DNA/Protein Binding, Molecular Docking and in Vitro Anti-cancer Activity of some Thioether-Dipyrrinato Complexes

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1 DNA/Protein Binding, Molecular Docking and in Vitro Anti-cancer Activity of some Thioether-Dipyrrinato Complexes Rakesh Kumar Gupta, Gunjan Sharma, ξ Rampal Pandey, Amit Kumar, Biplob Koch, ξ Pei- Zhou Li, Qiang Xu, and Daya Shankar Pandey * Departments of Chemistry, and ξ Zoology, Faculty of Science, Banaras Hindu University, Varanasi (U.P.) India National Institute of Advanced Industrial Science and Technology (AIST), , Midorigaoka, Ikeda, Osaka , Japan Contents 1. HRMS spectra of S3-S6 2. NMR spectra of the complexes S7-S10 3. Absorption titration spectra of 1, 3 and 4 with CT DNA..S11 4. Emission spectra from EtBr bound to the DNA with 1, 3 and 4 S12 5. Emission spectra of BSA with 1, 3 and S13 6. Stern-Volmer plot for complexes at different temperature... S14 7. Synchronous spectra of BSA with 1, 3 and 4 at λ = 60 nm...s15 8. Synchronous spectra of BSA with 1, 3 and 4 at λ = 15 nm...s D fluorescence spectra of BSA with 1, 3 and 4... S Evolution of the CV and DPV of 1, 3 and 4 with CT DNA.S18-S Molecular docking of 1, 3 and 4 with DNA and Protein..S20-S Anti-proliferative profiles of 1-4 S Fluorescence images (AO/EtBr) of DL cells after treatment with 2..S Absorption Spectral data of 1-4 Bound to CT DNA....S Quenching constant and binding constant (K bin ) of 1-4 with BSA. S31 1

2 16. 3D fluorescence spectral parameters of the BSA and BSA S Electrochemical data of S Log P values for the complexes S Binding site of protein (HSA)....S35-S36 2

3 Figure S1. ESI-MS spectra of 1 3

4 Figure S2. ESI-MS spectra of 2 4

5 Figure S3. ESI-MS spectra of 3 5

6 Figure S4. ESI-MS spectra of 4 6

7 Figure S5. 1 H (top) and 13 C NMR (bottom) spectra of 1 in CDCl 3. 7

8 Figure S6. 1 H (top) and 13 C NMR (bottom) spectra of 2 in CDCl 3. 8

9 Figure S7. 1 H (top) and 13 C NMR (bottom) spectra of 3 in CDCl 3. 9

10 Figure S8. 1 H (top) and 13 C NMR (bottom) spectra of 4 in CDCl 3. 10

11 (a) (b) (c) Figure S9. Absorption titration spectra of 1 (a), 3 (b) and 4 (d) (EtOH:H 2 O, c, 10 µm; 1:1, v/v; ph ~7.3) in the absence (black line) and in presence (other lines) of CT DNA to complex (1-20 µm) at rt. Arrow shows the absorbance changes upon increasing the CT DNA concentration. 11

12 (a) (b) (c) Figure S10. Emission spectra of EtBr (black dotted line) EtBr bound to the DNA (Red solid line) and in the presence (other lines) of 1 (a), 3 (b) and 4 (c) with increasing amounts 0-50 µm. [EtBr] = 10 µm, [DNA] = 10 µm. Arrow shows changes in the emission intensity upon addition of increasing complex concentration. 12

13 (a) (b) (c) (d) Figure S11. Emission spectrum of BSA (black line) (0.5 µm; λ ex = 280 nm; λ em = 343 nm) in presence (other lines) of 1 (a), 3 (b), 4 (c) (0-50 µm). Arrow shows the emission intensity changes upon increasing complex concentration. Stern-Volmer plot for quenching of the complexes 1-4 with BSA. 13

14 (b) (a) (c) (d) Figure S12. Stern-Volmer plot for quenching of the complexes 1 (a), 2 (b), 3 (c) and 4 (d) to BSA at 300 (black) and 310 K (red). 14

15 (a) (b) (c) Figure S13. Synchronous spectra of BSA (black line) (0.5 µm) in presence (other lines) of 1 (a), 3 (b) and 4 (c) with increasing amounts of complexes (0-50 µm) at the wavelength difference of λ = 60 nm. Arrows show decrease in the emission intensity accompanied by blue shift upon increasing concentration of the complexes 1, 3 and 4. 15

