Carbon-1 versus carbon-3 linkage of D-galactose to porphyrins: Synthesis, uptake, and photodynamic efficiency

Transcription

1 Supporting information for Carbon-1 versus carbon-3 linkage of D-galactose to porphyrins: Synthesis, uptake, and photodynamic efficiency Patrícia M. R. Pereira #, Waqar Rizvi #, N. V. S. Dinesh K. Bhupathiraju, Naxhije Berisha, Rosa Fernandes +, João P.C. Tomé Τ, Charles Michael Drain * QOPNA, Department of Chemistry, University of Aveiro, Aveiro, Portugal Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal + Centre for Neuroscience and Cell Biology - Institute for Biomedical Imaging and Life Sciences (CNC.IBILI), Research Consortium, University of Coimbra, Coimbra, Portugal Department of Chemistry and Biochemistry, Hunter College of the City University of New York, New York, NY 10065, USA Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, NY 10016, USA Τ CQE, Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal # These authors contributed equally to this work. Contents Page 1. Synthesis scheme S2 2. NMR spectra S3 3. UV-visible absorbance spectra S11 4. Fluorescence emission spectra S13 5. Mass spectra S15 6. DLS S20 7. Uptake S23 8. Dark toxicity S25 * cdrain@hunter.cuny.edu S1

2 1. Synthesis Scheme Scheme S1. Synthesis of porphyrin-galactose conjugates. S2

3 2. NMR spectra Figure S1. 1 H NMR spectrum of the free base of compound 1 in CDCl 3, * = CHCl 3 Figure S2. 13 C NMR spectrum of the free base of compound 1 in CDCl 3, * = CHCl 3 S3

4 Figure S3. 1 H NMR spectrum of 1, in CDCl 3, * = CHCl 3 Figure S4. 1 H NMR spectrum of 2 in CDCl 3 S4

5 Figure S5. 13 C NMR spectrum of 2 in CDCl 3, * = CDCl 3 Figure S6. 1 H NMR spectrum of (C1-Gal)4-ZnPor (3) in DMSO d 6, * water, ** DMSO. S5

6 Figure S7. 13 C NMR spectrum of (C1-Gal) 4-ZnPor (3) in DMSO d 6. Figure S8. 1 H NMR spectrum of (C1-Gal)4-Por (4) in DMSO-d 6. * water, ** DMSO S6

7 Figure S8A. Expansion of 1 H NMR spectrum of (C1-Gal)4-Por (4) in DMSO-d 6. * water, ** DMSO. Inner pyrrolic -NH broad peak is at Figure S9. 13 C NMR spectrum of (C1-Gal)4-Por (4) in DMSO-d 6. S7

8 Figure S10. 1 H NMR spectrum of 5 in CDCl 3, * = CHCl 3 Figure S C NMR spectrum of 5 in CDCl 3, * = CDCl 3 S8

9 Figure S12. 1 H NMR spectrum of (C3-Gal)4-ZnPor (6) in CD 3OD, * = CH 3OH Figure S C NMR spectrum of (C3-Gal)4-ZnPor (6) in CD 3OD. S9

10 Figure S14. 1 H NMR spectrum of (C3-Gal)4-Por (7) in DMSO-d 6, * water, ** DMSO. Figure S C NMR spectrum of (C3-Gal)4-Por (7) in DMSO-d 6. S10

11 3. UV-visible absorbance spectra Figure S16.UV-visible spectra of the compounds, 0.1 μm in ethanol, green = (C1-Gal) 4-ZnPor (3), red = (C3-Gal) 4-ZnPor (6), blue = (C1-Gal) 4-Por (4), black = (C3-Gal) 4-Por (7). Figure S17.UV-visible spectra of the compounds, 0.1 μm in DMSO, green = (C1-Gal) 4-ZnPor (3), red = (C3-Gal) 4-ZnPor (6), blue = (C1-Gal) 4-Por (4), black = (C3-Gal) 4-Por (7). S11

12 Figure S18.UV-visible spectra of the compounds, 0.1 μm in PBS, green = (C1-Gal) 4-ZnPor (3), red = (C3-Gal) 4-ZnPor (6), blue = (C1-Gal) 4-Por (4), black = (C3-Gal) 4-Por (7). S12

13 4. Fluorescence emission spectra Figure S19. Fluorescence emission spectra of the compounds in ethanol; excitation at 425 nm where the absorbance is ca for each compound, green = (C1-Gal) 4-ZnPor (3), red = (C3- Gal) 4-ZnPor (6), blue = (C1-Gal) 4-Por (4), black = (C3-Gal) 4-Por (7). Figure S20. Fluorescence emission spectra of the compounds in DMSO; excitation at 425 nm where the absorbance is ca for each compound, green = (C1-Gal) 4-ZnPor (3), red = (C3- Gal) 4-ZnPor (6), blue = (C1-Gal) 4-Por (4), black = (C3-Gal) 4-Por (7). S13

14 Figure S21. Fluorescence emission spectra of the compounds in PBS buffer; excitation at 425 nm where the absorbance is ca for each compound, green = (C1-Gal) 4-ZnPor (3), red = (C3- Gal) 4-ZnPor (6), blue = (C1-Gal) 4-Por (4), black = (C3-Gal) 4-Por (7). S14

