[60]Fullerenyl amino acids and peptides: a review of their synthesis and applications

Abstract

This review reports on the latest progress in the synthesis of fullerenyl amino acids and related derivatives, and categorises the molecules into functional types for different uses: these include directly attached fullerenyl amino acids, fullerenyl N- and C-capping amino acids, and those amino acids in which the [60]fullerene group is attached to the amino acid side chain. These first and last mentioned derivatives have the potential to be incorporated into non-terminal positions of peptides. The applications of these substrates, by integration into different biological and materials chemistry programs, are also highlighted.

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Sreenu Jennepalli

Sreenu Jennepalli was born in Burahanpally, in the Warangal district of Telangana, India. He has received a B. Sc degree from Kakatiya University in 1998 and M.Sc (Organic Chemistry) from Osmania University in 2001, before joining Dr Reddy's Research Foundation (DRF), Drug Discovery, Discovery Chemistry, Miyapur, Hyderabad, India. He worked on various projects including Anti-cancer, Metabolic disorders and Cardiovascular diseases from 2001–2008 as Associate Scientist, after which he moved to Hetero Drugs Pty Ltd, Hyderabad, India, working as a Team Leader-Scientist in New Drug Discovery from 2008–2010, on Antivirals and Metabolic disorders. In 2010, he undertook a PhD candidature at the University of Wollongong, Australia, working under the supervision of Assoc. Prof. Paul A. Keller and Prof. Stephen G. Pyne. His research interests are [60]fullerene chemistry, medicinal chemistry, synthesis of heterocycles and total synthesis of natural products.

Stephen G. Pyne

Prof. Stephen Pyne received his PhD in 1979 from the Australian National University under the supervision of Prof. Lew Mander, working on the chemical synthesis of the plant growth hormone, gibberellic acid. After postdoctoral research fellowships at Purdue University (with P. Fuchs) and Harvard (with E.J. Corey) he started his first academic appointment as a lecturer at the University of Wollongong in 1985. In 2008 he was appointed to Professor of Chemistry. He was the Young Researcher of the Year for 1992 (offered by the Australian Research Council and the von Humboldt Foundation), a Rhone Poulenc Fellow (1994), an ARC Senior Fellow (1994–1998) and won the A.J. Birch Medal for excellence in Organic Chemistry Research, from the Organic Division of the Royal Australian Chemical Institute in 2012. His main areas of research include, natural product synthesis (especially alkaloids), phytochemistry (including plants from SE Asia), the discovery of new methods for the synthesis of heterocycles, fullerene chemistry and antibacterial drug discovery.

Paul A. Keller

Assoc. Prof. Paul Keller completed his BSc(Hons) (1985) and PhD at the University of New South Wales, Australia, before undertaking an Alexander von Humboldt funded post-doctoral fellowship at the University of Wuerzburg, Germany, working in collaboration with Gerhard Bringmann. Since 1994, he has worked at the University of Wollongong, Australia, and is currently Associate Professor in Organic and Medicinal Chemistry. His interests lie in the drug design and development of new generation anti-infectives with a particular emphasis on new generation anti-bacterials and anti-virals, new chemistry of indigo, chiral ligand design for sterically hindered reactions and new fullerene chemistry.

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Introduction

A large portion of synthetic [60]fullerene chemistry is directed towards its functionalization, often providing biologically active derivatives and molecules with applications in materials science.¹ The impetus for such work was to exploit the physical properties of [60]fullerene (such as sensitization of singlet oxygen and electron acceptor characteristics) and combine this with the properties of biomolecules (water solubility and precise secondary and tertiary structure).^1e,2 For example, [60]fullerene derivatives covalently linked to peptides and proteins has been the goal of a number of research groups concerned with the application of [60]fullerene–peptide conjugates to biological problems.³ It was anticipated that the addition of biologically active compounds to [60]fullerene in a strict regioselective fashion would aid the activity and specificity of future therapies. From a material science and a medicinal chemistry perspective, fullerenyl amino acids are important targets potentially serving as central hubs in architecturally defined nanostructures or 3D-templates in drug design.^1e,4 To date fullerenyl amino acids and peptide derivatives have been prepared by the initial attachment of a handle to the fullerene followed by coupling to a protected amino acid or peptide.⁵ In recent times,^1f,5c the field has matured significantly such that there are many different types of fullerenyl amino acids that can be categorised according to the properties of the amino acid itself, allowing for a broader perspective of how they could be incorporated into different research programs. This review not only reports on latest progress in the synthesis of fullerenyl amino acids and related derivatives, but attempts to categorise the molecules into functional types for different uses: these include directly attached fullerenyl amino acids, fullerenyl N- and C-capping amino acids, and those amino acids in which the [60]fullerene group is attached to the amino acid side chain. These first and last mentioned derivatives have the potential to be incorporated into non-terminal positions of peptides. The applications of these substrates by integration into different biological and materials chemistry programs are then highlighted.

1 Synthesis of derivatives with fullerenyl substituents directly substituted to the amino acid α-carbon

There are surprisingly few examples of fullerenyl amino acids that are direct analogues of α-amino acids, i.e. the α-carbon is bonded directly to a C₆₀ carbon atom. There are two examples of this type of molecule, the well-established fullerenoprolines, and there are reports of protected versions of amino acids whereby the fullerenyl substituent is directly coupled to the α-carbon.

Fullerenoproline derivatives. The first synthesis of α-substituted fullerenyl amino acids, the fullerenoprolines (Fpr) (4) was achieved by the addition of azomethine ylides to [60]fullerene.⁶ The azomethine ylide intermediate can be generated in two different ways, either via a thermal ring-opening of aziridines 1 or via tautomerisation of iminium salts formed by the condensation of α-amino esters 2 with aldehydes 3 (Scheme 1). These reactions allow for a significant number of Fpr derivatives to be generated by using different combinations of aldehydes and amino esters.

Scheme 1 The synthesis of fulleropyrrolidines 4 can be achieved by thermal addition of aziridines or iminium salts to [60]fullerene.

Fpr analogues can be prepared with the pyrrolidine nitrogen protected (Scheme 2) or unprotected (Scheme 3).⁷ Addition of the aziridine 5 to [60]fullerene under thermal conditions formed the protected N-protected-Fpr 6. Subsequent treatment with TFA provided the secondary amine salt 7, which could then be acylated with acetic anhydride to provide 8 (Scheme 2).⁷

Scheme 2 Synthesis of 8 was achieved through thermal addition of aziridine 5 to [60]fullerene.

Scheme 3 Synthesis of Fpr peptide derivatives 16–19.

