Bioorganometallic chemistry of osmium carbonyl clusters

BIOORGANOMETALLIC CHEMISTRY OF OSMIUM CARBONYL CLUSTERS KONG KIEN VOON NATIONAL UNIVERSITY OF SINGAPORE 2008 BIOORGANOMETALLIC CHEMISTRY OF OSMIUM CARBONYL CLUSTERS KONG KIEN VOON B.Sc (Hons),2004 University of Malaya A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements I express my gratitude to my research supervisor, A/P Prof Leong Weng Kee for his encouragement, patience, understanding and invaluable guidance, throughout the project with me and reading through the drafts of the thesis. Next, I would like to thank my co-supervisor Dr. Chew Wee and Dr Lim Hsui Kim, Lina for their guidance and help during my postgraduate studies. I would like to thank the following who have given me assistance in various ways during my postgraduate studies: Chong Thiam Siong, Garvin Mak, Chan Pekke, Alaric Koh Chun Wai, Venugopal Shanmugham Sridevi, Li Chunxiang, Moawia Omer Elhag Ahmed, Kuan Seah Ling, Woo Chang Hong, Oh Suat Ping, Tan Wen Ling, Florence Ng, Nguyen Thanh Hung, Emily Ang, Tan Yuwen, Leow Shi Chi, and Sim Huiling. I would like to thank the following laboratory staff: Mdm Han Yanhui and Ms Peggy Ler from NMR lab; Mdm Wong Lai Kwai and Mdm Lai Huiyi from MS lab; Miss Soong Foong Yee, Joanne, Mdm Leng Lee Eng, Miss Tan Tsze Yin from Elemental Analysis Lab and Thermal analytical lab; Ms Doreen Lai Mei Ying from IMRE. The Research Scholarship from MedChem Programme, National University of Singapore over the last four years is gratefully acknowledged. Last but not least, I would like to thank my parents, wife, and my friends for their encouragement and moral support. i Summary This thesis describes two potential applications of osmium carbonyl clusters. The original aim was to examine the use of such clusters as tags in the mid-infrared in the imaging of cells but the investigations eventually culminated in the discovery of the anticancer properties of some of these clusters. Chapter describes the current state of bioorganometallic chemistry, in particularly research into the application of organometallic compounds in bioimaging and as pharmaceuticals. Also covered is a brief introduction to cellular biology and apoptosis of relevance to carcinogenesis. The synthesis of the water-soluble osmium clusters, [Na]+2a- and 3, which are a fatty acid and a phosphotidylcholine analogue, respectively, are described in chapter 2. These clusters were capable of permeating into cells, and their employment in bioimaging of whole cells was demonstrated using oral mucosa cells. The cells were visualized via IR microscopy coupled with a focal plane array detector, through the metal-carbonyl stretches in the mid-infrared (~2000 cm-1) region at concentrations at which the cytotoxicity was low. On the basis of cellular translocation and in vivo stability studies with a cancer cell line, it was shown that [Na]+2a- and accumulated in the organelles and the cell membrane, with no significant decomposition of the osmium clusters. The cytotoxicity of [Na]+2a- and at higher concentrations, however, prompted us to investigate the anticancer properties of osmium clusters. The cytotoxicity test on a series of osmium clusters are described in chapter 3, and three triosmium clusters, viz., Os3(CO)10(NCCH3)2, 5a, Os6(CO)18, 7a, and Os6(CO)16(NCCH3)2, 7b, were found to exhibit good anticancer potential. A number of assays, viz., morphological assay, phosphatidylserine inversion and membrane ii integrity analysis (annexin V-FITC and PI staining), DNA fragmentation, caspase inhibition and caspase activation (PARP cleavage), are described and the results showed conclusively that the mode of action was the induction of apoptosis. Investigations into the cellular mechanism by which the osmium clusters, especially 5a, induces apoptosis are presented in chapter 4. This includes in vivo studies as well as attempts at identifying the molecular species formed. Evidence is