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1 Electronic Supplementary Material (ESI) for Medicinal Chemistry Communications. This journal is The Royal Society of Chemistry 2015 BODIPY appended copper(ii) complexes of curcumin showing mitochondria targeted remarkable photocytotoxicity in visible light Arnab Bhattacharyya, a Akanksha Dixit, b Koushambi Mitra, a SamyaBanerjee, a Anjali A. Karande,* b and Akhil R. Chakravarty* a a Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore , India b Department of Biochemistry, Indian Institute of Science, Bangalore , India arc@ipc.iisc.ernet.in; anjali@biochem.iisc.ernet.in Electronic Supplementary Information (ESI) 1

2 Table of contents Experimental Section Materials and methods Synthetic procedures- Preparation of ligands (L 1 and L 2 ) Schemes Scheme S1 - Preparation of ligands L 1 and L 2 Scheme S2 - Preparation of complexes 1 and 2 Tables Table S1 - Selected physiochemical data Table S2 - Crystal data selected crystallographic parameters Table S3 - Selected bond lengths and angles Table S4 List of coordinates for energy minimized structure of complexes 1 and 2 Figures Fig. S1-1 H - NMR of ligands L 1 and L 2 Fig. S2-13 C NMR of ligands L 1 and L 2 Fig. S3-11 B NMR of ligands L 1 and L 2 Fig. S4-19 F NMR of ligands L 1 and L 2 Fig. S5 - Mass of ligands L 1 and L 2 Fig. S6 - Cyclic Voltammograms of ligands L 1 and L 2 Fig. S7 - ORTEP and unit cell packing diagram of Crystal structure of ligand L 2 Fig. S8 - Energy minimized structures of L 1 and L 2 Fig. S9 - HOMO-LUMO diagrams of the complexes 1 and 2 Fig. S10 - Mass of complexes 1 Fig. S11 - Mass of complexes 2 Fig. S12 - IR spectra of complexes 1 and 2 Fig. S13 - Cyclic Voltammograms of complexes 1 and 2 Fig. S14 - Time dependent absorption spectra for complexes 1 and 2 and free curcumin Fig. S15 - Fluorescence microscopic images (2h incubation data) for complexes 1 and 2 2

3 Experimental Section Materials and measurements: All reagents and chemicals were from commercial sources (Polysales, India; TCI chemicals, India; Aldrich, USA) and used without any further purification unless stated. Solvents were purified by given procedures. S1 Tetrabutylammonium perchlorate (TBAP) was obtained by reacting tetrabutylammonium bromide and perchloric acid. The elemental analysis was performed using a Thermo Finnigan FLASH EA 1112 CHNS analyzer. The IR and UV/Vis spectra were recorded on Perkin Elmer Lambda 35 and Perkin Elmer spectrum one 55, respectively. Cyclic voltammograms were recorded at room temperature on an EG&G PAR model 253 VersaStat potentiostat/galvanostat with electrochemical analysis software 270 using a three electrode setup consisting of a glassy carbon working, platinum wire auxiliary and a saturated calomel reference electrode (SCE) in DMF using tetrabutylammonium perchlorate (TBAP) (0.1 M) as a supporting electrolyte. Electrospray ionization (ESI) mass spectral measurements were done using Agilent Q-TOF instrument. The NMR spectra were recorded with Bruker Avance 400 NMR spectrometer. X-ray crystallographic procedures: All geometric and intensity data were collected using an automated Bruker SMART APEX CCD diffractometer equipped with a fine focus 1.75 kw sealed tube Mo-K α X-ray source (λ = Å) with increasing ω (width of 0.3 per frame) at a scan speed of 5 sec per frame. Intensity data were collected using ω-2θ scan mode and then corrected for the Lorentz polarization as well as for absorption effects. S2 The structures were solved and refined by WinGx suit of programs (Version a) with SHELXL-2013 method. S3,S4 Initially all non-hydrogen positions were spotted in the difference Fourier maps, but during the final refinement, the hydrogen atoms (placed in geometrically ideal positions) were refined in the riding mode. Atomic positions for all the atoms, anisotropic thermal parameters for all the non-hydrogen atoms and isotropic thermal parameters for all the hydrogen atoms also accounted for the final refinement. The crystal structures were represented using Diamond software. Selected 3