To obtain more useful Fpr derivatives for peptide synthesis, the azomethine ylides, which are generated in situ from the reaction of glycinate esters 9 and 10 with paraformaldehyde, can be added to [60]fullerene to produce the free Fprs 11 and 12, respectively. These were relatively unstable and had to be kept as dilute solutions in the dark.⁷ However, the amine group was readily functionalized using standard acylation procedures with acid anhydrides and acid chlorides (Scheme 3), to deliver diastereomeric mixtures of fullerenyl amino esters of the type 13. Alternatively the amine 11 was protected as the N-Fmoc derivative 14, and then the tert-butyl ester converted to the acid 15 by treatment with TFA. Subsequent coupling with ethyl L-alaninate under EDCI/HOAt conditions afforded the diastereomers 16 and 17. The diastereomeric ratio of 13 was determined by formation of the dioxopiperazines 18 and 19, thus eliminating the additional complexity in characterization imposed by amide rotamers.⁷

A second type of fullerenyl amino acid was initially envisaged as a methanofullerene of the type 21 (Scheme 4). Strategies towards such molecules include the addition of diphenyliminoglycinates to [60]fullerene under Bingel cyclopropanation reaction conditions which was originally reported to furnish the corresponding methano[60]fullerene derivatives 21 (Scheme 4).⁸ However subsequent extensive NMR studies revealed that the structure of the products was that of the dihydrofullerenoproline derivatives^8b 22, and not the more strained three-membered (cyclopropane) ring products 21. The dihydrofullerenoproline derivative 22b was hydrolysed to its carboxylic acid 23 using TFA yielded 23 in 90% yield.

Scheme 4 The addition of iminoglycinate to [60]fullerene under Bingel cyclopropanation reaction conditions provides the analogous [60] dihydrofullerenoproline derivative 22, with a proposed mechanism, and not the previously reported methano[60]fullerene 21.

The diacid⁹ 25 (Scheme 5) was synthesised from the previously reported bis-adduct 24 ^8b by hydrolysing the diester functionality with (CH₃)₃SnOH or BBr₃ in 65% and 50% yields respectively. This diacid 25 has been used to synthesise potential biologically useful fullerene amino acid derivatives.⁹

Scheme 5 Synthesis of diacid 25.

BF₃·Et₂O and NaCNBH₃ mediated reductive ring-opening of the dihydrofullerenoproline derivative^8b 26 yields ethyl N-benzhydrylfullerenyl[60]glycinate 27, which was N-deprotected to give ethyl fullerenylglycinate¹⁰ 28, a true fullerenyl α-amino ester, which was fully characterized as its more stable N-acetyl derivative 29. Therefore, the original structure 26 could be considered as a protected version of 28. The free amine of 28 reacted with a variety of aldehydes and ketones in a Mannich-type process to produce 5-substituted and 5,5-disubstituted fullerenoprolines 30–40. This process represents a versatile and general strategy to synthesise fullerenoprolines (Scheme 6).

Scheme 6 Synthesis of 5-substituted and 5,5-disubstituted fullerenoprolines 30–40.

The reactions of amino acid ester hydrochlorides and CS₂ with [60]fullerene in the presence of Et₃N yields novel [60]fullerene derivatives 42 (major) and 43 (minor) containing biologically active amino acids,¹¹ thioamide, and thiourea units. The thiolactam groups in compounds 42 are sensitive to moisture and can easily be hydrolyzed to the corresponding lactams (Scheme 7).

Scheme 7 Reactions of [60]fullerene with amino acid esters and CS₂.

2 Synthesis of [60]fullerenyl N-capped peptides and amino acids

The thermal addition of the diazomethane derivative 44 to [60]fullerene and subsequent deprotection of the ester of the resulting methano[60]fullerene 45 provided the acid 46.¹² This represented the first synthesis of a fullerenyl compound which had a synthetic handle to readily allow for peptide functionalization (Scheme 8). The acid 46 was converted to the reactive acid chloride 47 which underwent amide coupling with a pentapeptide under basic conditions to provide the first reported fullerenyl peptide 48.¹²

Scheme 8 Synthesis of 48 through a diazo-addition of 44 to [60]fullerene.

This work was extended to alkyl diazoacetates of the type 49, which was added to [60]fullerene forming the methano[60]fullerene 50, which was deprotected to the carboxylic acid 51 and then coupled to amino acids under standard DCC coupling reaction conditions to form fullerenyl amino acid esters of the type 52.¹³ Alternatively, a more efficient route was developed, that allowed the direct addition of diazoamides to [60]fullerene. For example, a solution of [60]fullerene in toluene was treated with the diazoamide 53 at reflux for 48 h providing the fullerenyl amino ester 52 (R = Bn) in 30% yield (Scheme 9).¹⁴

Scheme 9 Synthesis of 51 through a diazo-addition of 49 or 53 to [60]fullerene.

Thermal reactions of [60]fullerene with other diazo compounds such as diazomethanes,¹⁵ diazoacetates,¹⁶ diazoamides, diazomethylphosphonates,¹⁷ and diazoketones, provides a broad variety of methano[60]fullerenes, having handles for further functionalization. The methano[60]fullerene 51 can be accessed from either the tert-butyl ester 54 (Scheme 10) or the O-glycolic ester 49 (Scheme 9), followed by ester deprotection of the fullerenyl adducts 55 and 50, respectively. The synthetic utility of the methano[60]fullerenyl carboxylic acid 51 was demonstrated by its DCC/HOBt mediated amine coupling with peptides to produce the fullerenyl peptides 56 and 52.¹⁸

Scheme 10 Synthesis of peptide 56 was achieved through addition of 54 to [60]fullerene. (i) H-Thr-Thr-Asn-Tyr-Thr-OH, DCC, HOBt, C₆H₅Br–DMSO (6 : 1).

The Bingel reaction conditions have been modified to work, in moderate to excellent yields, with ketones, esters and iminoglycinates. This modification has allowed for the generation of the analogous α-haloanion in situ rather than the previous isolation of the halogenated intermediate.¹⁹ This efficient and reliable one-pot reaction has been used extensively with malonic esters 57 and derivatives, as illustrated in Scheme 11.^19a,20

Scheme 11 Malonates add to [60]fullerene under Bingel cyclopropanation conditions to provide methanofullerenes 58.

Thermolysis of sulfones 59 in the presence of [60]fullerene in 1,2,4-trichlorobenzene at reflux under a nitrogen atmosphere afforded the fullerene derivatives 60 (via the corresponding pyrimidine o-quinodimethanes) (Scheme 12). A similar reaction with 61 did not give the expected adduct but the dehydration product 63 (Scheme 12).²¹ Acetylation of 61 to 62 before the cycloaddition reaction did not prevent the formation of compound 63.

Scheme 12 Synthesis of [60]fullerene based tetrahydroquinazolines.