16 (a) (b) (c) Figure S14. Synchronous spectra of BSA (black line) (Tris-HCl buffer, c, 0.5 µm, ph ~7.5) in presence (other lines) of 1 (a), 3 (b) and 4 (c) with increasing amounts of complexes (0-50 µm) at wavelength difference of λ = 15 nm. Arrows show the emission intensity decrease is accompanied by blue shift upon increasing concentration of the complexes 1, 3 and 4. 16

17 (a) (b) (c) Figure S15. The 3D fluorescence spectra of BSA + 1 (a), BSA + 3 (b), BSA + 4 (c). c(bsa) = moll 1, c(2) = moll 1. 17

18 (a) (b) Figure S16. Evolution of the CV (a) and DPV (b) of 1 (c, 100 µm, MeCN) in absence (black) and presence (other lines) of increasing the amounts of CT DNA ( µm), at rt. (a) (b) Figure S17. Evolution of the CV (a) and DPV (b) of 3 (c, 100 µm, MeCN) in absence (black) and presence (other lines) of increasing the amounts of CT DNA ( µm), at rt. 18

19 (a) (b) Figure S18. Evolution of the CV (a) and DPV (b) of 4 (c, 100 µm, MeCN) in absence (black) and presence (other lines) of increasing the amounts of CT DNA ( µm), at rt. 19

20 Figure S19. Molecular docked model of complex 1 with DNA (PDB ID: 1BNA). 20

21 Figure S20. Molecular docked model of complex 3 with DNA (PDB ID: 1BNA). 21

22 Figure S21. Molecular docked model of complex 4 with DNA (PDB ID: 1BNA). 22

23 Figure S22. Most probable 10 (ten) binding sites of HSA (PDBID: 1h9z). 23

24 (a) (b) Figure S23. Molecular docked model of 1 located within the hydrophobic pocket of HSA (a) (PDB ID: 1h9z) (b) the interaction mode between 1 (stick) and HSA (cartoon). (a) (b) Figure S24. Molecular docked model of 3 located within the hydrophobic pocket of HSA (a) (PDB ID: 1h9z) (b) the interaction mode between 3 (stick) and HSA (cartoon). 24

25 Figure S25. Molecular docked model of 4 located within the hydrophobic pocket of HSA (a) (PDB ID: 1h9z) (b) the interaction mode between 4 (stick) and HSA (cartoon). Figure S26. Interaction of complex 1 with HSA subdomain IIA. 25

26 Figure S27. Interaction of complex 2 with HSA subdomain IIA. Figure S28. Interaction of complex 3 with HSA subdomain IIA. 26

27 Figure S29. Interaction of complex 3 with HSA subdomain IIA. 27

28 (a) (b) (c) (d) Figure S30. The anti-proliferative profiles of 1 (a), 2 (b), 3 (c) and 4 (d) against Dalton s Lymphoma cells after 24 h of treatments. Cytotoxicity and Cell proliferation was determined by MTT assay. Results are expressed as a percentage with respect to control. 28

29 (a) (b) Figure 31. Fluorescence images (AO/EtBr) showing blebbing in Dalton s Lymphoma cells treated with complex 2 (c, 2 µg/ml) after 24 h of incubation (a) 20X and (b) 40X. 29

30 Table S3. Absorption Spectral Properties of 1-4 Bound to CT DNA Complexes λ max (nm) Changes in ε Red shift K b (M -1 ) Site Size absorbance M 1 cm 1 (nm) (s) hyperchromism hypochromism No shift 490 hypochromism hyperchromism hypochromism No shift 489 hypochromism hyperchromism hypochromism No shift 501 hyperchromism hyperchromism hypochromism No shift 499 hyperchromism

31 Table S4. Quenching constant (K q ), binding constant (K bin ) and number of binding sites (n) for the interactions of complexes with BSA Complexes Temperature (K) K q (M -1 ) K bin (M -1 ) n R

32 Table S5. Characteristic 3D fluorescence spectral parameters of the BSA and BSA Complex Peaks Rayleigh Peaks Peak 1 Intensity Peak 2 Intensity [λ ex /λ em (nm/nm)] [λ ex /λ em (nm/nm)] [λ ex /λ em (nm/nm)] BSA 270/ / / / / / / / / / / / / / / / / / / /

33 Table S6. Electrochemical data of 1-4 (c, 100 µm, MeCN) (a) Changes in cyclic voltammetry after addition of CT DNA (1.0 µm) to a solution of 1-4. Compound E pa, dpm/dpm + (V), E pa, Ru 2+ /Ru 3+ (V) or M 3+ /M 4+ (M = Rh, Ir) Ε pa = Complex + CT DNA (V) CT DNA Positive shift Disappear CT DNA Positive shift Disappear CT DNA CT DNA (b) Changes in differential pulse voltammetry after addition of CT DNA (1.0 µm) to a solution of 1-4. Compound E pa, dpm/dpm + (V), E pa, Ru 2+ /Ru 3+ (V), Ε pa = Complex + CT DNA (V) CT DNA Positive shift Disappear CT DNA Positive shift Disappear CT DNA CT DNA