presented that 5a interacted with intracellular carboxylate and sulfhydryl residues, that it bind to the sulfhydryl residues of tubulins, to stabilize the microtubules and resulting in apoptosis and cell cycle arrest. This demonstrated that 5a resembled the taxanes in that it acted via stabilization of the microtubule structure. iii Table of Contents Acknowledgements i Summary ii Table of contents iv List of figures ix List of schemes xiv List of Tables xv Abbreviations xvi Molecular Numbering Scheme xviii Chapter Bioorganometallic chemistry 1.1 Organometallic compounds in bioanalysis and imaging 1.1.1 Electrochemical methods 1.1.2 Fluorescence methods 1.1.3 Radioactive methods 1.1.4 Infrared methods Organometallic anticancer drugs 1.2.1 The cell cycle 1.2.2 Apoptosis 1.2.3 Cisplatin 10 1.2.4 Organometallic tamoxifen derivatives 11 1.2.5 Ruthenium-based pharmaceuticals 12 1.3 Objective 15 1.4 References 16 1.2 iv Chapter Organometallic Carbonyl Tags for Infrared Imaging of Cells 2.1 Synthesis of phosphotidylcholine osmium carbonyl clusters 23 analogue 2.2 Cytotoxicity assay 28 2.3 Whole cell imaging in the mid-Infrared with Na+[2a]- and 29 2.4 Absorbance profile Across a single cell 34 2.5 Uptake and washout studies 36 2.6 Translocation study of Na+[2a]- and in cells 38 2.7 Intracellular stability study 40 2.8 Conclusion 41 2.9 Experimental 41 2.9.1 General Experimental 41 2.9.2 Synthesis of Os3(CO)10(-H)-S(CH2)10COOH, 2a 42 2.9.3 Synthesis of Os3(CO)10(-H)[-S(CH2)10COO]-Na+, 43 Na+[2a]2.9.4 Protection of solketal 43 2.9.5 Synthesis of benzylated glycerol, C 44 2.9.6 Synthesis of BzOCH2CHCH2-1,2 {Os3(CO)10(-H) 44 [-S(CH2)10COO]}2, D 2.9.7 Deprotection of BzOCH2CHCH2-1,2{Os3(CO)10(-H) 45 [-S(CH2)10COO]}2, E 2.9.8 Synthesis of 1,2-{Os3(CO)10(-H) 47 [-S(CH2)10COO]}2CH2CHCH2OPO3CH2CH2NMe3, 2.9.9 Cell culture and viability assay 49 2.9.10 FT-IR imaging experiments 49 v 2.10 2.9.11 Cell fractional assay 50 2.9.12 Washout studies and ICP-MS analysis 51 2.9.13 Stability of Na+[2a]- in vivo 51 2.9.14 Estimation of cell size 51 References 52 Chapter Osmium Carbonyl Clusters: A New Class of Apoptosis Inducing Agents 3.1 Cytotoxicity of osmium carbonyl clusters 56 3.2 Induction of apoptosis 63 3.2.1 Cell nuclear staining 63 3.2.2 Annexin V and propidium iodide (PI) staining analysis 65 3.2.3 Cell cycle analysis 67 3.2.4 PARP cleavage with caspase cascade pathway 70 3.3 Conclusion 73 3.4 Experimental 74 3.4.1 General Experimental 74 3.4.2 Preparation of Os3(CO)10(SbPh3)2, 5d 75 3.4.3 Drug treatment 76 3.4.4 Proliferation assay 76 3.4.5 Determination of intracellular osmium level 77 3.4.6 Morphological analysis 77 3.4.7 Analysis of nuclear staining with DAPI 77 3.4.8 Annexin V and PI for flow cytometry 78 3.4.9 Cell cycle by flow cytometry 78 vi 3.4.10 Cellular protein extraction 78 3.4.11 SDS gel electrophoresis and Western immunoblot analysis 79 3.5 References 80 Chapter Intracellular Mechanism of Apoptosis Induction by Osmium Carbonyl Clusters 4.1 Interaction of 5a with intracellular residues 84 4.2 Interaction of 5a with tubulin 94 4.3 Conclusion 99 4.4 Experimental 100 4.4.1 General Experimental 100 4.4.2 Infrared spectroscopy of treated cells and cell fractions 100 4.4.3 NMR spectroscopy of treated cells 101 4.4.4 Effect of FBS and cysteine on drug efficacy 101 4.4.5 Annexin-V and PI staining for flow cytometry 102 4.4.6 Determination of cell sulfhydryl with DTNB 102 4.4.7 Fluorescence labeling 103 4.4.8 Immunofluorescence 103 4.4.9 Cell cycle analysis by flow cytometry 104 4.4.10 Tubulin polymerization assay 104 4.5 References 105 vii Chapter Conclusion 108 Appendix CD: IR chemical mapping images, Optical images of cells, Cell cycle analysis, Annexin V staining analysis, Fluorescence confocal microscopic images, IR, NMR, MS spectra. viii Chapter microtubule (Figure 4.10c). Taxol is proposed to gain access to its binding sites by diffusing through small openings in the microtubule. 