Organic azides act as 1,3-dipoles and can undergo [3 + 2]-cycloaddition reactions (either photochemically or thermally) with [60]fullerene to yield fulleroaziridines (Scheme 13).²² Of these organic azides, photochemical or thermal reactions of [60]fullerene with perfluorophenylazide 64 produces the fulleroaziridine 65 with an activated N-hydroxysuccinamide ester. This active ester allows further functionalization, such as peptide coupling resulting in the formation of 66.

Scheme 13 Synthesis of fulleroaziridine.

The reaction of [60]fullerene with organolithium and Grignard reagents have been exploited to incorporate a protected carboxylic acid functionality onto the C₆₀ sphere.²³ The organometallic reagent made from the stable bicyclic orthoester 67, prepared by following the general procedure described by Corey,²⁴ has been successfully added to [60]fullerene to give adduct 68. This was converted to the acid 70 by two sequential hydrolysis steps and then coupled with L-alanine ethyl ester hydrochloride to give 71 (Scheme 14).

Scheme 14 Synthesis of 1-hydro-2-[3′-(L-Ala-OEt)propyl]-1,2-dihydro[60]fullerene 71.

Methano[60]fullerene carboxylic acid derivatives 51 and 72–74 were converted to their active esters of N-hydroxysuccinimide (NHS) 75–78 and pentafluorophenol (PFP)²⁵ 79–82 and coupled with amino acids and peptides. These active esters have been reacted selectively with the amine group of the amino acid or peptide, or the hydroxy group but only under DMAP catalysis (Scheme 15).

Scheme 15 Derivatization of 51 and 72–74 with amino acids and peptides.

3 Synthesis of [60]fullerenyl C-capped peptides and amino acids

Alternative methods to generate methano[60]fullerene derivatives via addition/elimination mechanisms have employed sulfonium and phosphonium ylides.²⁶ Deprotection of the ester moieties in these methano[60]fullerenes, as well as the malonate derivatives, has provided access to versatile handles, including the carboxylic acid 51 which was generated by the addition of the sulfonium ylide 87 to [60]fullerene, followed by cleavage of the tert-butyl ester 55 with p-TsOH (Scheme 16).²⁷ The acid 51 was then converted to its acid chloride 88, which was subsequently treated with tributyltin azide to deliver the acyl azide 89 in good yield. Exposure of 89 to o-xylenes at reflux was expected to have afforded the isocyanate 90, which was not isolated but trapped as the tert-butyl carbamate derivative 91. Treatment of 91 with TfOH provided the amine salt 92, which was coupled to various acyl chlorides 93 to generate the corresponding amide derivatives 94 (Scheme 16).²⁸

Scheme 16 Synthesis of amides 94 was achieved through a Curtius rearrangement.

Thermal ring opening of methylenecyclopropanone ketal 95 in chlorobenzene in the presence of [60]fullerene followed by silica gel hydrolysis of the resulting ketene acetal gave a mixture of cycloadducts 96 and 97.²⁹ The corresponding fullerenyl amino ester derivatives 98 and 99 were subsequently prepared from 96 by DCC coupling (Scheme 17).³⁰

Scheme 17 Synthesis of fullerenyl amino ester derivatives via [3 + 2]-cycloaddition reactions.

Protected bis-fulleropyrrolidine amino acids derivatives 103 and 104 have been synthesised and characterised from two successive [3 + 2]-cycloaddition reactions of azomethine ylides to [60]fullerene, prepared in situ from the reactions of N-glycine derivatives and formaldehyde (Prato reaction). These reactions resulted in two different fullereopyrrolidine couples having unsymmetrical, orthogonally protected amino and acid functional groups.³¹ These peptides could be used as models for fullerene-based peptidomimetics with the carbon sphere inserted into the peptide backbone (Scheme 18).

Scheme 18 Synthesis of mono-fulleropyrrolidines and unsymmetrical bis-fulleropyrrolidines.

The 1,3-dipolar cycloaddition reaction of the N-substituted glycine derivative 105 to [60]fullerene produced the fulleropyrrolidine 106 having a hydroxyl group at the terminus of the N-substituent (Scheme 19). Subsequent esterification of this alcohol with the C-terminus of the protected amino acids gave 107 and 108. Deprotection of 108 to its TFA salt 109 and then coupling with a hexapeptide followed by final deprotection with TFA resulted in the fullereno peptide 110, having an oxidised methionine residue.³²

Scheme 19 Synthesis of [60]fulleropeptides.

4 Synthesis of fullerenyl amino acids that could potentially be incorporated into peptide chains

The synthesis of highly functionalized benzo-annulated indane-based α-amino acid (AAA) derivatives was reported via a [4 + 2]-cycloaddition strategy using the sultine derivative 111, containing an AAA ester moiety, as a reactive diene component. By adopting this strategy, a new α,α-dialkylatedindane-based [60]fullerene containing a rigid AAA unit 112 was realized (Scheme 20).³³

Scheme 20 Synthesis of α,α-dialkylatedindane-based [60]fullerene containing AAAs.

A Diels–Alder approach has been developed to prepare dicarba analogues of cystine. Treatment of diene 113 with [60]fullerene in toluene at reflux furnished the stable cycloadduct 114 in good yield (Scheme 21).³⁴

Scheme 21 [60]Fullerene-based dicarba analogue of cysteine 114.

Ketone 117 has been used to synthesise [60]fullerene substituted D-phenylalanine derivatives (Scheme 22).^3d The para-amino substituted phenylalanine derivatives 115 and 116 readily undergo condensation reactions with 117 forming the analogous imines 118 and 119, which were converted to their corresponding amines 120 and 121 by borane reduction. The N-Boc-glycine derivative 120 was coupled to ethyl glycinate under HBTU mediated coupling conditions to provide the peptide 122. Treatment of the methyl glycinate derivative 121 with BBr₃ led to the isolation of the free glycine analogue 123.

Scheme 22 Synthesis of [60]fullerenyl amino acids 122 and 123.

A rhodium catalyzed reaction of [60]fullerene with the boronic acid derivative of phenylalanine 124 is shown in Scheme 23. This reaction gave the fullerene-tagged amino acid 125 in 47% yield.³⁵

Scheme 23 Synthesis of 1-substituted 1,2-dihydro[60]fullerene derivatives.

A direct route was developed to produce fullerenyl peptides 127 by prolonged thermolysis of various diazoamides (e.g. 126) in the presence of [60]fullerene (Scheme 24).¹⁴

Scheme 24 Thermal addition of diazoamide to [60]fullerene provides direct access to fullerenyl amino esters.

