Supporting Information for

Biophysical analysis of cancer stem cell-potent copper(II)

coordination complexes

Puyi Zheng,a‡ Arvin Eskandari,a‡ Chunxin Lu,a,b Kristine Laws,a Leigh Aldous,a* and

Kogularamanan Suntharalingama*

a Department of Chemistry, King’s College London, London, SE1 1DB, United Kingdom b College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing,

314001, China.

Email: kogularamanan.suntharalingam@kcl.ac.uk; leigh.aldous@kcl.ac.uk

‡ These authors contributed equally to this work

Table of Content

Experimental Details

Fig. S1. IR spectrum of (A) 1 and (B) 2 in the solid form.

Fig. S2 X-ray structure of the copper(II) complex, 2. Ellipsoids are shown at 30%

probability, O atoms are shown in red, C in black, N in dark blue, and Cu in

light blue. H atoms have been omitted for clarity.

Table S1. Crystallographic data for the copper(II) complex, 2.

Table S2. Selected bond lengths (Å) and angles (°) for the copper(II) complex, 2.

Fig. S3 Cyclic voltammograms of normalised scan rate studies of (A) 1 and (B) 2.

Fig. S4 UV-Vis spectrum of 1 (50 μM) in the presence of ascorbic acid (500 μM) in

PBS (pH 7.4) over the course of 24 h at 37 oC.

Fig. S5 UV-Vis spectrum of 1 (50 μM) in the presence of glutathione (500 μM) in

PBS (pH 7.4) over the course of 24 h at 37 oC.

Fig. S6 UV-Vis spectrum of 2 (50 μM) in the presence of ascorbic acid (500 μM) in

PBS (pH 7.4) over the course of 24 h at 37 oC.

Fig. S7 UV-Vis spectrum of 2 (50 μM) in the presence of glutathione (500 μM) in

PBS (pH 7.4) over the course of 24 h at 37 oC.

Fig. S8 UV-Vis spectrum of indomethacin (50 μM), naproxen (50 μM), 4,7-diphenyl-

1,10-phenanthroline (50 μM) in PBS (pH 7.4).

Fig. S9 UV-Vis spectrum of 1 (50 μM) in the presence of ascorbic acid (500 μM) and

bathocuproine disulfonate, BCS (100 μM) in PBS (pH 7.4) over the course of

24 h at 37 oC.

Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2019

Fig. S10 UV-Vis spectrum of 2 (50 μM) in the presence of ascorbic acid (500 μM) and

bathocuproine disulfonate, BCS (100 μM) in PBS (pH 7.4) over the course of

24 h at 37 oC.

Fig. S11 UV-Vis spectrum of (A) 1 and (B) 2 (25 μM) in sodium acetate (pH 5.2) over

the course of 48 h at 37 oC.

Fig. S12 Normalised ROS level in PBS:DMSO (200:1) (pH 7.4, 37 °C) for 1 (50 µM),

1 in the presence of ascorbic acid (AA, 500 µM), 1 in the presence of ascorbic

acid (AA, 500 µM) and N-acetylcysteine (NAC, 1 mM), 2 (50 µM), 2 in the

presence of ascorbic acid (AA, 500 µM), and 2 in the presence of ascorbic

acid (AA, 500 µM) and N-acetylcysteine (NAC, 1 mM). Error bars represent

standard deviations and Student t-test, * = p < 0.05. Fig. S13 ESI mass spectrum (positive mode) of 3 in DMSO.

Fig. S14 IR spectrum of 3 in the solid form.

Fig. S15 Emission spectrum (λex =395 nm) of coumarin-3-carboxylic acid (50 µM) and

3 (50 µM) in degassed Tris‐HCl (5 mM, pH 7.4) buffer.

Fig. S16 UV-Vis spectrum of 3 (50 μM) in the presence of ascorbic acid (500 μM) and

bathocuproine disulfonate, BCS (100 μM) in PBS (pH 7.4) over the course of

24 h at 37 oC.

