Transcript Slide 1
C60: Synthesis and
Biological Activity of
Water-Soluble Fullerenes
Matthew D. Shoulders
Raines Group
October 5, 2006
1
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.
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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. Nature 1994, 368, 229-231.
http://www.ifw-dresden.de/iff/14/Equipment/fullerene/index.htm
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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.
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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
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Low Solubility of C60
Highly hydrophobic molecule
Limited solubility in many
organic solvents
Completely insoluble in water
Solvent
Solubility (mg/mL)
Water
--
hexane
40
Dioxane
41
cyclohexane
51
carbon tetrachloride
447
Benzene
1,440
toluene
2,150
carbon disulfide
5,160
Sivaraman, N. et al. J. Org. Chem. 1992, 57, 6077-6079.
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Outline
Approaches to water-soluble C60
Biological applications of C60 derivatives
HIV-1 protease (HIVP) inhibition
Neuroprotective properties
Antibacterial properties
Gene transfection and related properties
Toxicity of C60 and derivatives
Non-covalent
Covalent
Pristine C60 (unmodified)
Functionalized C60
Conclusions and Outlook
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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
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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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, 1254312549.
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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.
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Approaches to Methanofullerenes
Carbene addition exclusively
at [6,6]-ring junctions
Bingel cyclopropanation
Tsuda, M. et al. Tetrahedron Lett. 1993, 34, 6911-6912; Bingel, C. Chem. Ber. 1993, 126, 1957-1959. 20
Approaches to Methanofullerenes
Addition of diazo compounds
Suzuki, T.; Li, Q.; Khemani, K.C.; Wudl, F.; Almarsson, Ö. Science. 1991, 254, 1186-1188.
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[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.
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[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.
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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.
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Bis, Tris, and Higher Adducts of C60
Complex product mixtures and poor yields obtained from nonselective syntheses of multiple adducts
Hirsch, A. et al. Angew. Chem. Int. Ed. Engl. 1994, 33, 437-438.
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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.
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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.
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Outline
Approaches to water-soluble C60
Biological applications of C60 derivatives
HIV-1 protease (HIVP) inhibition
Neuroprotective properties
Antibacterial properties
Gene transfection and related properties
Toxicity of C60 and derivatives
Non-covalent
Covalent
Pristine C60 (unmodified)
Functionalized C60
Conclusions and Outlook
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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…
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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
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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.
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Improving HIVP Inhibitors
Zhu, Z. et al. Biochemistry 2003, 42, 1326-1333.
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Improving HIVP Inhibitors
Marcorin, G.L. et al.Org. Lett. 2000, 2, 3955-3958.
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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.
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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-tobatch
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.
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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.
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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 radicalinduced cell death
Dugan, L.L et al. Proc. Natl. Acad. Sci. USA 1997, 94, 9434-9439.
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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.
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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.
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C60 Derivatives as Antibacterials
Photodynamic therapy for
treatment of localized
bacterial infections
Screened 10-12 compounds
Effective against grampositive 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.
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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.
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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.
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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.
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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.
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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. 45
Outline
Approaches to water-soluble C60
Biological applications of C60 derivatives
HIV-1 protease (HIVP) inhibition
Neuroprotective properties
Antibacterial properties
Gene transfection and related properties
Toxicity of C60 and derivatives
Non-covalent
Covalent
Pristine C60 (unmodified)
Functionalized C60
Conclusions and Outlook
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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
Oberdorster E. Environ. Health Perspect. 2004, 112, 1058-1062.
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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.
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Early Studies on 14C-labeled C60
14C-labeled,
water-soluble C60 derivative was prepared
and toxicity to mice investigated
Oral administration
Intraperitoneal injection (500 mg/kg)
Rapid excretion of C60 in the feces
No acute toxicity
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.
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Dependence of Toxicity on Functionalization
Fullerene
Live Stain
Dead Stain
Fullerene
Live Stain
Sayes, C.M. et al. Nano Lett. 2004, 4, 1881-1887.
Dead Stain
50
Dependence of Toxicity on Functionalization
Cytotoxicity dependent on specific chemical
characteristics of fullerene derivatives
Tested on human dermal fibroblasts (48 h exposure)
Derivative
LC50 (ppb)
pristine C60
20
C3
10,000
fulleroxide
40,000
fullerol
>5,000,000
Sayes, C.M. et al. Nano Lett. 2004, 4, 1881-1887.
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Summary
Unusual properties of the buckyball have generated
interest in broadly ranging fields
Biological applications of fullerenes are broad and
rapidly evolving
Water-solubility issues have been addressed synthetically
Enzyme inhibition, gene transfection, neuroprotection, and other
biological applications may become commercially viable
52
Outlook
Continued improvement of synthetic techniques
Biological activities must be fine-tuned
Specific enzyme inhibition
Prevent membrane disruption/DNA cleavage
Toxicity issues must be more fully addressed
Selective methods for preparation of complex bis- and trisadducts
Breakdown
Pristine versus derivatized C60
Commercial interest in biological applications is growing
C Sixty focuses on neuroprotective properties--recently merged
with Carbon Nanotechnologies, Inc.
Moving C60 drugs toward clinical trials
Other companies also researching biological applications of
fullerenes (both C60 and higher fullerenes)
53
Acknowledgements
Ron Raines
Practice talk attendees
Funding Agencies
Joe
Binder
Daniel Gottlieb
Jeet Kalia
Luke Lavis
Raines group members
54