1 / 54

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

C 60 : Synthesis and Biological Activity of Water-Soluble Fullerenes. Matthew D. Shoulders Raines Group October 5, 2006. Carbon Allotropes. Diamond Graphite Fullerene. Buckminsterfullerenes discovered in 1985

arleen
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.

More Related