34 Table S7. Log P Values for Complexes 1-4 a Log P Complex Mean SD a Results are the means of three independent experiments and are expressed as means ± SDs. 34

35 Table S8. 5 most probable binding sites of HSA (PDB ID: 1h9z; Q-site finder) and preferential binding site of complexes from docked structure. SITE 1 LYS 195, GLN 196, LEU 198, LYS 199, SER 202, LEU 203, PHE 206, GLY 207, GLU 208, ARG 209, ARG 209, ALA 210, PHE 211, LYS 212, ALA 213, TRP 214, VAL 216, ARG 218, GLN 221, VAL 235, HIS 242, ASN 295 LYS 323, ASP 324, LEU 327, GLY 328, LEU 331, PRO 339, TYR 341, SER 342, VAL 343, VAL 344, LEU 345, LEU 346, LEU 347, ARG 348, ALA 350, LYS 351, GLU 354, GLU 383, Pro 384, LEU 397, ILE 388, LYS 389, ASN 391, CYS 392, PHE 395, PHE 403, LEU 407, ARG 410, TYR 411, LEU 430, GLY 431, Val 433, GLY 434, CYS 437, CYS 438, ARG 445, MET 446, PRO 447, CYS 448, ALA 449, GLU 450, ASP 451, LEU 453, SER 454, VAl 455, LEU 457, VAL 455, LEU 457, ASN 458, SER 480, LEU 481, VAL 482, ARG 484, ARG 485, PRO 486, SER 489 SITE 2 VAL 7, ARG 10, LEU 14, PHE 19, LEU 22, VAL 23, ALA 26, PHE 27, TYR 30, GLU 45, VAL 46, PHE 49 ASN 61, LEU 66, HIS 67, THR 68, LEU 69, PHE 70, ASP 72, LYS 73, THR 76, ASN 99, LRU 103, TYR 150, ALA 151, PRO 152, GLY 248, ASP 249, LEU 250, LEU 251, ALA 254, ASP 255, ARG 257, ALA 258, ALA 261, LEU 283, LEU 284, GLU 285, LYS 286, SER 287, HIS 288 SITE 3 LEU 115, VAL 116, ARG 117, PRO 118, MET 123, PHE 134, LEU 135, TYR 138, LEU 139, ILE 142, HIS 146, PHE 149, LEU 154, PHE 157, ALA 158, TYR 161, LYS 162, PHE 165, LEU 182, ASP 183, LEU 185, ARG 186, GLY 189, LYS 190, SER 193 SITE 4 TYR 401, ASN 405, PHE 502, PHE 507, PHE 509, LYS 524, LYS 525, GLN 526, ALA 528, LEU 529, LEU 532, HIS 535, LYS 536, VAL 547, MET 548, PHE 551, ALA 552, LEU 575, VAL 576, SER 579, GLN 580 SITE 5 GLN 29, LYS 106, ASP 108, HIS 146, PRO 147, TYR 148, PHE 149, TYR 150, ALA 151, GLU 153, SER 192, SER 193, ALA 194, LYS 195, GLN 196, ARG 197, LYS 199, CYS 200, ALA 201, HIS 242, GLU 244, CYS 245, CYS 246, HIS 35

36 247, GLY 248, ASP 249, LEU 250, CYS 253, ARG LYS 195, TRP 214, ARG 218, GLN 221, ARG 222, ASN 295, PRO 339, ASP 340, TYR 341, SER 342, VAL 343, ALA 443, LYS 444, PRO 447, CYS 448, GLU 450, ASP LYS 195, ARG 218, ARG 222, ALN 291, GLU 292, VAL 293, GLU 294, ASN 295, PRO 339, ASP 340, VAl 343, ALA 443, LYS 444, PRO 447, CYS 448, GLU 450, ASP GLU 153, SER 192, LYS 195, GLN 196, LEU 198, LYS 199, SER 202, TRP 214, ARG 222, HIS 288, ALA 291, GLU 292, ASP LYS 195, TRP 214, ARG 218, GLN 221, ARG 222, GLU 292, GLU 294 ASN 295, TYR 341, VAL 343, LYS 444, PRO 447, CYS 448, GLU 450, ASP