13 They are effective in the treatment of breast and lung carcinomas.14 (3) The third class, typified by colchicines, comprises a structurally diverse collection of small molecules that are related by the fact that all bind to a common site on tubulin known as the colchicine site and thereby prevent the normal polymerization of microtubules (Figure 4.10b). 15 No representative of this third class has yet been approved for clinical use. Figure 4.10. Antimitotic drugs bind to microtubules at diverse sites. When MDA-MB-231 cells treated with 5a or 7b were stained with tubulinFITC antibody, the confocal microscopic images showed disruption of the morphology of the tubulins (Figure 4.11), suggesting that 5a or 7b interfered with the intracellular tubulin function. The treated cells also showed a condensed DNA and tubulin spindle structure at higher concentration. This is also consistent with the fractional DNA content analyses, which showed an accumulation of cells in the G2phase upon treatment with 5a or 7b (Figure 4.12); no significant block in other phases 95 Chapter was observed. This induction of G2 arrest by 5a or 7b is consistent with its reaction with tubulin sulfhydryl groups. Figure 4.11. Fluorescence confocal microscopic images of MDA-MB-231 cells stained with tubulin-FITC antibody after they have been incubated (24 h) with (a) DMSO (control), (b) 20 M solution of 5a, (c) 20 M solution of 7b, (d) 40 M solution of 5a, (e) 40 M solution of 7b. 96 Chapter Figure 4.12. Effect on cell cycle progression: a) control, and incubation (24 h) with solutions (10M) of b) 1, c) 5a, and d) 7b. An attempt was also made at identifying the type of interaction between 5a and tubulins through ToF-SIMS analysis of tubulins from treated cells. The ToFSIMS spectrum (Figure 4.13) showed mass fragments that could be assigned to [Os3H(CO)n(S)]− and [Os3H(CO)n(O2C)]− (n ≤ 6) thus suggesting again the interaction of 5a with both intracellular carboxylic acid groups and sulfhydryl groups. 97 Chapter Figure 4.13. ToF-SIMS spectrum of protein (a) and tubulin (b) of MDA-MB-231 cells treated for 24 h with 40 M solution of 5a. The effect of 5a and 7b on tubulin polymerization was also examined in comparison with Taxol (control) via the turbidity assay. 16 It has been shown that turbidity is a reliable measure of the mass of tubulin assembled into higher molecular weight structures. Turbidity is an approximation to the total light scattering from the higher molecular weight structures of tubulin; an increase in absorbance indicates an increase in polymerization. Wavelengths between 340-405 nm have been used to measure the turbidity of solutions of tubulin; shorter wavelengths (< 340 nm) could not be used because of possible interference from the absorption bands of tubulin. The results of the assay show that, like Taxol, 5a induced an increase in tubulin polymerization at both 10 M and 100 M concentrations (Figure 4.14). The tubulin polymerization ability of 7b, on the other hand, appeared to be similar to that of the control. The induction of tubulin polymerization with 5a is consistent with the 98 Chapter confocal images above. Our studies thus showed that the G2 phase cell cycle arrest induced by 5a is due to microtubule polymerization. Figure 4.14. Tubulin polymerization assays with 5a and 7b. Plot of absorbance (340 nm) vs treatment time. 4.3 Conclusion In this chapter, we have established via spectroscopic and biochemical studies that 5a interacts with intracellular carboxylate and sulfhydryl residues. Its ability at inducing apoptosis and cell cycle arrest is the result of its binding to the sulfhydryl residues of tubulins. In particular, 5a stabilizes microtubules by inducing their polymerisation. Thus 5a represents a new class of antimitotic agent that resembles the taxanes in that it acts via stabilization of the microtubule structure, but it differs from the taxanes in that it appears to so via binding to sulfhydryl residues. 