Lisa Tanzi
email: lisa.tanzi01@universitadipavia.it

Marco Terreni
email: marco.terreni@unipv.it.

Yongmin Zhang
email: yongmin.zhang@upmc.fr.

Synthesis and biological application of glyco-and peptide derivatives of fullerene C 60

Keywords: Fullerene, C60, Carbohydrate, Peptide, Synthesis, Biological Application

Fullerenes have attracted considerable attention for their possible use in human therapy. Pure C60 is soluble only in some organic solvents, but this could be overcome by chemical modifications. This review investigates the derivatization strategies and biological applications of fullerene C60 by using polar "active" molecules as sugars and amino acids/peptides that allow the increase of solubility in water.

The effect of glycosylation on biological activity of fullerene can be divided in indirect and direct action. The "indirect action" of sugars correlates with their ability to make fullerene soluble in water but glycosylation can be also exploited for the target delivery; accordingly, glyco-derivatives of fullerenes have been investigated in PDT (photodynamic therapy), as inhibitors of in HIV-1 protease inhibition or against neurodegenerative diseases.

The "direct action" involves fullerenes conjugated with sugars having a defined therapeutic role and the "multivalency" is the properties that ensure a good biological activity of glycofullerene derivatives. Increasing the sugars attached to fullerene intensifies the multivalency needed to efficiently use these glycosylated nanoparticles as potential ligands for receptors and enzymes that mediate the infection of viruses and bacteria (e.g. E. Coli, Ebola or Dengue viruses) for the infection. Also, amino acids-derivatives of fullerenes have been studied as anti-infective agents (against viruses such as cytomegalovirus and HIV), thanks to their immunological properties; derivatives as fullerenol or by linking tuftsin on a C60 core could be exploited as immunogenic nano-carriers. Alternatively fullerene conjugated with amino acids or peptides is investigated in the treatments of pathologies that request new approaches (Alzheimer, cancer, mixed connective tissue disease, lupus).

In 1985 was discovered C60 Fullerene, a new carbon allotrope defined as a soccer ball shape pure carbon structure. This new material due to its size and composition has attracted attention of scientist belonging to different fields and homologous with higher number of carbons were also discovered.

Fullerenes are generally represented by a formula Cn, where n is the number of carbons that makes up the cage but until 1990 fullerene structure remained a matter of theory because the spectroscopy analysis was the only method to detect it. After some years also experimental studies confirmed this structure (by using analytical instruments as DCC, mass spectrometry and NMR) [START_REF] Yannoni | Characterization of fullerenes and doped fullerenes[END_REF].

Since C60 is the most representative and the most studied compound within the fullerene family, in this review the word fullerene is always attributed to C60. This is the smallest stable fullerene, has high symmetry, as confirmed by 13 C-NMR analysis and studies performed on crystalline C60 [2]. The diameter is 10.34 Å. There are two different types of bonds: the 5,6 bond that is a single bond of 1,45 Å length between pentagon and hexagon; the 6,6 that is a double bond of 1,38 Å between two hexagons that correlates with the fullerene reactivity (Figure 1).

Solubility is an important parameter to evaluate fullerene for possible chemical transformations and reactions. Ruoff R.S. et al. [START_REF] Ruoff | Solubility of C60 in a variety of solvents[END_REF] checked the solubility of C60 in different solvents: it is completely insoluble in polar solvents, sparingly soluble in alkanes, appreciably soluble in aromatic solvents.

These data were also confirmed by computational studies [START_REF] Sivaraman | QSPR Modeling for Solubility of Fullerene (C60) in Organic Solvents[END_REF]. Thus, pure C60 is highly hydrophobic and soluble only in organic solvents, in particular in aromatic ones. This is incompatible with a possible pharmacological application. Moreover, the lack of solubility in aqueous solvent leads to the consequent formation of aggregates when C60 is used in water. This problem can be solved with chemical or supramolecular approach: by using cyclodextrins, by co-solving C60 with polyvinylpyrrolidone (PVP) in chloroform or by using artificial lipids membrane [START_REF] Bosi | Fullerene derivatives: An attractive tool for biological applications[END_REF][START_REF] Ajrin | Review on fullerene: A cutting edge trend in drug delivery[END_REF]. However, the most common method regards modifications of surface by addition of molecules that change the physical and chemical property of the fullerene. Depending on the choose of the attached compounds, the derivatization increases the solubility in aqueous solvent and reduces aggregation opening to a possible application in medicinal chemistry.

