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C 60 : Synthesis and Biological Activity of Water-Soluble Fullerenes

C 60 : Synthesis and Biological Activity of Water-Soluble Fullerenes

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C 60 : Synthesis and Biological Activity of Water-Soluble Fullerenes

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  1. C60: Synthesis and Biological Activity of Water-Soluble Fullerenes Matthew D. Shoulders Raines Group October 5, 2006

  2. Carbon Allotropes Diamond Graphite Fullerene • Buckminsterfullerenes discovered in 1985 • Prepared in microscopic quantities via laser vaporization of graphite • Soccer ball structure proposed based on MS results • Chemistry Nobel prize awarded in 1996 Kroto, H.W.; Heath, J. R.; O’Brien, S.C.; Curl, R.F.; Smalley, R.E. Nature 1985, 318, 162-163.

  3. Preparation and Purification of C60 • Production Difficulties • Problem solved in 1990 by evaporating graphite electrodes in He(g) atmosphere • Resulted in production of >95% pure C60 • Prompted an explosion of experimental results • Further purification of C60 via chromatography or calixarene complexation Krätschmer, W. et al. Nature 1990, 347, 354-358; Atwood, J.L. et al. Nature1994, 368, 229-231. http://www.ifw-dresden.de/iff/14/Equipment/fullerene/index.htm

  4. The Structure of C60 • 12 pentagons surrounded by 20 hexagons (corannulene substructure) • Two types of ring junctions (6,6 and 5,6) • Isolated pentagon rule (pyracylene subunits) Wudl, F. Acc. Chem. Res. 1992, 25, 157-161.

  5. Important Properties of C60 • Structural • Unique geometry • High symmetry • Closed, spherical structure • 7 Ǻ diameter—can encapsulate other atoms • Electronic • Small HOMO-LUMO bandgap (3 degenerate orbitals form LUMO) • Easily reduced by up to 6 electrons • Strongly electronegative • Highly conjugated, but not “superaromatic” • Bent p bonds reduce conjugation • Photosensitizer

  6. Highly hydrophobic molecule Limited solubility in many organic solvents Completely insoluble in water Low Solubility of C60 Sivaraman, N. et al. J. Org. Chem. 1992, 57, 6077-6079.

  7. Outline • Approaches to water-soluble C60 • Non-covalent • Covalent • Biological applications of C60 derivatives • HIV-1 protease (HIVP) inhibition • Neuroprotective properties • Antibacterial properties • Gene transfection and related properties • Toxicity of C60 and derivatives • Pristine C60 (unmodified) • Functionalized C60 • Conclusions and Outlook

  8. Water-Soluble C60 • Pristine C60 can be suspended in water • Biological uses of fullerenes require genuine water solubility and little or no aggregation • Complexation with water-soluble supramolecules is one effective approach • Surfactants • Polyvinylpyrrolidone (PVP) • Cyclodextrins

  9. Non-Covalent Methods: C60-PVP Solutions • PVP is a dispersant used in cosmetics and medicines. • C60-toluene mixed with PVP-chloroform, solvents evaporated, and residue dissolved in water • Highest [C60] obtained was 400 mg/mL, using 100:0.8 PVP:C60 w/w Yamakoshi, Y.N. et al. Chem. Comm. 1994, 517-518; Sera, N. et al. Carcinogenesis 1996, 17, 2163-2169; Ungurenasu, C.; Airinei, A. J. Med. Chem. 2000, 43, 3186-3188.

  10. C60-Cyclodextrin Complexes • Non-covalent or covalent complexes enhance water solubility • Aggregation phenomena encountered with 1:1 complexes g-cyclodextrin b-cyclodextrin Andersson, T. et al. Chem. Comm. 1992, 604-606; Filippone, S. et al. Chem Comm. 2002, 1508-1509; Liu, Y. et al. Tetrahedron Lett. 2005, 46, 2507-2511; Chen, Y. et al. Tetrahedron 2006, 62, 2045-2049.

  11. Covalent Approaches: Principles of C60 Reactivity • Generally that of any electron-poor polyene • C60 can be reduced by up to 6 electrons • Most reactions occur at the 6,6-ring junction forming the thermodynamically stable product • Electron-poor nature of neutral C60 • Excellent substrate for nucleophilic attack • Electrophilic additions are less common but have been observed (halogenation, nitronium chemistry) Xie, Q.; Perez-Cordero, E.; Echegoyen, L. J. Am. Chem. Soc. 1992, 114, 3978-3980. Diederich, F.; Thilgen, C. Science 1996, 271, 317-323.