References

Experimental Details

Materials and Methods. All synthetic procedures were performed under normal

atmospheric conditions. High resolution electron spray ionisation mass spectra were

recorded on a BrukerDaltronics Esquire 3000 spectrometer by Dr. Lisa Haigh

(Imperial College London). Fourier transform infrared (FTIR) spectra were recorded

with a IRAffinity-1S Shimadzu spectrophotometer. Elemental analysis of the

compounds prepared was performed commercially by London Metropolitan

University. Coumarin-3-carboxylic acid was purchased from Sigma Aldrich and used

as received. The copper(II) complexes, 1 and 2 were prepared according to our

previously reported protocol.1 For all biophysical and cellular studies, a 10 mM stock

solution in DMSO was initially prepared. The stock solution was diluted in the

appropriate biological solution to the working concentration(s).

Synthesis of Cu(4,7-diphenyl-1,10-phenanthroline)(coumarin-3-carboxylic acid)2

(3). KOH (78 mg, 1.38 mmol) was added to a solution of coumarin-3-carboxylic acid

(240 mg, 1.26 mmol) dissolved in methanol (8 mL). This solution was stirred at room

temperature for 1 h, after which 4,7-diphenyl-1,10-phenanthroline (210 mg, 0.63

mmol) was added, followed by a methanolic solution (4 mL) of CuCl2•2H2O (85.4 mg,

0.63 mmol). The resulting cloudy cyan suspension was stirred at 50 °C for 72 h. The

solution obtained was evaporated to dryness and the resulting solid was washed

thoroughly with water (3 x 20 mL) and diethyl ether (3 x 20 mL). The product isolated

was a cyan solid. (56.2 mg, 12 %); IR (solid, cm-1): 1736, 1598, 1557, 1516, 1494,

1449, 1423, 1393, 1277, 1229, 1162, 1082, 1016, 998, 923, 848, 810, 762, 732, 699,

665, 632, 594, 576, 546; HR ESI-MS Calcd. for C44H25CuN2O8 [M-H]+: 772.7064

a.m.u. Found [M-H]+: 772.1918 a.m.u.; Anal. Calcd. for 3, C44H26CuN2O8: C, 68.26;

H, 3.38; N, 3.62. Found: C, 68.11; H, 3.45; N, 3.78.

X-ray Single Crystal Diffraction Analysis. Crystallographic data of 2 was collected

on a Gemini diffractometer with graphite-monochromated Cu-Kα radiation (λ =

1.54184 Å) at 296 K. The crystal structure was resolved using direct methods in the

SHELXS program and refined by full-matrix least-squares routines based on F2, using

the SHELXL package.2 All the H atoms were placed in geometrically idealised

positions and constrained to ride on their parent atoms. The structure has been

deposited with the Cambridge Crystallographic Data Centre (CCDC 1513414). This

information can be obtained free of charge from

www.ccdc.cam.ac.uk/data_request/cif.

Electrochemical Studies. Electrochemical studies were performed using 5 mM

solutions of 1 and 2 in DMSO and with tetrabutylammonium hexafluorophosphate

(100 mM) as the supporting electrolyte. Studies were conducted using a 3mm glassy

carbon working electrode, a platinum counter electrode and an Ag/AgCl (3 M NaCl)

reference electrode (all from BASi Analytical, USA). Prior to each scan, the solution

was degassed for 3 min using nitrogen and was obtained using a Metrohm Autolab

Potentiostat.

Fluorescence Spectroscopy Studies. Fluorescence studies were performed using 50

μM solutions of 3 and coumarin-3-carboxylic acid, in 5 mM pH 7.4 Tris buffer

(degassed) at 37 oC. Fluorescence spectra were recorded on a Perkin Elmer

Fluorescence Spectrometer using an excitation wavelength (λex) of 395 nm, a slit

width of 20 nm, and a PMT of 600 V. The fluorescence spectra were recorded from

425-650 nm.

ROS Assay. The copper(II)-NSAID complexes, 1 and 2 (50 µM) were incubated with 10

equivalents of ascorbic acid for 2 h in PBS (pH 7.4):DMSO (200:1) at 37 °C, and the

resultant reduced products were treated with 6-carboxy-2’,7’-dichlorodihydrofluorescein

diacetate (DCFH-DA) for a further for 30 min. The ROS level was then determined by

measuring the fluorescence of the solutions at 529 nm (λex = 495 nm).