Even if fullerene was discovered more than thirty years ago, it is still considered a "new material" because chemical modification and the introduction of fullerene derivatives in biomedical application is recent [START_REF] Riley | Recent advances in carbon nanomaterials for biomedical applications: A review[END_REF][START_REF] Td | A Dyanamic Organic Ball : Pharmaceutical Application[END_REF][START_REF] Sharoyko | Biologically active water-soluble fullerene adducts: Das Glasperlenspiel (by H. Hesse)?[END_REF]. Consequently, the toxicity is one of the characteristics that are still under investigation. It seems that the toxicity decreases with increasing of functionalization of fullerene [START_REF] Sayes | The differential cytotoxicity of water-soluble fullerenes[END_REF]. The pure C60 toxicity is due to three factors [START_REF] Sergio | Fullerenes toxicity and electronic properties[END_REF]:

 Its dominant apolar character that collate with its ability to penetrate and merge with biological membrane.

 The propensity to form aggregates that can have an impact mainly on the environment [START_REF] Park | No bioavailability of 17αethinylestradiol when associated with nC60 aggregates during dietary exposure in adult male zebrafish (Danio rerio)[END_REF][START_REF] Malhotra | An Update Report on the Biosafety and Potential Toxicity of Fullerene-Based Nanomaterials toward Aquatic Animals[END_REF].

 The capacity of reacting with a lot of biological relevant compounds.

Accordingly, Colvin et al. hypothesized that toxicity decreases as much as fullerene derivatives are soluble in water [START_REF] Sayes | The differential cytotoxicity of water-soluble fullerenes[END_REF], as confirmed by other studies [START_REF] Youn | Multifunctional nano-sized fullerenes for advanced tumor therapy[END_REF][START_REF] Wilson | Metallofullerene drug design[END_REF][START_REF] Monteiro-Riviere | Lack of Hydroxylated Fullerene Toxicity after Intravenous Administration to Female Sprague[END_REF].

Actually the toxicity depends on several features as dose and the method of administration but also the size of fullerene (C60, C70 etc..) and the compounds used for its derivatization, [START_REF] Lalwani | Multifunctional Fullerene-and Metallofullerene-Based Nanobiomaterials[END_REF]. So the crucial aspect is conducting a specific toxicity assay on the final product of interest [START_REF] Partha | Biomedical applications of functionalized fullerene-based nanomaterials[END_REF][START_REF] Kolosnjaj | Toxicity studies of fullerenes and derivatives[END_REF].

Chemical modification

The geometry, the high symmetry and shape of fullerene are at the base of its reactivity. Its particular shape forces carbons to have no planarity changing the hybridation of sp2 orbitals [START_REF] Haddon | Electronic structure and bonding in icosahedral C60[END_REF]; this leads to a sort of deactivation of the aromatic nature inducing a different reactivity of fullerene with respect to a normal aromatic compounds. Thus, It's better to use the term "pseudo-aromaticity" for fullerene that is considered an electronegative molecule, proper for addition at 6,6 bond and redox reactions.

The addition can be obtained in different positions: 1,2 position (for no bulky reagents) and 1,4 position (principally for bulky reagents because of their steric hindrance) [START_REF] Dixon | Patterns for addition to c60[END_REF]. (Figure 1)

Main strategies of conjugation

Different strategies have been explored to make fullerene suitable for pharmaceutical application. In order to make fullerene soluble in water and aqueous solvents, a lot of different functional group have been used (OH,-NH2, -COOR). There are three main synthetic approaches to obtain these derivatives (Scheme 1): the cyclopropanation, polyhydroxylation and cycloaddition azomethine ylides (known also as Prato reaction) [START_REF] Prato | Synthesis and electrochemical properties of substituted fulleropyrrolidines[END_REF].

