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.
2
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
3
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.
4
Important Properties of C60

Structural

Unique geometry


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
High symmetry
Closed, spherical structure
7 Ǻ diameter—can encapsulate other atoms
Electronic

Small HOMO-LUMO bandgap (3 degenerate orbitals form LUMO)
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Easily reduced by up to 6 electrons
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Strongly electronegative
 Highly conjugated, but not “superaromatic”


Bent p bonds reduce conjugation
Photosensitizer
5
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.
6
Outline

Approaches to water-soluble C60
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
Biological applications of C60 derivatives

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HIV-1 protease (HIVP) inhibition
Neuroprotective properties
Antibacterial properties
Gene transfection and related properties
Toxicity of C60 and derivatives
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Non-covalent
Covalent
Pristine C60 (unmodified)
Functionalized C60
Conclusions and Outlook
7
Water-Soluble C60
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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
8
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.
9
C60-Cyclodextrin Complexes
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Non-covalent or covalent complexes enhance water solubility
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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.
10
Covalent Approaches: Principles of
C60 Reactivity

Generally that of any electron-poor polyene
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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.
11
Reactivity of C60

Cycloaddition chemistry

Diels-Alder
 1,3-Dipolar cycloadditions
 Carbene additions
 Bingel cyclopropanation
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Radical reactions
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C60 is stable to:
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
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.
12
Synthesis of Fullerols
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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.
13
Well-Defined, Covalent C60 Adducts

Essential for biological applications

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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
14
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.
15
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.
16
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.
17
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.
19
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.
21
[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.
22
[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.
23
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.
24
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.
25
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.
26
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
28
Overview of Biological Activities
of C60 Derivatives

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
Antioxidant
DNA cleavage
Membrane disruption
Photodynamic therapy
Drug delivery (e.g. paclitaxel)
X-ray contrast agents
Inhibition of b-amyloid aggregation
Free radical sponge
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

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.
31
Improving HIVP Inhibitors
Zhu, Z. et al. Biochemistry 2003, 42, 1326-1333.
32
Improving HIVP Inhibitors
Marcorin, G.L. et al.Org. Lett. 2000, 2, 3955-3958.
33
Bis-Adduct, Nanomolar HIVP Inhibitors


Screened 10-12 cationic fullerenes
Cationic functionalities near the fullerene backbone

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
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.
35
Carboxyfullerenes

Properties:


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
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.
38
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
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

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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.
40
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.
41
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.
42
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.
43
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.
44
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
46
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.
47
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.
48
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.
49
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.
51
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