S.A.), and then

the medium was replaced with 100 μl fresh incomplete medium containing various

concentrations of L1 (0 to 10 μM). The assay was performed in quadruplet for each

concentration. Cells were then incubated for 12h, after which the culture medium was

removed, and 100 μl of 1 mg/ml MTT reagent in PBS was added to each well. Thereafter, it

was incubated for 4 hrs; during this period active mitochondria of viable cells reduce MTT to

purple formazan. Unreduced MTT were then discarded and DMSO (100 μl) was added into

each well to dissolve the formazan precipitate, which was then measured

spectrophotometrically using a microplate reader at 570 nm. The cytotoxic effect of each

treatment was expressed as percentage of cell viability relative to the untreated control

cells. [MTT= (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow

tetrazole].

Reference: L. V. Rubinstein, K. D. Paull, R. M. Simon, P. Skehan, D. A. Scudiero, A. Monks and M. R. Boyd, J. Natl. Cancer Inst., 1990, 82, 1113.

0

20

40

60

80

100

01

25

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