SCHEME 1: Main synthetic approaches

The cyclopropanation strategy is the most used. It can be obtained by different methods as the addition of frees carbenes, the terminal addition of diazo compounds and the nucleophilic cyclopropanation with addition-elimination mechanism. The last one is the most common strategy (best yields) and it's also known as Bingel reaction [START_REF] Biglova | Nucleophilic cyclopropanation of [60]fullerene by the addition-elimination mechanism[END_REF] discovered in 1993. Bingel described a kind of Michael reaction between an α-halo carbanion and fullerene followed by intramolecular substitution of halogen in order to form a methanofullerene [START_REF] Bingel | Cyclopropanierung von Fullerenen[END_REF]. Initially NaH was used as base for the reaction, afterwards also other bases were employed (pyridine, triethylamine and LDA) [START_REF] Benito | Synthesis and Characterisation of the methanofullerenes, C60(CHCN) and C60(CBr2)[END_REF].

In order to improve the yields, some other strategies were tested, such as a one-step reaction, performed using α-halomalonates generated in situ (in presence of I2 or CBr4) and DBU (1,8diazabicyclo[5.4.0]undec-7-ene) as base [START_REF] Nakamura | Synthesis of [60]fullerene adducts bearing carbazole moieties by bingel reaction and their properties[END_REF][START_REF] Abellán Flos | Potent Glycosidase Inhibition with Heterovalent Fullerenes: Unveiling the Binding Modes Triggering Multivalent Inhibition[END_REF][START_REF] Camps | Efficient cyclopropanation of C 60 starting from malonates[END_REF][START_REF] Thilgen | Spacer-Controlled Multiple Functionalization of Fullerenes[END_REF]. The use of I2 is preferable in mono-addition (maximum three groups conjugation), otherwise tetrabromomethane can be used for higher adducts [START_REF] De La Puente | Fullerenes: Principles and Applications[END_REF].

The mechanism is always the same: the base removes an acid proton from malonate forming a carbanion which reacts with the halogen building an α-halomalonate. Again, the base removes the other malonate acid proton and the new carbanion attacks the electron deficient double bond of fullerene. The carbanion just formed on fullerene removes the halogen by intramolecular nucleophilic substitution (Scheme 2).

SCHEME 2: Bingel reaction mechanism

The different strategies proposed for fullerene derivatization were considered for developing different products. For example by using Prato reaction peptides can be introduced on fullerene: Aroua S. et al., starting from Bis-t-butyl ester, prepared a scaffold for obtaining water-soluble fullerene derivatives such as peptide or PEG conjugates [START_REF] Aroua | C60 pyrrolidine bis-carboxylic acid derivative as a versatile precursor for biocompatible fullerenes[END_REF].

Otherwise by using cyclopropanation it is possible to functionalize fullerene with two different groups in order to derivatize it with two different molecules: one group can be propargyl, therefore suitable for reacting with azido group by click chemistry; the other one is a thiol maleimide that can be used to link also amino acids or peptides via Michael addiction [START_REF] Fensterbank | Sequential Copper-Catalyzed Alkyne-Azide Cycloaddition and Thiol-Maleimide Addition for the Synthesis of Photo-and/or Electroactive Fullerodendrimers and Cysteine-Functionalized Fullerene Derivatives[END_REF].

There are also other functionalizing methods based on some specific characteristics of fullerene. They include radical additions, metal complex formations, oxidations and reactions with electrophiles [START_REF] Hirsch | The Chemistry of the Fullerenes[END_REF].

Conjugation of carbohydrates

Conjugation of fullerene with carbohydrates for biological application has been already investigated [START_REF] Zhou | Sugar-Functionalized Fullerenes[END_REF]. The main way to link sugars with C60 is through a multiple cyclopropanation: six malonate ester chains having azido terminal groups are bonded in one step on fullerene by Bingel reaction. Now it is easy to link carbohydrates (mainly monosaccharides) by click chemistry, once sugar has a terminal propargyl group. It works also vice versa, with propargyl on fullerene chain and azide on the carbohydrate [START_REF] Cecioni | Synthesis of dodecavalent fullerene-based glycoclusters and evaluation of their binding properties towards a bacterial lectin[END_REF].