[60]Fullerene functionalized amino acids with 4–6 methylene spacers from the α-carbon to the nitrogen atom of fulleropyrrolidine 128, and their corresponding fullerene peptides 130, have been synthesized from the reactions of N-glycine derivatives, formaldehyde and [60]fullerene under Prato's reaction conditions as shown in Scheme 25.³⁶

Scheme 25 Synthesis of [60]fullerene functionalized amino acids with 4–6 methylene spacers.

The [4 + 2]-cycloaddition of the 2-silyloxybutadiene 131 to [60]fullerene provided the ketone 117, after hydrolysis. Subsequent reduction with DIBAL-H afforded the racemic alcohol 132, a versatile synthon for the generation of fullerenyl amino acid derivatives (Scheme 26). For example, the DCC mediated esterification of 132 with alanine and glutamate derivatives provided the protected fullerenyl amino acids 133.^3c

Scheme 26 Synthesis of [60]fullerenyl amino acid 133.

[60]Fullerene pyrrolidine derivative (139), bearing two carboxylic acid functional groups has been reported (Scheme 27).³⁷ The crucial glycine precursor 137, was synthesised from commercially available starting materials and reacted with [60]fullerene and formaldehyde under Prato's reaction conditions to yield the diester 138. The tert-butyl groups of 138 were deprotected under acidic conditions, which efficiently provided the stable bis-acid derivative 139. This diacid derivative was readily functionalized under solid phase conditions to yield the derivatives dipeptide 140 and PEG 141. In addition, the reaction of 138 with TFA and TFAA provide an anhydride intermediate, which was used to synthesise [60]fullerene derivatives bearing two different amide moieties 142.

Scheme 27 Synthesis of bis-carboxylic acid group functionalized [60]fullerene derivatives.

The [60]fullerene derivative 143 was deprotected with TFA to give a free amino functional linker containing [60]fullerene derivative 144. This was then reacted with N-Fmoc-L-glutamic acid α-tert-butyl ester to yield a [60]fullerene functionalized amino acid. The tert-butyl ester was deprotected under acidic conditions to give the carboxylic acid 145, and this acid was subjected to solid-phase peptide synthesis with peptide 146. The product 147 was cleaved from the resin support and was found to be was water soluble (Scheme 28).³⁸

Scheme 28 Solid phase synthesis of fullereno peptide 147.

Applications of [60]fullerene amino acids and peptides

The potential uses of amino acid and peptide derivatives of fullerenes range from the biological to the material sciences. This section will outline the major areas of achievement.

Biological applications

The product 110 was biologically active against sera from Mixed Connective Tissue Disease (MCTD) and Systemic Lupus Erythematosus (SLE) patients (ELISA experiments).³²

The peptide 146 itself was not active against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), whereas the analogous [60]fullerenopeptide 147 showed antimicrobial activity against S. aureus and E. coli (Table 1).³⁸

Table 1 Antimicrobial activity of peptides 146 and 147, reported as MIC (Minimum Inhibitory Concentrations). Results are the mean of a minimum four independent evaluations run in duplicate (na = nonactive)

Peptide	146	147
S. aureus	Na	8 μM
E. coli	Na	64 μM

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The antioxidant properties of the water soluble amino acid derivatives of the sodium salts of fullerenylaminobutyric acid (C₆₀-γ-ABNa, 148) and fullerenylaminocaproic acid (C₆₀-ω-ACNa, 149) as well as that of the hybrid structure based on a N-fullerenoproline and the natural oxidant carnosine (C₆₀-Pro-carnosine, 150) (Fig. 1) have been studied. Their roles in the inhibition of herpes virus infection have also been described.³⁹ The amino acid fullerene derivatives 148 and 149 were found to have significant antioxidant activities and were not cytotoxic (IC₅₀ 1000 and 1200 μg mL⁻¹ (mln. cells), for 149 and 148, respectively). These derivatives were also studied as inhibitors of cytomegalovirus infection (CMVI). The introduction of C₆₀-γ-ABNa 148, into infected human embryonic fibroblasts (HEF) reduced the concentration of virus proteins in the cells to values approaching that in non-infected cells (Table 2). The malonic dialdehyde (MDA) concentration in the infected HEF culture also decreased to the concentration in intact HEF. The drug ganciclovir also decreased the lipid peroxidation (LPO) level in the HEF culture but had significantly lower antioxidant and inhibitory effects than 148 (Table 2). These results indicated that the antioxidant activity of these fullerene derivatives played an important role in their antiviral effect against CMVI. Compound 148 was considered as a potential chemotherapeutical drug against CMVI. Its chemotherapeutical index (CTI) was found to be 5000, which fivefold exceeds that of ganciclovir.

Fig. 1 [60]Fullerene based water soluble amino acids.

Table 2 Contents of the protein and MDA in the HEF cultures^a

Sample	Protein content/mg (mln. cells)^−1	[MDA] × 10^−5/mol L^−1 (mln. cells)^−1
a Note: the concentration of ganciclovir and C60-γ-ABNa 148, in the cell culture was 2 × 10^−5 mol L^−1(mln. cells)^−1. The average values of five measurements were presented.
HEF	0.07 ± 0.02	3.75 ± 0.03
HEF-CMV	0.284 ± 0.03	6.8 ± 0.01
HEF-CMV-gancyclovir	0.22 ± 0.04	5.4 ± 0.03
HEF-CMV-C60-γ-ABNa (148)	0.072 ± 0.02	3.8 ± 0.03

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As part of an ongoing antibacterial drug research project a series of mono substituted and disubstituted peptide fullerene derivatives (Fig. 2) were synthesised.⁹ These were prepared from the acid 23 and diacid 25, respectively. Although these derivatives did not show any substantial anti-bacterial activity, their synthesis has progressed the development of the important field of fullerenyl amino acids and peptide derivatives, in particular, their synthesis and isolation as peptide salts (156–160) and the high incorporation of basic amino acids with side chains. This is highlighted by the reasonable solubility of the deprotected compounds (156–160) in DMSO/water and the relative ease of purification of the protected fullerenyl peptides (151–155 and 161) by column chromatography. Further, these fullerenyl amino acids have a more rigid, and thus more defined, tethered structure than many other fullerenyl peptides reported, which traditionally employ a flexible fullerene–peptide linker. These more rigid properties could become increasingly important in the fields of medicinal chemistry or materials science.

Fig. 2 [60]Fullerene based novel peptides (151–155 and 161) and dicationic peptoids (156–160).