Fig. S1 IR spectrum of (A) 1 and (B) 2 in the solid form.

Fig. S2 X-ray structure of the copper(II) complex, 2. Ellipsoids are shown at 30% probability,

O atoms are shown in red, C in black, N in dark blue, and Cu in light blue. H atoms have

been omitted for clarity.

Table S1. Crystallographic data for the copper(II) complex, 2.

Copper(II) complex, 2

formula C52H42CuN2O6

Fw 854.42

crystal system Monoclinic

space group P2(1)

a, Å 9.9496(8)

b, Å 12.4616(9)

c, Å 17.5712(16)

α, deg. 90

β, deg. 97.452(8)

γ, deg. 90

V, Å3 2160.2(3)

Z 2

Dcalcd, Mg/m3 1.314

Reflections collected 8026

Reflections independent (Rint) 5244 (0.0294)

Goodness-of-fit on F2 1.031

R(I> 2σI) 0.0505, 0.1344

Table S2. Selected bond lengths (Å) and angles (°) for the copper(II) complex, 2.

Cu(1)-O(4) 1.945(3) Cu(1)-O(1) 1.962(4)

Cu(1)-O(2) 2.597(4) Cu(1)-O(5) 2.530(5)

Cu(1)-N(2) 1.990(4) Cu(1)-N(1) 2.021(4)

O(4)-Cu(1)-O(1) 96.52(18) O(4)-Cu(1)-N(2) 91.46(18)

O(1)-Cu(1)-N(2) 165.25(17) O(4)-Cu(1)-N(1) 165.51(15)

O(1)-Cu(1)-N(1) 93.17(18) N(2)-Cu(1)-N(1) 81.56(18)

Fig. S3 Cyclic voltammograms of normalised scan rate studies of (A) 1 and (B) 2.

Fig. S4 UV-Vis spectrum of 1 (50 μM) in the presence of ascorbic acid (500 μM) in PBS (pH

7.4) over the course of 24 h at 37 oC.

Fig. S5 UV-Vis spectrum of 1 (50 μM) in the presence of glutathione (500 μM) in PBS (pH

7.4) over the course of 24 h at 37 oC.

250 300 350 400 450 500 5500

1

2

3

4

UV Stability of PZ15B in PBS With AA (10 eqv)

Ab

so

rba

nce

/ a

.u.

Wavelength/ nm

0 h

2 h

4 h

6 h

8 h

10 h

12 h

14 h

16 h

18 h

20 h

22 h

24 h

250 300 350 400 450 500 5500.0

0.5

1.0

1.5

Wavelength/ nm

UV Stability of PZ15B in PBS With GSH (10 eqv)

Ab

so

rba

nce

/ a

.u.

0 h

2 h

4 h

6 h

8 h

10 h

12 h

14 h

16 h

18 h

20 h

22 h

24 h

Fig. S6 UV-Vis spectrum of 2 (50 μM) in the presence of ascorbic acid (500 μM) in PBS (pH

7.4) over the course of 24 h at 37 oC.

Fig. S7 UV-Vis spectrum of 2 (50 μM) in the presence of glutathione (500 μM) in PBS (pH

7.4) over the course of 24 h at 37 oC.

250 300 350 400 450 500 5500.0

0.2

0.4

0.6

0.8

1.0

UV Stability of PZ9 in PBS With AA (10 eqv)

Ab

so

rba

nce

/ a

.u.

Wavelength/ nm

0 h

2 h

4 h

6 h

8 h

10 h

12 h

14 h

16 h

18 h

20 h

22 h

24 h

250 300 350 400 450 500 5500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

UV Stability of PZ9 in PBS With GSH (10 eqv)

Ab

so

rba

nce

/ a

.u.

Wavelength/ nm

0 h

2 h

4 h

6 h

8 h

10 h

12 h

14 h

16 h

18 h

20 h

22 h

24 h

Fig. S8 UV-Vis spectrum of indomethacin (50 μM), naproxen (50 μM), 4,7-diphenyl-1,10-

phenanthroline (50 μM) in PBS (pH 7.4).