  12. Reactivity of C60 • Cycloaddition chemistry • Diels-Alder • 1,3-Dipolar cycloadditions • Carbene additions • Bingel cyclopropanation • Radical reactions • C60 is stable to: • Weak acid/base • Mild oxidizing agents • Some mild reducing agents • Other common reaction conditions including peptide coupling conditions Yamago, S. et al. J. Org. Chem. 1993, 58, 4796-4798. Diederich, F.; Thilgen, C. Science 1996, 271, 317-323.

  13. Synthesis of Fullerols • The first water-soluble, non-aggregating C60 derivatives • Structure remains ill-defined and number of hydroxyls added is variant Chiang, L.Y. et al. Chem. Comm. 1992, 1791; Chiang, L.Y. et al. J. Am. Chem. Soc. 1992, 114, 10154-10157; Li, J. et al. Chem. Comm. 1993, 1784-1785.

  14. Well-Defined, Covalent C60 Adducts • Essential for biological applications • Mono-adducts can suffer from aggregation phenomena in polar solvents • Multi-adducts can display altered properties • Covalent approaches remain the most important and developed method for solubilizing C60

  15. First Synthesis of Fulleropyrrolidines • 1,3-dipolar cycloaddition of azomethine ylides • Cycloaddition is irreversible under standard reaction conditions • Addition of up to nine pyrrolidines is possible Maggini, M.; Scorrano, G.; Prato, M. J. Am. Chem. Soc. 1993, 115, 9798-9799.

  16. Diversity of Prato’s Reaction • Starting materials commercially available or easily prepared • Wide variety of products can be obtained • Can start with N-substituted glycines or functionalized aldehydes Da Ros, T. et al. J. Org. Chem. 1996, 61, 9070-9072.

  17. Diversity of Prato’s Reaction Da Ros, T. et al. J. Am. Chem. Soc. 1998, 120, 11645; Maggini, M. et al. Chem. Comm. 1994, 305; Cusan, C. et al. Eur. J. Org. Chem. 2002, 3, 2928.

  18. C60 Peptides by SPPS • Synthesis of fulleropeptides via Fmoc protocols • Tyr-Gly-Gly-Fgu-Leu • Fgu-Gly-Gly-Phe-Leu • Gly-Orn-Gly-Fgu-Gly-Orn-Gly • Complicated by properties of the fullerene, but good yields can be obtained • DBU in DMF in the dark under Ar for deprotections Pellarini, F. et al. Org. Lett. 2001, 3, 1845-1848; Pantarotto, D. et al. J. Am. Chem. Soc. 2002, 124, 12543-12549.

  19. Stucture of a Fulleropeptide • The water-soluble fulleropeptide Fgu-(Gly-Orn)6-Gly-NH2 • Antimicrobial activity against S. aureus and E. coli Pellarini, F. et al. Org. Lett. 2001, 3, 1845-1848.

  20. Carbene addition exclusively at [6,6]-ring junctions Bingel cyclopropanation Approaches to Methanofullerenes Tsuda, M. et al. Tetrahedron Lett. 1993, 34, 6911-6912; Bingel, C. Chem. Ber. 1993, 126, 1957-1959.

  21. Approaches to Methanofullerenes • Addition of diazo compounds Suzuki, T.; Li, Q.; Khemani, K.C.; Wudl, F.; Almarsson, Ö. Science. 1991, 254, 1186-1188.

  22. [5,6] versus [6,6] additions • 4 possible adducts from single addition of a diazo compound Prato, M. et al.. J. Am. Chem. Soc. 1993, 115, 8479-8480.

  23. [5,6] versus [6,6] additions • [5,6]-open and [6,6]-closed are formed initially • [5,6]-open converts to [6,6]-closed at moderate temperatures Prato, M. et al.. J. Am. Chem. Soc. 1993, 115, 8479-8480.

  24. Diazo Additions for Fullero-Amino Acids • Wide range of diazo derivatives is accessible Isaacs, L.; Diederich, F. Helv. Chim. Acta 1993, 76, 2454-2464; Siebe, A.; Hirsch, A. Chem. Comm. 1994, 335-336.

  25. Bis, Tris, and Higher Adducts of C60 • Complex product mixtures and poor yields obtained from non-selective syntheses of multiple adducts Hirsch, A. et al. Angew. Chem. Int. Ed. Engl. 1994, 33, 437-438.

  26. Tether-Directed Remote Functionalization • Diastereoselectivity in multi-adduct formation is essential to achieve reasonable yields and purity • Methodology has been expanded to enable selective synthesis of nearly all bis- , tris-, and some higher adducts of C60 Nieregarten, J.-F. et al. Angew. Chem. Int. Ed. Engl. 1996, 35, 1719-1723. Thilgen, C.; Diederich, F. C. R. Chimie 2006, 9, 868-880.