17 99 Chapter 4.4 Experimental 4.4.1 General Experimental The general cell culture techniques and conditions for the biochemical studies are as described in chapter 3. All infrared spectra were recorded in the solid state with a Shimadzu Prestige-21 FTIR spectrometer at a spectral resolution of cm-1 on CaF2 windows (25mm dia., 4mm thick, Perkin Elmer). For the compartment spectra, A Chemicon compartment separation kit (Chemicon International Inc) was used for cell compartment separation. 1H NMR spectra were recorded on a Bruker ACF300 NMR spectrometer as CDCl3 solutions; chemical shifts reported were referenced against the residual proton signals of the solvents. Annexin V and PI staining were analyzed using a FACScan instrument equipped with a FACStation running Cell Quest software (Becton Dickinson, San Jose, CA). For the fluorescence staining, cells were analyzed using a Leica SP5 fluorescence confocal microscope. 4.4.2 Infrared spectroscopy of treated cells and cell fractions. MDA-MB-231 cells (2 x 107) cells were incubated (24 h) with 40 M of 5a and serum-free DMEM, washed with PBS (2 ml), and then scraped in the presence of 200 l of lysis buffer (Triton x-100, 5M NaCl, 0.5 M EDTA, 10% NP40 (Sigma)) supplemented with x protease inhibitor (Pierce Biotechnology) and phosphatase inhibitors (50 M okadaic acid (Sigma) and 200 mM sodium vanadate (Sigma)). The mixture was vortexed (1 every half hour) for h and centrifuged (13,000 g) at o C. The supernatant and pellet were saved as protein and non-protein fractions, respectively. For the compartment spectra, the cells (2 x 10 7) were into four fractions, namely, cytoplasmic, nuclear, membrane and organelles. Briefly, the treated cells 100 Chapter were homogenized in sucrose gradient buffer at moderate speed and centrifuged (20,000 g) at °C for 20 min. The supernatant was saved as the cytoplasmic fraction, while the pellet was washed with cold sucrose gradient buffer and centrifuged again as above. The supernatant so obtained was saved as the nuclear fraction, and the procedure was repeated to yield the membrane fraction as the supernatant and a pellet which contained organelles, granules and cytoskeleton, which was labelled as the organelles fraction. All these compartments were then freeze dried. 4.4.3 NMR spectroscopy of treated cells Approximately x 1010 cells (MDA-MB-231) treated with 40 M of 5a for 24 h was accumulated. They were rinsed with PBS (3 x 20 ml, each), resuspended in chloroform (10 ml) and vortexed (5 min) before homogenizing. The chloroform layer was then collected and vacuum dried, and CDCl3 (3 ml) was added to the dried residues and transferred to an NMR tube. The 1H NMR spectrum was recorded on a Bruker ACF300 NMR spectrometer; chemical shifts reported are referenced against the residual proton signals of the solvents. 4.4.4 Effect of FBS and cysteine on drug efficacy The compound 5a was dissolved in dimethylsulfoxide (DMSO) with final concentration used for treatment being 0.1%. Cells were seeded in growth medium at the same initial density, allowed to adhere and grow for 24 h to 80% confluence, washed once with serum-free DMEM medium and then serum-starved for h before treatment with a solution of 5a at the indicated concentrations together with DMEM and 10% FBS. These served as controls. For treatment under FBS-free condition, cells were seeded, grown and treated using growth medium without FBS. Cysteine-free 101 Chapter treatment condition was achieved using cysteine-free DMEM (Invitrogen). After 24 h incubation, to each well was added 20% of Cell Titer 96® Aqueous One Cell Proliferation Assay (Promega) which were then incubated in a 37 oC incubator with 5% CO2 for h. The absorbance intensities at 490 nm were then measured and the cell proliferation relative to the control sample was calculated. Each sample was analyzed in triplicates. 