Antioxidative/anti-inflammatory activity of the fullerene–polyamine (FUL–PA) conjugates 162–169 has been described.⁴⁰ Conjugation of [60]fullerene with spermine (SPM) or spermidine (SPD) resulted in a large improvement of both the anti-lypoxygenase (LOX) and the anti-lipid peroxidation activity of the unconjugated molecules. An enhancement in the anti-inflammatory potency of [60]fullerene was observed only with the conjugate 163 (42%), which was comparable in activity to the reference compound indomethacin (47%). Most of the FUL–SPD conjugates, and especially compounds 163 and 165 were of comparable toxicity, even at the highest concentration tested (50 mM), therefore they could be potentially safely used for possible biomedical applications (Fig. 3).⁴⁰

Fig. 3 Biologically active novel fullerene–polyamine (FUL–PA) conjugates.

The glycopeptide antibiotic derivative teicoplanin ψ-aglycone has been covalently attached to a fullerenopyrrolidine derivative using azide–alkyne “click chemistry”. The aggregation of the resulting antibiotic–fullerene conjugate 170 (Fig. 4) in aqueous solution has been studied resulting in nano-sized clusters.⁴¹ The conjugate exhibited antibacterial activity against Enterococci resistant to teicoplanin (Table 3).

Table 3 Antibacterial activity of compound 170^a

Bacteria	Teicoplanin	Compound 170
	MIC (μg mL^−1)
a Note: MIC: Minimum Inhibition Concentration, ATCC: American Typed Culture Collection, B. subtilis: Bacillus subtilis; E. faecalis: Enterococcus faecalis, MRSA: methicillin resistant Staphylococcus aureus, vanA: vanA gene positive, vanB: vanB gene positive (Fig. 4).
B. subtilis ATCC 6633	0.5	16
S. aureus MSSA ATCC 29213	0.5	8
S. aureus MRSA ATCC 33591	0.5	4
S. epidermidis	4	2
ATCC 35984 biofilm
S. epidermidis mec A	16	1
E. faecalis ATCC 29212	1	6
E. faecalis 15376 VanA	256	16
E. faecalis ATCC 51299 VanB	0.5	16

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Fig. 4 Teicoplanin ψ-aglycon–fullerene conjugate 170.

The water-soluble dendrofulleropyrrolidine 171 (synthesised from 101) and its bis-analogues 172 and 173 have been prepared.⁴² These cationic species can efficiently complex plasmid DNA as demonstrated by gel electrophoresis studies. These molecules showed excellent DNA binding efficiencies and were therefore considered to be potential candidates for DNA binding therapies (Fig. 5).

Fig. 5 Monoadduct 171 and bis adducts 172 and 173 of highly water soluble polycationic [60]fullerenes.

The fulleropyrrolidine peptides containing only GABA (γ-aminobutyric) residues, having zero or one glycine moiety have been reported (Fig. 6).⁴³ The authors claim that these may be used as nanobioparticles. However, no results related to these properties were reported.

Fig. 6 Fulleropeptides 175–185 synthesized from fulleropyrrolidinic acid 174.

Mitsutoshi et al. reported the inhibitory effects of [60]fullerene derivatives 186 ([60]fullerene-proline-N-acetic acid) and 187 ([60]fullerene-ethylenediamine-N,N′-diacetic acid) on acetylcholine-induced (endogenous NO) relaxation in endothelium-intact rabbit thoracic aorta precontracted by phenylephrine (10⁻⁶ M).⁴⁴ The [60]fullerene derivatives 186 (10⁻⁵ M) and 187 (10⁻⁵ M) reduced the maximum amplitude of the acetylcholine-induced relaxation without significantly changing the pD2 values obtained from the concentration response curves (Fig. 7).

Fig. 7 [60]Fullerene derivatives 186 ([60]fullerene-proline-N-acetic acid) and 187 ([60]fullerene-ethylenediamine-N,N′-diacetic acid).

Water soluble [60]fullerene derivatives 188–190 (WSFD), have been studied to show the potential to prevent NO-induced cytotoxicity without obvious toxicity.⁴⁵ These fullerene derivatives apply protective activities by direct interaction with NO and neutralization of O₂⁻. The folacin fullerene derivative 190 (FFD) self-assembles to form spherical aggregates because of hydrogen bonding, and the aggregate ion morphology impacts on the NO-scavenging activities of the WSFD. Pretreatment of the cells with WSFD prior to the sodium nitroprusside (SNP) exposure blocks NO-induced cellular events including oxidation of membrane lipids, depolarization of mitochondrial membranes, reduction of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidise (GSH-Px) activities and caspase-mediated apoptotic cell death. Apart from the biological studies, the authors have revealed the synthesis of novel fullerene derivatives with anti-apoptotic properties (Fig. 8).^45,46

Fig. 8 Chemical structures of (188) AFD, (189) VFD and (190) FFD.

Oxidized [60]fullerene glutathione derivatives have been synthesized and characterized.^46,47 The [60]fullerene glutathione derivative 192 was soluble in dimethylsulfoxide, dimethylformamide and dimethylacetamide. Rat pheochromocytoma (PC12) cells were treated with hydrogen peroxide, cytotoxicity and apoptotic death was estimated by the MTT assay, flow cytometry analysis, PI/Hoechst 33342 staining and glutathione assay. These results indicated that the glutathione [60]fullerene derivative 192 has the potential to prevent oxidative stress-induced cell death without toxicity (Fig. 9).⁴⁶

Fig. 9 Cystein (191) and oxidized glutathione (192) [60]fullerene derivatives.

Materials chemistry applications

A new class of peptide nanotubes from α,γ-cyclic peptides (CPs) 193 tethered to a methanofullerene moiety have been reported.⁴⁸ These CPs are able to form nanotubes in which the fullerenes point outward from the nanotube on both sides (180° orientation). The fullerenes form two parallel wires separated by the insulating peptide nanotube. The authors suggest that these materials may have applications as nanowire components and/or in optical and electronic devices (Fig. 10).

Fig. 10 Structure of tethered α,γ-cyclic peptide (CP) 193.

Two molecules having a pyrrolidino[60]fullerene tethered through an intramolecular hydrogen bonding peptide linker to a nitroxide radical have been synthesised.⁴⁹ These peptide moieties have rigid 3₁₀-helical structures that possess a strong molecular dipole. The direction of the molecular dipole moment can be reversed by switching the position of fullerene and nitroxide with respect to the peptide nitrogen and carbon terminus. The fullerene-peptide-radical systems 194 and 195 were compared to the behaviours of otherwise identical peptides but without either [60]fullerene or the free radical moiety with retaining the same number of intramolecular hydrogen bonds (Fig. 11).

Fig. 11 Structures of [60]fullerene based peptides with intramolecular hydrogen bonds and dipole moments illustrated.