4 crystallographic parameters, bond distances/angles, list of coordinates for the ligand L 2 are provided in Tables S2-S4. Computational details: The complexes 1 and 2 were optimized by using B3LYP/LanL2DZ functional by Density Functional Theory (DFT) method. S5,S6 The initial coordinates for geometry of the ligand were adapted from the obtained crystal structure of L 2. All the jobs were run using Gaussian09 package. S7 The coordinates of the optimized geometries of the complexes 1 and 2 are given in a list and HOMO and LUMO orbital diagrams are given in Fig. S9. Synthetic procedures Synthesis of Ligands (Scheme 1) Synthesis of 4,4-difluoro-8-(4-chloromethylphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diazas-indacene (A): The precursor species A was prepared using previous literature reports. S8 Synthesis of L 1 : The precursor (1 g, 2.67 mmol, 1.0 eq.) was taken in a dry 100 ml round bottom flask in 50 ml CH 3 CN. K 2 CO 3 (739 mg, 5.35 mmol, 2.0 eq.) and KI (890 mg, 5.35 mmol, 2.0 eq.) dissolved in 10 ml of water were subsequently added to the mixture. Bis-(2- pyridylmethyl)amine (5.32 g, 26.7 mmol, 10.0 eq.) dissolved in 10 ml CH 3 CN was added and the resulting solution was refluxed for 6 h. The solvent was evaporated under reduced pressure and resulting residue was dissolved in 50 ml dichloromethane. The organic layer was washed with water and brine solution, dried over anhydrous Na 2 SO 4, filtered and concentrated under reduced pressure to give the crude product. It was then purified by silica gel column chromatography using EtOAc-MeOH as eluent (9:1) to afford an orange solid [L 1 ] (1.05 g, 1.96 mmol, 73% yield). Characterization data: 1 H NMR (400 MHz, d 4 -MeOD, TMS) δ: (d, 2H), (td, 2H), (d, 2H), (dd, 2H), 7.33 (d, 2H), (dd, 2H), 6.05 (s, 2H), 3.82 (s, 4H), 3.80 (s, 2H), 2.49 (s, 6H), 1.36 (s,6h). 13 C NMR (d 4 -MeOD) δ: , , , , , , , , , , , , , , , , , 59.58, 58.40, 29.91, 22.74, 13.62, B NMR (d 4 -MeOD) δ: (t); 4

5 19 F NMR (d 4 -MeOD) δ: (m). ESI-MS (m/z in MeOH) calculated for C 32 H 33 BF 2 N 5 [M+H] ; found Synthesis of L 2 : L 1 (0.536 g, 1.0 mmol, 1.0 eq.) was taken in a 100 ml dry round bottom flask and dissolved in 50 ml of deoxygenated CH 2 Cl 2 and N-iodo succinimide (NIS) (1.35 g, 6.0 mmol, 6.0 eq.) was added to it under N 2 atmosphere. It was then stirred at room temperature until full consumption of L 1 was found by thin layer chromatography (TLC). The reaction mixture was then washed with water, dried over anhydrous Na 2 SO 4, filtered and the solvent was removed under reduced pressure. The crude product thus found, was then purified by silica gel column chromatography using EtOAc/MeOH as eluent (8:2) to afford a pink red solid [L 2 ] (0.62 g, 0.79 mmol, 78% yield). Characterization data: 1 H NMR (400 MHz, d 6 -DMSO, TMS) δ: (d, 2H), (td, 2H), (d, 2H), (d, 2H), (d, 2H), (dd, 2H), 3.77 (s, 6H), 2.50 (s, 6H), 1.27 (s, 6H). 11 B NMR (d 6 -DMSO) δ: (t); 13 C NMR (d 6 -DMSO) δ , , , , , , , 59.53, 57.86, 31.54, 17.54, F NMR (d 6 -DMSO) δ: (m). ESI-MS (m/z in MeOH) calculated for C 32 H 31 BF 2 I 2 N 5 [M+H] ; found References S1. D. D. Perrin, W. L. F. Armarego and D. R. Perrin; Purification of Laboratory Chemicals; Pergamon Press:Oxford, S2. N. Walker and D. Stuart; Acta Crystallogr., 1983, A39, S3. G. M. Sheldrick; Acta Crystallogr. Sect. A, 2008, 64, S4. J. L. Farrugia; J. Appl. Crystallogr., 1999, 32, S5. C. Lee, W. Wang, R. G. Parr, Phys. Rev. B: Condens Matter, 1988, 37, S6. W. J. Hehre, L. Radom, P. v. R. Schleyer and J. A. Pople, Ab Initio Molecular Orbital Theory, Wiley, New York, 1986, p S7. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, 5