4.4.5 Annexin-V and PI staining for flow cytometry The percentage of cells actively undergoing apoptosis was determined using annexin V-PE-based immunofluorescence, as described previously. 18 Briefly, cells were plated in 6-well culture plates at concentrations determined to yield 80% confluence within 24 h, and treated under the conditions described above. After 24 h of treatment, the cells were harvested and then double-labeled with annexin V-FITC and PI (Becton, Dickinson and Company), as described by the manufacturer. All experiments were performed in duplicate and yielded similar results. 4.4.6 Determination of cell sulfhydryl with DTNB The amount of sulfhydryl in cells was determined according to the literature method.6 MDA-MB-231 cells were seeded in triplicates in 6-well plates and allowed to adhere, serum starved for h, and then supplemented with the indicated concentrations of 5a in serum-free DMEM for 24 h. The cells were washed with serum-free DMEM (3 x ml) and PBS (2 ml), harvested, dispersed in PBS (800 ml), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, 200 mL of a 10 mM solution) was then added. After 40 at room temperature, the solution and its absorbance at 412 nm was determined. All readings were relative to the control. 102 Chapter 4.4.7 Fluorescence labeling Cells were grown as described above and treated with the indicated concentrations of 5a or 7b for 24 h, fixed with formaldehyde (10%, 30 at -20 oC), and then permeabilized with Triton X-100 (0.1% in PBS, min) (USB Corp). They were then incubated in PBS solution containing tetramethylrhodamine-5-maleimide (TRM, 10 M, 25 at 22 ºC) (Invitrogen). Nuclei were stained concomitantly with 4,6-diamidino-2-phenylindole (DAPI, mg ml-1 in methanol, at 37 oC). The samples were then washed with PBS (3 x ml), mounted with 100 l Fluorosave (Calbiochem) mounting medium and analyzed under fluorescence confocal microscope. 4.4.8 Immunofluorescence Cells were grown on coverslips (60% confluence), incubated (24 h) with solutions (40 M) of 5a or 7b, washed with PBS (3 x ml), fixed with 5% formaldehyde in PBS (20 min, room temperature), aspirated, rinsed with PBS (3 x ml), and permeabilized with 0.1% Triton X-100 solution (100 ml). The cells were blocked with blocking buffer (2 ml, 5% goat serum, h at room temperature) and then the cell monolayers were incubated (overnight at oC) with monoclonal antibody tubulin conjugated with FITC (100 l, mg ml-1) (Sigma-Aldrich). After removal of the antibody solution, the cells were mounted with Fluorosave mounting medium (100 l). The cover slip slides were sealed with commercial nail polish. 103 Chapter 4.4.9 Cell cycle analysis by flow cytometry Cells were plated in 6-well flat bottom plates with 80% confluence after 24 h. They were then incubated with or 5a for 24 h, trypsinized, washed with cold PBS (2 x ml), resuspended in 70% ethanol and then incubated (30 at room temperature). After this, they were spun down at 2000 rpm (400 g) for min, washed once and resuspended again with 500 l PBS containing 1% FBS. This was then incubated (37 C for 15 min) after the addition of 20 l x RNase, after which 50l PI was added o and the sample analyzed by Dako CyAn™ ADP high-performance flow cytometer within h. 4.4.10 Tubulin polymerization assay A Cytoskeleton tubulin polymerization assay kit (Cat.#CDS03 and BK006) was used for the tubulin polymerization study. Briefly, (10 l of General Tubulin buffer (80 mM PIPES pH6.9, 2mM MgCl2, 0.5 mM EGTA) containing 5a, 7b or taxol was pipetted into the pre-warmed 96-well plate. Tubulin (defrosted to room temperature from -80 oC, and then placed on ice before use) was diluted with tubulin polymerization buffer (750 l General Tubulin buffer, 250 l Tubulin Glycerol Buffer [15% glycerol in General Tubulin buffer], 10 l of 1mM GTP) to a final concentration of mg ml-1. Diluted tubulin (100 l) was added into the wells containing or taxol. The absorbance at 340 nm was read immediately with a Biotek Synergy microplate reader. 