A strategy of photocurrent generation out of thin layers composed by the amphiphilic [60]fullerene and pyrene derivatives was investigated by Shunsaku's group.⁵⁰ Three different types of compounds namely [60]fullerene-cyclic peptide–PEG conjugate 198 (cyclo8–C₆₀–PEG), [60]fullerene–PEG conjugate 197 (C₆₀–PEG) without a cyclic peptide scaffold, and the pyrene derivative 196 (R-Pyr) having a diethylene glycol and a long alkyl chain were synthesised. The study of photoinduced electron transfer concluded that a bilayer structure with desired orientation of functional units is important for efficient photoinduced electron transfer and that cyclic peptide scaffold is useful to locate hydrophobic functional groups in a thin layer (Fig. 12).

Fig. 12 Chemical structures of R-Pyr 196, C₆₀–PEG 197 and cyclo8–C₆₀–PEG 198.

[60]Fullerene amino acid monomer 199 and the bis-fullerene 200 been studied experimentally using spectroelectrochemical measurements. The bis-fullerene-substituted peptide⁵¹ 200 provided experimental support for density-functional theory (DFT) calculations which indicate that van der Waals fullerene dimers can form between adjacent fullerenes in semiconductor layers resulting in interstitial electron traps. The fullerenes behave like “super atoms” and the interstitial electron traps represent one-electron intermolecular σ-bonds. The proposed electron traps are relevant for all organic electronics applications in which non-covalently linked fullerenes in van der Waals contact with one another are used as n-type semiconductors (Fig. 13).⁵²

Fig. 13 Structures [60]fullerene amino acid monomer 199 and bis-fullerene 200.

The stereoselectivity of the formation of hybrid amino acid derivatives of fullerene (AADF) C₆₀ was studied by Dolinina et al.⁵³ The energies of the model addition reactions of two different reactions were calculated by the DFT method B3LYP/631G*. The most stable products of these reactions are hexamethylated fullerene derivatives in which five Me groups are arranged in the form of a regular pentagon and AADF. Among the AADF obtained by reactions, 1,4-disubstituted fullerene isomers are most stable. The molecular structures of such isomers were calculated for six biologically active hybrid AADF's 201–206 (Fig. 14); the solvent contact areas of these molecules were evaluated (Table 4).

Fig. 14 Structures of AADF 201–206.

Table 4 Calculated water contact areas (S/Ų) of hybrid AADF^a

Entry	X	Y	Stot (Sful)^b
a The equilibrium conformations were calculated at the B3LYP/631G*//B3LYP/631G* level.b Stot and Sful are the contact areas of the whole molecule and the fullerene fragment, respectively. The radius of the sphere of the water molecule is 1.4 Å.
C60	—	—	530 (530)
201	C2H4OH	Me	711 (443)
202	C2H4ONO2	Me	783 (440)
203	C2H4OH	CH2ONO2	818 (437)
204	C2H4ONO2	CH2ONO2	831 (412)
205	CH2CH(NO3)CH2NO3 (S)	Me	838 (423)
206	C2H4–carnosine	Me	968 (406)

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Induction of optical activity of fullerene C₆₀ by covalently bound amino acid moieties and peptide fragments (207–215) was studied by Babievsky et al.⁵⁴ using optical circular dichroism (CD) (Table 5). Significant asymmetric induction transfer was found when a stereogenic atom is adjacent to fullerene cage (Fig. 15).⁵⁴

Table 5 Circular dichroism spectra of fullerene derivatives 207–215

Entry	M	C × 10^3/mol L^−1	ΔA × 10^−6 (rel. units)	λmax/nm	Δε × 10^−2/mol L^−1 cm^−1
207	847	1.77	350	530	19.7
208	823	2.43	370	540	15.2
209	918	1.04	330	556	10.1
210	904	1.77	180	550	32.0
211	874	1.80	—	—	—
212	918	0.44	150	450	34.0
213	867	0.35	78	505	22.3
214	937	1.82	123	460	6.7
215	1027	0.30	155	499	51.6

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Fig. 15 Structures of compounds 207–215.

Investigation of energy transfer interactions between C₆₀ fullerenes and four different quantum dots (QDs), composed of CdSe/ZnS (type I) and CdSe/CdS/ZnS (quasi type II), with emission maxima ranging from 530 to 630 nm was performed by Stewart et al.⁵⁵ C₆₀–pyrrolidine tris-acid was first coupled to the N-terminus of a hexahistidine terminated peptide via carbodiimide chemistry to yield a C₆₀-labeled peptide 216 (pep-C₆₀). This peptide was studied for the ratiometric self-assembly of the QD-(pep-C₆₀) nanoheterostructures by exploiting metal affinity coordination to the QD surface. Photoinitiated energy transfer and competition between electron transfer and Förster resonance energy transfer from the QD to the C₆₀ were also studied with the aim of potential use in optoelectronic and biosensing applications (Fig. 16).⁵⁵

Fig. 16 Structure of C₆₀-labelled peptide 216.

Conclusions

In conclusion, fullerenyl amino acids and related derivatives have been classified into functional types for different uses: these include directly attached fullerenyl amino acids, fullerenyl N- and C-capping amino acids, and those amino acids in which the [60]fullerene group is attached to the amino acid side chain. These first and last mentioned derivatives have the potential to be incorporated into non-terminal positions of peptides. The applications of these substrates, by integration into different biological and materials chemistry programs, have also been highlighted.

Acknowledgements

SJ thanks the ARC Centre of Excellence for Electromaterial Science for the provision of a Scholarship.