  27. Fullerodendrimers • Methanofullerene formed by nucleophilic cyclopropanation • Most water-soluble C60 mono-adducts to date (65 mg/mL of C60 at pH = 10) • Anti-HIV activity Brettreich, M.; Hirsch, A. Tetrahedron Lett. 1998, 39, 2731-2734.

  28. Outline • Approaches to water-soluble C60 • Non-covalent • Covalent • Biological applications of C60 derivatives • HIV-1 protease (HIVP) inhibition • Neuroprotective properties • Antibacterial properties • Gene transfection and related properties • Toxicity of C60 and derivatives • Pristine C60 (unmodified) • Functionalized C60 • Conclusions and Outlook

  29. Overview of Biological Activities of C60 Derivatives • Antioxidant • DNA cleavage • Membrane disruption • Photodynamic therapy • Drug delivery (e.g. paclitaxel) • X-ray contrast agents • Inhibition of b-amyloid aggregation • Free radical sponge • Neuroprotection • Antibacterial • Gene transfection • Enzyme inhibition (HIVP, etc.) • And more…

  30. Life Cycle of the HIV Retrovirus http://www.ovc.uoguelph.ca/BioMed/Courses/Public/Pharmacology/pharmsite/98-409/HIV/AIDS_images/HIV_life_cycle.gif

  31. First Discovery of Biological Activity of a Fullerene • Hydrophobic, 7-8 Å binding pocket of HIV-1 protease (HIVP) is an attractive target for fullerene inhibition • Computational analysis suggested C60 fits snugly in the active site of HIVP • Properties • Ki = 5.3 mM (Best inhibitors are nanomolar or lower) • Toxic even against drug-resistant HIV-variants Kenyon G.L. and co-workers. J. Am. Chem. Soc. 1993, 115, 6506-6509 and 6510-6512.

  32. Improving HIVP Inhibitors Zhu, Z. et al. Biochemistry 2003, 42, 1326-1333.

  33. Improving HIVP Inhibitors Marcorin, G.L. et al.Org. Lett. 2000, 2, 3955-3958.

  34. Bis-Adduct, Nanomolar HIVP Inhibitors • Screened 10-12 cationic fullerenes • Cationic functionalities near the fullerene backbone • High nanomolar inhibition of HIVP (210 nM and 350 nM) • Low cytotoxicity • Non-toxic to other DNA- and RNA-viruses Marchesan, S. et al. Bioorg. Med. Chem. Lett. 2005, 15, 3615-3618.

  35. C60 Derivatives Scavenge Free Radicals • C60 reacts with multiple alkyl radicals (5-6 or more per fullerene) • Fullerols exhibit free radical scavenging activity • Not useful for medicine due to variant properties from batch-to-batch • C60 entrapped in PVP has the registered name Radical Sponge • Cytoprotective activity toward UV radiation (Vitamin C60 BioResearch Corp.) McEwen, C.N. et al. J. Am. Chem. Soc. 1992, 114, 4412-4414; Chiang, L.Y et al. Chem. Comm.1995, 1283-1284; Xiao, L. et al. Bioorg. Med. Chem. Lett. 2006, 16, 1590-1595.

  36. Carboxyfullerenes • Properties: • Well-defined structures • High water solubility • Strong radical scavengers (as good or better than commonly used scavengers) • Non-aggregating Dugan, L.L. et al. Proc. Natl. Acad. Sci. USA 1997, 94, 9434-9439.

  37. Neuroprotective Properties of Carboxyfullerenes • ·OH and ·O2- radicals were scavenged effectively in vitro • Protective effects on cortical neurons were studied • C3 derivative enters brain lipid membranes better than D3 derivative • Glutamate receptors were overstimulated in cortical neurons • Causes increase in free radical concentration and cell death • C3 derivative provided complete protection from free radical-induced cell death Dugan, L.L et al. Proc. Natl. Acad. Sci. USA 1997, 94, 9434-9439.

  38. In Vivo Neuroprotection • C3 derivative administered to mice intraperitoneally beginning at 2 mths. of age • Slowed neural deterioration • Delay of death Dugan, L.L. et al. Proc. Natl. Acad. Sci. USA 1997, 94, 9434-9439.

  39. C60 Derivatives as Antibacterials • Antibacterial activity of C3-carboxyfullerene • Inhibitory to gram-positive bacteria including Streptococcus B. at < 50 mg/L culture dose Tsao, N. et al J. Antimicrob. Chemother. 2002, 49, 641-649.

  40. C60 Derivatives as Antibacterials • Photodynamic therapy for treatment of localized bacterial infections • Screened 10-12 compounds • Effective against gram-positive and gram-negative bacteria • Low dark toxicity • Selective for bacterial cells • Mechanism not clear • Anionic fullerenes not as effective Tegos, G.P. et al. Chem. Biol.2005, 12, 1127-1135.