6 K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, GAUSSIAN 09 (Revision A.1), Gaussian, Inc., Wallingford, CT, S8. B. W. Michel, A. R. Lippert and C. J. Chang; J. Am. Chem. Soc. 2012, 134,

7 Schemes Scheme S1. Synthesis of the BODIPY ligands. Scheme S2. Synthesis of the complexes (8-{[bis(2-pyridylmetyhl)amino]metyhlphenyl}-4,4-difluoro- 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene){(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6- heptadiene-3,5-dionato}copper(ii) chloride (1) and (8-{[bis(2-pyridylmetyhl)amino]metyhlphenyl}- 4,4-difluoro-1,3,5,7-tetramethyl-2,6-diiodo-4-bora-3a,4a-diaza-s-indacene){(1E,6E)-1,7-bis(4- hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dionato}copper(ii) chloride (2). 7

8 Tables Table S1 Selected physicochemical data of the complexes and ligands Entry λ abs /nm ( ε/dm 3 mol -1 cm -1 ) a λ em /nm ( λ exc /nm) a [φ] b E f / V ( E p / mv) c IR (cm -1 ) (υ C=O, υ C=C ) L 1 500(24580) 510(465) [0.55] L 2 534(22370) 565(510) [0.01] 1 501(20750), 435(18650) 512(465) [0.12] (133) (103) (107) 1600, (17350), 432(18600) 510(430) [0.01] , 1505 a In (1:1) DMSO-phosphate buffer (ph= 7.4). b Fluorescence quantum yield values in DMSO using fluorescein in 0.1M NaOH as standard (φ = 0.79). c Cathodic peaks of cyclic voltammograms in DMF-0.1M TBAP, E f = 0.5(E pa + E pc ), Ep = (E pa E pc ), where E pa and E pc are the anodic and the cathodic peak potentials, respectively. The potentials are vs. SCE. Scan rate = 100 mv s -1. 8

9 Table S2 Selected crystallographic data Entry L 2 Empirical formula C 32 H 30 BF 2 I 2 N 5 Fw / g M Crystal system Orthorhombic Space group P a / Å (8) b / Å (8) c / Å (2) α / 90 β / 90 γ / 90 U / Å (4) Z 4 2θ max / 52 T, K h,k,l max 298(2) 12,12,36 ρ calcd / g cm λ / Å (Mo-K α ) µ / mm Data / restraints / parameters 6306 / 0 / 379 F(000) 1544 Goodness-of-fit R (F o ) a, I > 2σ (I) [wr (F o )] [0.1190] R (all data)[wr (all data)] [0.1190] a R = Σ F o - F c / F o. b wr = {R [w (F o 2 F c 2 ) 2 ] / R [w (F o ) 2 ]} 1/2, w = [σ 2 (F o ) 2 + (AP) 2 + BP] 1, where P = (F o 2 + 2F c 2 )/3 9

10 Table S3 Selected bond distances (Å) and angels ( o ) for L 2 B(1)-F(1) 1.379(11) B(1)-F(2) 1.378(11) B(1)-N(1) 1.555(10) B(1)-N(2) 1.543(11) C(1)-N(1) 1.346(9) C(4)-N(1) 1.399(9) C(6)-N(2) 1.399(9) C(9)-N(2) 1.312(10) C(2)-I(1) 2.055(7) C(8)-I(2) 2.076(7) C(20)-N(3) 1.465(9) C(21)-N(3) 1.457(10) C(27)-N(3) 1.455(9) C(22)-N(4) 1.327(11) C(26)-N(4) 1.344(17) C(28)-N(5) 1.390(12) C(32)-N(5) 1.376(15) F(1)-B(1)-F(2) 110.5(7) N(1)-B(1)-N(2) 107.3(6) B(1)-N(1)-C(4) 125.3(6) B(1)-N(2)-C(6) 125.1(6) C(4)-C(5)-C(14) 118.5(6) C(6)-C(5)-C(14) 119.9(6) C(17)-C(20)-N(3) 111.4(6) C(20)-N(3)-C(21) 110.9(6) C(20)-N(3)-C(27) 111.7(5) C(22)-C(21)-N(3) 111.0(6) C(28)-C(27)-N(3) 113.1(6) 10