Notes and references

1. (a) A. Bianco, T. Da Ros, M. Prato and C. Toniolo, J. Pept. Sci., 2001, 7, 346–347; (b) T. Da Ros and M. Prato, Chem. Commun., 1999, 663–669; (c) M. Satoh and I. Takayanagi, J. Pharmacol. Sci., 2006, 100, 513–518; (d) B. C. Braden, F. A. Goldbaum, B. X. Chen, A. N. Kirschner, S. R. Wilson and B. F. Erlanger, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 12193–12197; (e) N. Tagmatarchis and H. Shinohara, Mini-Rev. Med. Chem., 2001, 1, 339–348; (f) A. Bianco, T. Da Ros, M. Prato and C. Toniolo, J. Pept. Sci., 2001, 7, 208–219.
2. (a) C. S. Foote, Top. Curr. Chem., 1994, 169, 347–363; (b) E. Nakamura and H. Isobe, Acc. Chem. Res., 2003, 36, 807–815; (c) S. Bosi, T. Da Ros, G. Spalluto and M. Prato, Eur. J. Med. Chem., 2003, 38, 913–923.
3. (a) A. Bianco, M. Maggini, G. Scorrano, C. Toniolo, G. Marconi, C. Villani and M. Prato, J. Am. Chem. Soc., 1996, 118, 4072–4080; (b) A. Bianco, F. Gasparrini, M. Maggini, D. Misiti, A. Polese, M. Prato, G. Scorrano, C. Toniolo and C. Villani, J. Am. Chem. Soc., 1997, 119, 7550–7554; (c) Y. Z. An, L. J. Anderson and Y. Rubin, J. Org. Chem., 1993, 58, 4799–4801; (d) J. Yang and A. R. Barron, Chem. Commun., 2004, 2884–2885; (e) S. Bosi, L. Feruglio, T. Da Ros, G. Spalluto, B. Gregoretti, M. Terdoslavich, G. Decorti, S. Passamonti, S. Moro and M. Prato, J. Med. Chem., 2004, 47, 6711–6715; (f) D. Pantarotto, A. Bianco, F. Pellarini, A. Tossi, A. Giangaspero, I. Zelezetsky, J. P. Briand and M. Prato, J. Am. Chem. Soc., 2002, 124, 12543–12549.
4. (a) A. Bianco, T. Bertolini, M. Crisma, G. Valle, C. Toniolo, M. Maggini, G. Scorrano and M. Prato, J. Pept. Res., 1997, 50, 159–170; (b) A. Kurz, C. M. Halliwell, J. J. Davis, H. A. O. Hill and G. W. Canters, Chem. Commun., 1998, 433–434; (c) L. L. Dugan, D. M. Turetcky, C. Du, D. Lobner, M. Wheeler, C. R. Almli, C. K. Shen, T. Y. Luh, D. W. Choi and T. S. Lin, Proc. Natl. Acad. Sci. U. S. A., 2000, 94, 9434–9439; (d) G. Pastorini, S. Marchesan, J. Hoebeke, T. Da Ros, L. Ehret-Sabatier, J.-P. Briand, M. Prato and A. Bianco, Org. Biomol. Chem., 2006, 4, 2556–2562; (e) D. M. Guldi, G. M. A. Rahman, V. Sgobba and C. Ehli, Chem. Soc. Rev., 2006, 35, 471–487; (f) T. Y. Zakharian, A. Seryshev, B. Sitharaman, B. E. Gilbert, V. Knight and L. J. Wilson, J. Am. Chem. Soc., 2005, 127, 12508–12509; (g) T. Da Ros, G. Spalluto and M. Prato, Croat. Chem. Acta, 2001, 74, 743–755; (h) N. Martin, Chem. Commun., 2006, 2093–2104.
5. (a) L. A. Watanabe, M. P. I. Bhuiyan, B. Jose, T. Kato and N. Nishino, Tetrahedron Lett., 2004, 45, 7137–7140; (b) G. A. Burley, P. A. Keller and S. G. Pyne, Fullerene Sci. Technol., 1999, 7, 973–1001; (c) D. Pantarotto, N. Tagmatarchis, A. Bianco and M. Prato, Mini-Rev. Med. Chem., 2004, 4, 805–814.
6. (a) M. Maggini, G. Scorrano and M. Prato, J. Am. Chem. Soc., 1993, 115, 9798–9799; (b) M. Maggini, G. Scorrano, A. Bianco, C. Tiniolo, R. P. Sijbesma, F. Wudl and M. Prato, Chem. Commun., 1994, 305–306; (c) M. Prato and M. Maggini, Acc. Chem. Res., 1998, 31, 519–526.
7. A. Bianco, M. Maggini, G. Scorrano, C. Toniolo, G. Marconi, C. Villana and M. Prato, J. Am. Chem. Soc., 1996, 118, 4072–4080.
8. (a) G. A. Burley, P. A. Keller, S. G. Pyne and G. E. Ball, Chem. Commun., 1998, 2539–2540; (b) G. E. Ball, G. A. Burley, L. Chaker, B. C. Hawkins, J. R. Williams, P. A. Keller and S. G. Pyne, J. Org. Chem., 2005, 70, 8572–8574.
9. S. Jennepalli, K. A. Hammer, T. V. Riley, P. A. Keller and S. G. Pyne, Unpublished results.
10. R. Thayumanavan, B. C. Hawkins, P. A. Keller, S. G. Pyne and G. E. Ball, Org. Lett., 2008, 10, 1315–1317.
11. G.-W. Wang, J.-X. Li, Y.-J. Li and Y.-C. Liu, J. Org. Chem., 2006, 71, 680–684.
12. M. Prato, A. Bianco, M. Maggini, G. Scorrano, C. Toniolo and F. Wudl, J. Org. Chem., 1993, 58, 5578–5580.
13. (a) L. Isaacs and F. Diederich, Helv. Chim. Acta, 1993, 76, 2454–2464; (b) C. Toniolo, A. Bianco, M. Maggini, G. Scorrano, M. Prato, M. Marastoni, R. Tomatis, S. Spisani, G. Palu and E. D. Blair, J. Med. Chem., 1994, 37, 4558–4562.
14. A. Skiebe and A. Hirsch, Chem. Commun., 1994, 335–336.
15. (a) J.-F. Eckert, C. Bourgogne and J.-F. Nierengarten, Chem. Commun., 2002, 712–713; (b) N. Martin, L. Sanchez and D. M. Guldi, Chem. Commun., 2000, 113–114.
16. R. Pelliciari, D. Annibali, G. Constantino, M. Marinozzi and B. Natalini, Synlett, 1997, 1196–1198.
17. R. Pelliciari, B. Natalini, L. Amori, M. Marinozzi and R. Seraglia, Synlett, 2000, 1816–1818.
18. (a) L. Isaacs and F. Diederich, Helv. Chim. Acta, 1993, 76, 2454–2464; (b) C. Toniolo, A. Bianco, M. Maggini, G. Scorrano, M. Prato, M. Marastoni, R. Tomatis, S. Spisani, G. Palu and E. D. Blair, J. Med. Chem., 1994, 37, 4558–4562.