15 5. Mass spectra Figure S22. HRMS of 2. S15

16 Figure S23. HRMS of (C1-Gal)4-ZnPor (3). S16

17 Figure S24. HRMS of (C1-Gal)4-Por (4). S17

18 Figure S25. HRMS of 5. S18

19 Figure S26. HRMS of (C3-Gal)4-ZnPor (6). S19

20 Figure S27. HRMS of (C3-Gal)4-Por (7). S20

21 6. DLS 25 (C1-Gal) 4 -ZnPor (3) Number (%) E+03 Size (d.nm) EtOH PBS Figure S28. DLS of (C1-Gal) 4-ZnPor (3). 25 (C1-Gal) 4 -Por (4) Number (%) E+03 Size (d.nm) EtOH PBS Figure S29. DLS of (C1-Gal) 4-Por (4). S21

22 25 (C3-Gal) 4 -ZnPor (6) Number (%) E+03 Size (d.nm) EtOH PBS Figure S30. DLS of (C3-Gal) 4-ZnPor (6). 25 (C3-Gal) 4 -Por (7) Number (%) E+03 Size (d.nm) EtOH PBS Figure S31. DLS of (C3-Gal) 4-Por (7). S22

23 7. Singlet oxygen Figure S32. Photo-oxidation of DPBF (50 μm) in the presence of galactose-porphyrins (3, 4, 6, 7) or tetraphenylporphyrin (TPP) at a concentration of 5 μm in DMF, after irradiation with 0.49 mw/cm 2 (measured at the front of the cuvette) light from a xenon arc lamp in Horiba SPEX fluorometer with the monochonometer set at 550 nm and the band pass set at 10 nm to minimize absorption by DPBF. DPBF absorbance was obtained at 415 nm. Data are means of two independent experiments. 8. Cell and Spheroid Uptake Figure S33. Cellular uptake of galactose-porphyrins by MCF-7 monolayer cultures. The concentration of porphyrins was determined by fluorescence spectroscopy after incubation of cancer cells with 0, 2.25, 4.5 S23

24 or 9 μm of the galactose-porphyrins for 0, 0.5, 1, 2 or 4 h and the results were normalized to protein quantity. Data are the means ± S.D. of at least three independent experiments performed in triplicate. Figure S34. Cellular uptake of galactose-porphyrins by HCT-116 monolayer cultures. The concentration of porphyrins was determined by fluorescence spectroscopy after incubation of cancer cells with 0, 2.25, 4.5 or 9 μm of galactose-porphyrins for 0, 0.5, 1, 2 or 4 h and the results were normalized to protein quantity. Data are the means ± S.D. of at least three independent experiments performed in triplicate. Figure S35. Cellular uptake of galactose-porphyrins by HeLa monolayer cultures. The concentration of porphyrins was determined by fluorescence spectroscopy after incubation of cancer cells with 0, 2.25, 4.5 or 9 μm of galactose-porphyrins for 0, 0.5, 1, 2 or 4 h and the results were normalized to protein quantity. Data are the means ± S.D. of at least three independent experiments performed in triplicate. S24

25 Figure S36. Cellular uptake of galactose-porphyrins by UM-UC-3 monolayer cultures. The concentration of porphyrins was determined by fluorescence spectroscopy after incubation of cancer cells with 0, 2.25, 4.5 or 9 μm of galactose-porphyrins for 0, 0.5, 1, 2 or 4 h and the results were normalized to protein quantity. Data are the means ± S.D. of at least three independent experiments performed in triplicate. Figure S37. Representative fluorescence images of cancer cells monolayer cultures incubated with porphyrins (red) and cell nuclei stained with DAPI (blue). S25

26 Figure S38. Cellular uptake of galactose-porphyrins by UM-UC-3 monolayer cultures after pre-incubation with thiogalactose sugar. The concentration of porphyrins was determined by fluorescence spectroscopy after pre-incubation with thiogalactose sugar (100 or 1000 μm, 1h) and incubation with 0, 2 9 μm of galactose-porphyrins for 3 h. The results were normalized to the uptake values of cells with pre-incubation with thiogalactose sugar. Data are the means ± S.D. of at least three independent experiments performed in triplicate. *(p<0.05), **(p<0.001) significantly different from cells with pre-incubation of thiogalactose sugar. Note that the absolute uptake of the C1 compounds is about half that of the C3 (see figure 2 in the manuscript). 9. Dark toxicity of porphyrins in monolayers and spheroids S26

27 Figure S39. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in HCT-116 monolayer cultures as determined using the MTT assay. Figure S40. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in HCT-116 spheroid cultures as determined using the LHD assay. Figure S41. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in HeLa monolayer cultures as determined using the MTT assay. S27

28 Figure S42. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in HeLa spheroid cultures as determined using the LHD assay. Figure S43. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in MCF-7 monolayer cultures as determined using the MTT assay. Figure S44. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in MCF-7 spheroid cultures as determined using the LHD assay. S28

29 Figure S45. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in UM-UC-3 monolayer cultures as determined using the MTT assay. Figure S46. Dark toxicity assays of galactose-porphyrins at concentration of 9 μm and uptake time of 4 h, in UM-UC-3 spheroid cultures as determined using the LHD assay. S29