  41. C60 and DNA • Water-soluble fullerenes oxidatively cleave DNA when photo-excited • C60-oligonucleotide complexes enable site-selective cleavage (at G sites) and water solubility • Potentially applicable to photodynamic therapy (PEG derivatives) • Can water-soluble fullerene derivatives be synthesized that will bind DNA and transport it through cell membranes, without damaging it? Tokuyama, H. et al. J. Am. Chem. Soc. 1993, 115, 7918-7919; Boutorine, A.S. et al. Angew. Chem Int. Ed. Engl. 1994, 33, 2462-2465; Tabata, Y. et al. Jpn. J. Cancer Res. 1997, 88, 1108-1116.

  42. Gene Transfection • Common methods • Microinjection • Viral vectors (short DNA) • Chemical methods • Cationic lipids and polymers • Commercial reagents for transfection are available • Discovery of other methods could reduce cytotoxicity and enhance efficiency and reliability of transfection methods Isobe, H. et al. Mol. Pharm. 2006, 3, 124-134.

  43. Non-Viral Gene Delivery with C60 Derivatives Nakamura, E. et al. Angew. Chem. Int. Ed. Engl. 2000, 39, 4254-4257. Isobe, H. et al. Chem. Lett. 2001, 1214-1215.

  44. Non-Viral Gene Delivery with C60 Derivatives Optical micrographic analysis of transfection: A) Fullerene-DNA aggregates in buffer. B) Dispersed aggregates in buffer with serum. C) Incubated with COS-1 cells for 1 h. D) Overlayed with fluorescence micrograph showing expression of GFP in COS-1 cells after 2 d incubation. Isobe, H. et al. Mol. Pharm. 2006, 3, 124-134.

  45. Non-Viral Gene Delivery with C60 Derivatives • Fullerene transfection agents proved as good or better than traditional lipofection agents • Lower cytotoxicity • Higher transfection efficiency • Both transient and stable transfection possible • Fullerene does not appear to interfere with gene expression (esters cleaved in the cell?) • No problems with photo-induced DNA cleavage • Fullerene transfection agents could be an improvement over viral vectors • Not introducing a potentially harmful virus • Enable addition of larger nucleotide sequences • Methodology for large-scale synthesis of related amino-fullerene derivatives could enable commercialization Isobe H. et al. J. Org. Chem. 2005, 70 4826-4832; Isobe, H. et al. Mol. Pharm. 2006, 3, 124-134.

  46. Outline • Approaches to water-soluble C60 • Non-covalent • Covalent • Biological applications of C60 derivatives • HIV-1 protease (HIVP) inhibition • Neuroprotective properties • Antibacterial properties • Gene transfection and related properties • Toxicity of C60 and derivatives • Pristine C60 (unmodified) • Functionalized C60 • Conclusions and Outlook

  47. Fullerene Toxicity • Broad possibilities for applications of fullerenes precipitated a burst of studies on their toxicity • Study by Oberdorster showing pristine C60 toxic to fish • Earlier studies focused on in vivo localization and excretion of labeled fullerenes • Recent studies focus specifically on 2 classes of fullerenes • Pristine C60 • Dispersed in water • Solubilized by PVP • Water-soluble derivatives of C60 • Impact of functionalization on toxicity OberdorsterE. Environ. Health Perspect. 2004, 112, 1058-1062.

  48. Early Studies on 14C-labeled C60 • 14C-enriched C60 prepared and suspended in water • Suspension combined with culture medium containing human keratinocytes • Rapid uptake of C60 over 2 hours (in absence of light) • Demonstrated rapid particle-membrane association and passage into cells • No effect on proliferation rate of the cells Scrivens, W.A. et al.. J. Am. Chem. Soc. 1994, 116, 4517-4518.

  49. Early Studies on 14C-labeled C60 • 14C-labeled, water-soluble C60 derivative was prepared and toxicity to mice investigated • Oral administration • Rapid excretion of C60 in the feces • No acute toxicity • Intraperitoneal injection (500 mg/kg) • Some discomfort, but no acute toxicity • Intravenous injection • Very slow excretion (5.4% after 160 h) • Rapid accumulation in the liver (within 1 h), some in spleen and kidneys (30 h) • After 160 h, radioactivity in organs disappeared and distributed to muscle and hair • Still no acute toxicity, but accumulation in liver raises chronic toxicity concerns Yamago, S. et al. Chem. Biol. 1995, 2, 385-389.

  50. Dependence of Toxicity on Functionalization Fullerene Live Stain Dead Stain Fullerene Live Stain Dead Stain Sayes, C.M. et al. Nano Lett. 2004, 4, 1881-1887.