Fig. S9 UV-Vis spectrum of 1 (50 μM) in the presence of ascorbic acid (500 μM) and

bathocuproine disulfonate, BCS (100 μM) in PBS (pH 7.4) over the course of 24 h at 37 oC.

250 300 350 400 450 500 5500.0

0.2

0.4

0.6

0.8

1.0

1.2

UV Single Scans of Compounds and Ligands

Ab

so

rba

nce

/ a

.u.

Wavelength/ nm

indomethacin

naproxen

4,7-diphenyl-1,10-phenanthroline

400 450 500 550 600 650 700 750 8000.0

0.1

0.2

0.3

0.4

0.5

UV Study of PZ2B Reduction to Cu(I) Using BCS (2 eqv)

Ab

so

rba

nce

/ a

.u.

Wavelength/ nm

1

[CuI(BCS)

2]3-

+ AA + BCS 0h

+ AA + BCS 3h

+ AA + BCS 24h

Fig. S10 UV-Vis spectrum of 2 (50 μM) in the presence of ascorbic acid (500 μM) and

bathocuproine disulfonate, BCS (100 μM) in PBS (pH 7.4) over the course of 24 h at 37 oC.

Fig. S11 UV-Vis spectrum of (A) 1 and (B) 2 (25 μM) in sodium acetate (pH 5.2) over the

course of 48 h at 37 oC.

400 450 500 550 600 650 700 750 8000.0

0.1

0.2

0.3

0.4

0.5

2

[CuI(BCS)

2]3-

+ AA + BCS 0h

+ AA + BCS 3h

+ AA + BCS 24h

UV Study of PZ9 Reduction to Cu(I) Using BCS (2 eqv)

Ab

so

rba

nce

/ a

.u.

Wavelength/ nm

Fig. S12 Normalised ROS level in PBS:DMSO (200:1) (pH 7.4, 37 °C) for 1 (50 µM), 1 in

the presence of ascorbic acid (AA, 500 µM), 1 in the presence of ascorbic acid (AA, 500 µM)

and N-acetylcysteine (NAC, 1 mM), 2 (50 µM), 2 in the presence of ascorbic acid (AA, 500

µM), and 2 in the presence of ascorbic acid (AA, 500 µM) and N-acetylcysteine (NAC, 1

mM). Error bars represent standard deviations and Student t-test, * = p < 0.05.

Fig. S13 ESI mass spectrum (positive mode) of 3 in DMSO.

Fig. S14 IR spectrum of 3 in the solid form.

Fig. S15 Emission spectrum (λex =395 nm) of coumarin-3-carboxylic acid (50 µM) and 3 (50

µM) in degassed Tris‐HCl (5 mM, pH 7.4) buffer.

1800 1600 1400 1200 1000 800 60020

30

40

50

60

70

80

90

100

Tra

nsm

itta

nce

/ %

Wavenumber/ cm-1

450 500 550 600 6500

100

200

300

400

500

Flu

ore

sce

nce

In

ten

sity/

a.u

.

Wavelength/ nm

coumarin-3-carboxylic acid

3

Fig. S16 UV-Vis spectrum of 3 (50 μM) in the presence of ascorbic acid (500 μM) and

bathocuproine disulfonate, BCS (100 μM) in PBS (pH 7.4) over the course of 24 h at 37 oC.

References

1. Eskandari, A.; Boodram, J. N.; Cressey, P. B.; Lu, C.; Bruno, P. M.; Hemann, M. T.;

Suntharalingam, K., Dalton Trans. 2016, 45 (44), 17867-17873.

2. Sheldrick, G., Acta Cryst. 2008, A64 (1), 112-122.

400 450 500 550 600 650 700 750 8000.0

0.1

0.2

0.3

0.4

0.5

3

[CuI(BCS)

2]3-

+ AA + BCS 0h

+ AA + BCS 3h

+ AA + BCS 24h

UV Study of PZ31B Reduction to Cu(I) Using BCS (2 eqv)

Ab

so

rba

nce

/ a

.u.

Wavelength/ nm