104 Chapter 4.5 References (a) Leoni, L. M.; Hamel, E.; Genini, D.; Shih, H.; Carrera, C. J.; Cottam, H. B.; Carson, D. A. J. Natl. Cancer Inst. 2000, 92, 217. (b) Tahir, S. K.; Han, E. K-H.; Credo, B.; Jae, H. S.; Pietenpol, J. A.; Scatena, C. D.; Wu-Wong, J. R.; Frost, D.; Sham, H.; Rosenberg, S. H.; Ng, S. C. Cancer Research 2001, 61, 5480. (c) Jordan, M. A.; Wilson, L. Nature Rev. Cancer 2004, 1, 253. (d) Chen, Z. H.; Merta, P. J.; Lin, N. H.; Tahir, S. K.; Kovar, P. K.; Sham, H. L.; Zhang, H. Y. Mol. Cancer Ther. 2005, 4, 562. (a) Ainscough, E W.; Brodie, A. M.; Coll, R. K.; Mair, A. J. A.; Waters, J. M. J. Organomet Chem. 1996, 509, 259. (b) Anson, C. E.; Ditzel, E. J.; Fajardo, M.; Holden, H. D. Johnson, B. F. G.; Lewis, J.; Puga, J. Raithby, P. R. J. Chem. Soc., Dlaton Trans. 1984, 2723. Lee, S. M.; Cheung, K. K.; Wong, W. T. J. Cluster Sci. 1996. 7, 435. Li, C. X.; Lai, M. Y. D.; Leong, W. K. J. Organomet. Chem. 2005, 690, 3861. (a) Hanif, M. K.; Kabir, S. E.; Mottalib, M. A.; Hursthouse, M. B.; Malik, K. M. A.; Rosenberg, E. Polyhedron 2000, 19, 1073. (b) Monari, M.; Pfeiffer, R.; Rudsander, U.; Nordlander, E. Inorg. Chim. Acta 1996, 247, 131. Ellman, G. L. Arch Biochem Biophys. 1959, 82, 70. Sahaf, B.; Heydari, K.; Herzenberg, L. A. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 4001. Wilson, L.; Jordan, M. A. Nature Rev. Cancer 2004, 4, 253. (a) Tahis, S. K.; Trogadis, J. E.; Stevens, J. K.; Zimmerman, A. Biochem. Cell Biol. 1992, 70, 1159. (b) Wilson, R. G.; Jr-Tahir, S. K; Mechoulam, R.; Zimmerman, S.; Zimmerman, A. M. Cell Biol. Int. 1996, 20, 147. (c) Hamel, E. Biochemistry 1996, 16, 7. (d) Ponstingl, H.; Krauhs, E.; Little, M.; Kempf, T. 105 Chapter Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 2757. (e) Roychowdhury1, M.; Sarkar1, N.; Manna1, T.; Bhattacharyya1, S.; Sarkar1, T.; BasuSarkar1, P.; Roy, S.; Bhattacharyya, B. Eur. J. Biochem. 2000, 267, 3469. (f) Nogales, E.; Wolf, S. G.; Downing, K. H. Nature 1998, 391, 199. (g) Krauhs, E.; Little, M.; Kempf, T.; Hofer-Warbinek, R.; Ade, W.; Ponstingl, H. PNAS 1981, 78, 4156. (h) Jean Legault, L.; Gaulin, J. F.; Mounetou, E.; Bolduc, S.; Lacroix, J.; Poyet, R.; Gaudreault, R. Cancer Research 2000, 60, 985. (c) Yong, M. L.; John, B. D. Cancer Research 1999, 59, 776. Luduena, R. F.; Roach, M. C. Pharmacol. Ther. 1991, 49, 133. (i) Lee, Y. C.; Yaple, A.; Baldridge, R.; Kirsch, M.; Himes, R. H. Biochim Biophys Acta. 1981, 671, 71. (j) Ikeda, Y.; Iner, M. Biochemistry 1978, 17, 3454. (k) Bai, R.; Lin, C. M.; Nguyen, N. Y.; Liu, T.; Hamel, E. Biochemistry 1989, 28, 5606. 10 (a) Tahir, S. K.; Trogadis, J. E.; Stevens, J. K.; Zimmerman, A. M. Biochem. Cell Biol. 1992, 70, 1159. (b) Wilson, R. G.; Jr., Tahir, S. K.; Mechoulam, R.; Zimmerman, S.; Zimmerman, A. M. Cell Biol. Int. 1996, 20, 147. (c) Hamel, E. Biochemistry 1996, 16, 207. (d) Jordan, M. A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med. Res. Rev. 1998, 18, 259. 11 Bai, R. B.; Pettit, G. R.; Hamel, E. J. Biol. Chem. 1990, 265, 17141. 12 (a) Jordan, M. A.; Margolis, R. L.; Himes, R. H.; Wilson, L. J. Mol. Biol. 1986, 187, 61. (b) Singer, W. D.; Jordan, M. A.; Wilson, L.; Himes, R. H. Mol. Pharmacol. 1989, 36, 366. 13 (a) Nogales, E.; Wolf, S. G.; Khan, I. A.; Luduena, R. F.; Downing, K. A. Nature 1995, 375, 424. (b)Nogales, E. Annu. Rev. Biophys. Biomol. Struct. 2001,30, 397. 14 Rowinsky, E. K. Annu. Rev. Med. 1997, 48, 353. 106 Chapter 15 (a) Ikeda, Y.; Steiner, M. Biochemistry 1978, 17, 3454. (b) Li, Q.; Sham, H.; Rosenberg, S. Ann. Rep. Med. Chem. 1999, 34, 139. 16 (a) Gaskin, F. Methods Enzymol. 1982, 85, 433. (b) Gaskin, F.; Cantor, C. R.; Shelanski, M. L. J. Mol. Biol. 1974, 89, 737. (c) Berne, B. J. Mol.Biol. 1974, 89, 737. 17 Lowe, J.; Li, H.; Downing, K. H.; Nogales, E. J. Mol. Biol. 2001, 313, 1045. 18 Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C. J. Immunol. Methods 1995, 184, 39. 