11 Figures (a) (b) Fig. S1. 1 H NMR spectra of (a) L 1 (in d 4 -MeOD) and (b) L 2 (in d 6 -DMSO). 11

12 (a) (b) Fig. S2. 13 C NMR spectra of (a) L 1 (in d 4 -MeOD) and (b) L 2 (in d 6 -DMSO). 12

13 (a) (b) Fig S3. 11 B spectra of (a) L 1 (in d 4 -MeOD) and (b) L 2 (in d 6 -DMSO). (a) (b) Fig. S4 19 F spectra of (a) L 1 (in d 4 -MeOD) and (b) L 2 (in d 6 -DMSO). 13

14 (a) (b) Fig. S5. Mass spectra of (a) L 1 and (b) L 2 (in MeOH). 14

15 (a) (b) Fig. S6. Cyclic voltammograms of (a) L 1 and (b) L 2 in 0.1M TBAP-DMF using 1.0 mmol concentration at a scan rate of 100 mv sec

16 (a) 16

17 (b) Fig. S7 (a) ORTEP view of the crystal structure of L 2 showing labeling scheme for hetero atoms and thermal ellipsoids at 50% probability level. Hydrogen atoms are not shown for clarity. [Colour codes: black carbon, blue nitrogen, red boron, green fluorine, purple iodine]. (b) Unit cell packing diagram of the crystal structure of L 2. [Colour codes: black carbon, white - hydrogen, blue nitrogen, red- boron, green fluorine, purple iodine] 17

18 (a) (b) Fig. S8 Energy minimized structures of complexes (a) 1 and (b) 2. 18

19 (a) 19

20 (b) Fig. S9 HOMO - LUMO diagrams of complexes (a) 1 and (b) 2. (Contour value = 0.03) [HOMO of complexes 1 and 2 localizes on BODIPY unit, while LUMO involves orbitals from metal coordinated curcumin moiety] 20

21 (a) (b) Fig. S10. Mass spectra of the complexes (a) 1 and (b) 2 (in MeOH) Counts (%) vs. Mass-to-charge (m/z) Fig. S10. ESI-MS spectrum of complex 1 in MeOH showing prominant peak of [M-Cl] + in (a) and the isotopic distribution of that peak in (b). 21

22 (a) (b) Counts (%) vs. Mass-to-charge (m/z) Fig. S11. ESI-MS spectrum of complex 2 in MeOH showing prominant peak of [M-Cl] + in (a) and the isotopic distribution of that peak in (b). 22

23 (a) (b) Fig. S12. IR spectra of the complexes (a) 1 and (b) 2. 23

24 (a) (b) Fig. S13. Cyclic voltammograms of the complexes (a) 1 and (b) 2 in 0.1M TBAP-DMF using 1.0 mmol concentration at a scan rate of 100 mv sec

25 Fig. S14. Time dependent absorbance plot (434 nm) of curcumin and complexes 1 and 2 (concentration = 50 µm) in 1% DMSO DMEM-10% FBS buffer (ph = 7.4). The plot shows the instability of curcumin and its relative stability on binding to Cu(II). 25

26 Fig. S15. Fluorescence microscopic images of the HeLa cells treated with complexes 1 and 2 (10 µm) and nuclear staining Hoechst dye (5 µg ml -1 ) on 2 h incubation: panels (e, i) show the green emission of the complexes; panels (b, f, j) show the blue emission of the Hoechst dye; panels (c, g, k) are the merged images and panels (d, h, l) are the bright field images. Panel (a) is the control in absence of the complex or the Hoechst dye. Scale bar = 20 µm. 26

27 Table S4. List of coordinates for energy minimized structures: Complex 1 coordinates

28

29 Complex2 Coordinates

30

31