19. (a) X. Camps and A. Hirsch, J. Chem. Soc., Perkin Trans. 1, 1997, 1595–1596; (b) J.-F. Nierengarten, V. Gramlich, F. Cardullo and F. Diederich, Angew. Chem., Int. Ed., 1996, 35, 2101–2103; (c) J.-F. Nierengarten and J.-F. Nicoud, Tetrahedron Lett., 1997, 38, 7737–7740.
20. (a) C. Thilgen, A. Herrmann and F. Diederich, Helv. Chim. Acta, 1997, 80, 183–199; (b) C. F. Richardson, D. I. Schuster and S. R. Wilson, Org. Lett., 2000, 2, 1011–1014.
21. R. F. Enes, A. C. Tome and J. A. S. Cavaleiro, Tetrahedron, 2005, 61, 1423–1431.
22. (a) M. Prato, Q. Li, F. Wudl and V. Lucchini, J. Am. Chem. Soc., 1993, 115, 1148–1150; (b) M. Yan, S.-X. Cai and J. F. W. Keana, J. Org. Chem., 1994, 59, 5951–5954.
23. E. Champeil, C. Crean, C. Larraya, G. Pescitelli, G. Proni and L. Ghosez, Tetrahedron, 2008, 64, 10319–10330.
24. (a) E. J. Corey and N. Raju, Tetrahedron Lett., 1983, 24, 5571–5574; (b) E. Keinan, S. C. Sinha and S. P. Singh, Tetrahedron, 1991, 47, 4631–4638.
25. H. Tsumoto, K. Takahashi, T. Suzuki, H. Nakagawa, K. Kohda and N. Miyata, Bioorg. Med. Chem. Lett., 2008, 18, 657–660.
26. (a) Y. Wang, J. Cao, D. I. Schuster and S. R. Wilson, Tetrahedron Lett., 1995, 36, 6843–6846; (b) H. J. Bestmann, D. Hadawi, T. Roeder and C. Moll, Tetrahedron Lett., 1994, 35, 9017–9020.
27. H. Ito, T. Tada, M. Sudo, Y. Ishida, T. Hino and K. Saigo, Org. Lett., 2003, 5, 2643–2645.
28. T. Tada, Y. Ishida and K. Saigo, Org. Lett., 2005, 7, 5897–5900.
29. M. Prato, T. Suzuki, H. Foroudian, Q. Li, K. Khemani, F. Wudl, J. Leonetti, R. D. Little, T. White, B. Rickborn, S. Yamago and E. Nakamura, J. Am. Chem. Soc., 1993, 115, 1594–1598.
30. S. Yamago, H. Tokuyama, E. Nakamura, M. Prato and F. Wudl, J. Org. Chem., 1993, 58, 4796–4801.
31. D. Milic and M. Prato, Eur. J. Org. Chem., 2010, 476–483.
32. P. Sofou, Y. Elemes, E. Panou-Pomonis, A. Stavrakoudis, V. Tsikaris, C. Sakarellos, M. Sakarellos-Daitsiotis, M. Maggini, F. Formaggio and C. Toniolo, Tetrahedron, 2004, 60, 2823–2828.
33. S. Kotha and A. K. Ghosh, Tetrahedron Lett., 2004, 45, 2931–2934.
34. S. Kotha, K. Mandal, S. Banerjee and S. M. Mobin, Eur. J. Org. Chem., 2007, 1244–1255.
35. M. Nambo, Y. Segawa, A. Wakamiya and K. Itami, Chem.–Asian J., 2011, 6, 590–598.
36. L. A. Watanabe, M. P. I. Bhuiyan, B. Jose, T. Kato and N. Nishino, Tetrahedron Lett., 2004, 45, 7137–7140.
37. S. Aroua, W. B. Schweizer and Y. Yamakoshi, Org. Lett., 2014, 16, 1688–1691.
38. F. Pellarini, D. Pantarotto, T. Da Ros, A. Giangaspero, A. Tossi and M. Prato, Org. Lett., 2001, 3, 1845–1848.
39. R. A. Kotel'nikova, I. I. Faingol'd, D. A. Poletaeva, D. V. Mishchenko, V. S. Romanova, V. N. Shtol'ko, G. N. Bogdanov, A. Y. Rybkin, E. S. Frog, A. V. Smolina, A. A. Kushch, N. E. Fedorova and A. I. Kotel'nikov, Russ. Chem. Bull., 2011, 60, 1172–1176.
40. G. E. Magoulas, T. Garnelis, C. M. Athanassopoulos, D. Papaioannou, G. Mattheolabakis, K. Avgoustakis and D. Hadjipavlou-Litina, Tetrahedron, 2012, 68, 7041–7049.
41. S. Tollas, I. Bereczki, A. Sipos, E. Rőth, G. Batta, L. Daróczi, S. Kéki, E. Ostorházi, F. Rozgonyi and P. Herczegh, Eur. J. Med. Chem., 2012, 54, 943–948.
42. A. M. López, F. Scarel, N. R. Carrero, E. Vázquez, A. Mateo-Alonso, T. Da Ros and M. Prato, Org. Lett., 2012, 14, 4450–4453.
43. M. Bjelaković, N. Todorović and D. Milić, Eur. J. Org. Chem., 2012, 27, 5291–5300.
44. M. Satoh, T. Mashino, T. Nagano, M. Hirobe, I. Takayanagi and K. Koike, Fullerene Sci. Technol., 2001, 9, 141–151.
45. Z. Hu, Y. Huang, W. Guan, J. Zhang, F. Wang and L. Zhao, Biomaterials, 2010, 31, 8872–8881.
46. Z. Hu, W. Guan, W. Wang, L. Huang, H. Xing and Z. Zhu, Cell Biol. Int., 2007, 31, 798–804.
47. Z. Hu, S. Liu, Y. Wei, E. Tong, F. Cao and W. Guan, Neurosci. Lett., 2007, 429, 81–86.
48. C. Reiriz, R. J. Brea, R. Arranz, J. L. Carrascosa, A. Garibotti, B. Manning, J. M. Valpuesta, R. N. Eritja, L. Castedo and J. R. Granja, J. Am. Chem. Soc., 2009, 131, 11335–11337.
49. L. Garbuio, S. Antonello, I. Guryanov, Y. Li, M. Ruzzi, N. J. Turro and F. Maran, J. Am. Chem. Soc., 2012, 134, 10628–10637.
50. (a) S. Fujii, T. Morita and S. Kimura, Langmuir, 2008, 24, 5608–5614; (b) S. Fujii, T. Morita and S. Kimura, Bioconjugate Chem., 2007, 18, 1855–1859.
51. S. Jennepalli, P. A. Keller and S. G. Pyne, Unpublished results.
52. T. E. Shubina, D. I. Sharapa, C. Schubert, D. Zahn, M. Halik, P. A. Keller, S. G. Pyne, S. Jennepalli, D. Guldi and T. Clark, J. Am. Chem. Soc., 2014, 136, 10890–10893.
53. T. Y. Dolinina and V. B. Luzhkov, Russ. Chem. Bull., 2012, 61, 1631–1634.
54. K. K. Babievsky, V. S. Romanova, Y. A. Davidovich and I. A. Yamskov, Russ. Chem. Bull., 2013, 62, 2609–2611.
55. M. H. Stewart, A. L. Huston, A. M. Scott, E. Oh, W. R. Algar, J. R. Deschamps, K. Susumu, V. Jain, D. E. Prasuhn, J. Blanco-Canosa, P. E. Dawson and I. L. Medintz, ACS Nano, 2013, 7, 9489–9505.

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