107 Chapter 5.1 Conclusion We have demonstrated the successful synthesis of the water-soluble osmium carbonyl cluster analogues of a fatty acid salt, [Na]+2a-, and a phosphotidylcholine analogue, 3. The osmium carbonyl cluster moiety in both compounds have been shown to serve as a tag in the mid-infrared for cell imaging via IR microscopy coupled with a focal plane array detector. They are stable under physiological conditions, and exhibit low cytotoxicity at spectroscopically useful concentrations. We have also discovered that osmium carbonyl clusters may possibly represent a new class of pharmaceuticals for the treatment of cancers. Three clusters, viz., Os3(CO)10(NCCH3)2, 5a, Os6(CO)18, 7a, and Os6(CO)16(NCCH3)2, 7b, were found to exhibit good anticancer potential against ER+ breast carcinoma (MCF-7), ER- breast carcinoma (MDA-MB-231), metastatic colorectal adenocarcinoma (SW620) and hepatocarcinoma (Hepg2), with IC50 values between 5-10 M. In particular, 5a showed a significantly higher cytotoxicity against MDA-MB-231 compared to a normal epithelial cell line (MCF-10A). The cell growth inhibition has been demonstrated through a number of assays to be due to the induction of apoptosis. Through a combination of spectroscopic and biochemical evidence, it is also suggested that 5a interacted with intracellular carboxylate and sulfhydryl residues. In particular, it binds to the sulfhydryl residues of tubulins which in turn stabilizes the microtubule structure. We have thus demonstrated that 5a represents a promising new class of tubulin-binding compound with antimitotic activity. Our findings may be summarized pictorially below: 108 Chapter 109 Chapter 5.2 Future work Our results show that osmium carbonyl clusters can be used as a mid-IR tag for whole cell imaging. The natural extension can be in two directions: (a) selective imaging of organelles, and (b) tissue imaging. Both 5a and Na+[2a]- accumulated selectively in the organelle and membrane fractions. It is therefore of interest to examine if functionalized osmium carbonyl clusters targeting specific organelles, such as the nucleosome or vacuoles, may be synthesized. These compounds can aid us in understanding cellular events, such as intracellular trafficking of organelles. Similarly, osmium carbonyl cluster derivatives that can selectively accumulate in cancer cells, for example, those containing a thymine moiety, may be useful in the differentiation between cancer and normal cells via IR imaging. Our preliminary structure-activity relationship study and the cellular mechanism studies have shown that the key factor in inducing apoptosis and cell cycle arrest is the chemical reactivity of the clusters towards –SH residues on tubulin. Investigations should therefore be extended to the preparation of water-soluble, reactive clusters; the water solubility should enhance the uptake of the clusters. Unlike compared to 5a, the hexaosmium analogues 7a and 7b exhibited equal cytotoxicities, and they are as cytotoxic to cancer as to normal cells. This suggests that the mechanism for the hexaosmium clusters is different from that for the triosmium clusters, even though apoptosis is induced in both cases. Investigations into mode of action of the hexaosmium clusters may thus open up other possible targets in the apoptotic pathway. 110 [...]... cell 85 pellet of MDA-MB-231 treated with 40 M of 5a 4.2 1 H NMR spectrum (CDCl3 solution) of chloroform extract of 86 MDA-MB-231 cells treated for 24 h with 40 M solution of 5a 4.3 Diagrammatic representation of Os3(-H)(CO)10(-O2CR), 86 Os3(-H)(CO)10(-Cl) and Os3(-H)(CO)10(-SR) 4.4 ToF-SIMS spectrum of MDA-MB-231 cells treated for 24 h with 87 40 M solution of 5a 4.5 Effect of sulfhydryl biomolecules... 3.4 Schematic of cell cycle 68 3.5 Caspase activation induces PARP cleavage 71 4.1 Sulfhydryl group with Ellman’s reagent 90 4.2 Interaction of 5a or TRM with sulfhydryl groups 92 List of tables xiv Table caption 3.1 Inhibition of cell growth activity (IC50, M) of the series of Page 58 triosmium carbonyl clusters on the six cell lines after 24 h incubation, determined by MTS assay 3.2 Osmium content... Proposed mechanism of redox activation of ferrocifen 12 2.1 Retrosynthetic analysis for phosphotidylcholine osmium carbonyl 24 cluster 3 2.2 Synthetic pathway to compound 3 26 2.3 Synthesis of Na+[2a]- 28 2.4 Yellow MTT is reduced to purple formazan in the mitochondria 28 of living cells 3.1 Formation of formazan from MTS 56 3.2 Process of apoptosis and necrosis 63 3.3 Detection of membrane phospholipid... 2.10 IR spectra of cell fractions for HL-60 cells treated with 39 compound Na+[2a]- (top) and 3 (bottom) Boxed region shows carbonyl (CO) stretching of the organometallic fragments 2.11 The IR spectra of HL-60 cells after incubation in a solution of 40 Na+[2a]- for various periods of time Boxed region shows carbonyl (CO) stretching of the organometallic fragments 3.1 Optical images of (a) MDA-MB-231... stretches of lipids (middle); and at 1650 cm-1, corresponding to the amide I stretches 2.7 Absorbance profile of of Na+[2a]- (top) and 3 (bottom) at 2013 35 cm-1 across a single mucosa cell Inset shows an optical image (top) and the sampling points (bottom) 2.8 The intensity at 2010 cm-1 as a function of soaking time of 36 mucosa cells in an aqueous solution (0.032 M) of Na+[2a]- 2.9 Washout study of compound... incubation 97 with 10 M solution of b) 1, c) 5a, d) 7b 4.13 ToF-SIMS spectrum of protein (a) and tubulin (b) of MDA-MB- 98 231 cells treated for 24 h with 40 M solution of 5a 4.14 Tubulin polymerization assays with 5a and 7b Plot of 99 absorbance at 340 nm vs treatment time List of Scheme xiii Scheme caption 1.1 Page Vitamin B12 serves as a methylating agent for the synthesis of 1 methionine from homocysteine... 24 h 57 incubation with 10M solutions of (from left to right) control, 5a, 7a and 7b x 3.2 Time and concentration dependence effects of 5a on the 59 proliferation of the MDA-MB-231 and MCF-7 cell lines 3.3 Optical images of (a) MDA-MB-231 cells after 6, 12 and 24 h 59 incubation with various concentrations of 5a 3.4 Figure 3.4 Comparison of cell viability of MCF-7 ( ) and MDAMB-231 ( 60 ) after 24... methylating agent for the synthesis of methionine from homocysteine (Scheme 1.1).3 Figure 1.1 Vitamin B12 and related compounds.2 Scheme 1.1 Vitamin B12 serves as a methylating agent for the synthesis of methionine from homocysteine.3 1 Chapter 1 The majority of bioorganometallic compounds, however, are synthetic Nevertheless, the application of bioorganometallic chemistry and compounds now covers a... treatment of diseases, in particular cancer, requires an understanding of the cell life cycle and apoptosis Compounds which can regulate apoptosis and overcome the apoptotis deficiency of cancer cells are therefore of high medical significance This section will therefore begin with an examination of the fundamentals of the cell cycle and apoptosis This will be followed by an examination of the anticancer...List of Figure Figure caption Page 1.1 Vitamin B12 and related compounds 1 1.2 Schematic representation of the DNA detection system with 3 ferrocenyl derivatives of DNA 1.3 Resonance energy transfer occurs from Ru(dpp) 3Cl2-biotinylated 4 nanospheres to Alexa Fluor 633 (acceptor) 1.4 Estrogen derivative of technetium 5 1.5 Structure of estradiol analogue derivatized with Cr(CO)3 6 1.6 Organometallic carbonyl . BIOORGANOMETALLIC CHEMISTRY OF OSMIUM CARBONYL CLUSTERS KONG KIEN VOON NATIONAL UNIVERSITY OF SINGAPORE 2008 BIOORGANOMETALLIC CHEMISTRY OF OSMIUM CARBONYL. discovery of the anticancer properties of some of these clusters. Chapter 1 describes the current state of bioorganometallic chemistry, in particularly research into the application of organometallic. Stability of Na + [2a] - in vivo 51 2.9.14 Estimation of cell size 51 2.10 References 52 Chapter 3 Osmium Carbonyl Clusters: A New Class of Apoptosis Inducing Agents 3.1 Cytotoxicity of osmium