However, the CuAc (Cu(I) alkyne-azide cycloaddition reaction) (Figure 2A) is not the only way to link sugars once malonate chain is linked to C60. Ramos-Soriano et al. [START_REF] Ramos-Soriano | Maleimide and Cyclooctyne-Based Hexakis-Adducts of Fullerene: Multivalent Scaffolds for Copper-Free Click Chemistry on Fullerenes[END_REF] designed a cyclooctyne fullerene hexakis adducts suitable for cupper free click chemistry (SPAAC) [START_REF] Agard | A comparative study of bioorthogonal reactions with azides[END_REF] (Figure 2B). Otherwise, through cyclopropanation is possible to link just one malonate ester chain already presenting the carbohydrates [START_REF] Enes | Synthesis and solvent dependence of the photophysical properties of [60]fullerene-sugar conjugates[END_REF] (Figure 2C). According to literature there are also other approaches to conjugate sugars (Figure 3). The first is the cyclopropanation mediated by carbene addition (Figure 3A). In this case the reaction between C60 and glycosylidene carbenes, derived from diazirine, leads to spiro-linked C-glycosides with good yields. The reaction is enantioselective [START_REF] Vasella | Fullerene sugars: preparation of enantiomerically pure, spiro-linked C-Glycosides of c60[END_REF][START_REF] Uhlmann | Glycosylidene Carbenes. Part 20. Synthesis of deprotected, spiro-linked C-glycosides of C60[END_REF][START_REF] Jonas | Synthesis of a Fullerene[60] Cryptate and Systematic Langmuir-Blodgett and Thin-Film Investigations of Amphiphilic Fullerene Derivatives[END_REF].

There is also a method suitable for linking in one-step five carbohydrates, by using a thiolate/alkyl halide coupling reaction (Figure 3B): in the first step the pentathiol fullerene is prepared, followed by nucleophilic substitution reaction of thiolate with bromoalkyl glycosides. The aqueous solvent, in which these reactions take place, eliminates the protecting groups and accelerates the reaction [START_REF] Isobe | Synthesis of Fullerene Glycoconjugates through Sulfide Connection in Aqueous Media[END_REF]. A particular method uses iron salt and oxidant to link directly the sugar through two hydroxy groups (Figure 3C) [43].

The sugar conjugation can also occur between an anomeric alkyl-azido group and the double bond of fullerene via 1,3-dipolar cycloaddition [START_REF] Grösser | Ring Expansion of the Fullerene Core by Highly Regioselective Formation of Diazafulleroids[END_REF]: for the monoadducts obtained theoretically there are four possible isomers, but the most common are 5,6-open [START_REF] Yashiro | Fullerene glycoconjugates: A general synthetic approach via cycloaddition of per-O-acetyl glycosyl azides to [60]fullerene[END_REF] (Figure 3D) (adduct preferred by alkyl azide) and 6,6-close (Figure 3E) (preferred by acyl azide). It is also possible obtain bis-adduct involving the all-carbon framework of fullerene [START_REF] Kato | Sugar balls: Synthesis and supramolecular assembly of [60]fullerene glycoconjugates[END_REF] (Figure 3F).

Another conjugation reaction involves a mixture of sarcosine, sugar aldehydes and C60 in reflux [START_REF] Dondoni | Synthesis of [60]fulleropyrrolidine glycoconjugates using 1,3dipolar cycloaddition with C-glycosyl azomethine ylides[END_REF]. 1,3-dipolar sugar azomethine ylide intermediate is formed as firs step and after it is linked to fullerene (attacks 6,6-ring junction) forming a fulleropyrrolidine glycoconjugate (Figure 3G). Selecting the appropriate approach, different kinds of sugars can be linked to fullerene, for example cyclodextrins [START_REF] Chen | Synthesis of water-soluble 2alkylcyclodextrin-C60 conjugates and their inclusion complexation in aqueous solution[END_REF] or carbohydrates (as mannose)[49].

Preparation amino acids and peptide-fullerenes conjugates

Amino acids and peptides are another class of compounds with biological interest that once linked on fullerene could be exploited for several pharmaceutical applications. The preparation of this type of fullerene derivatives could be classified depending on the chemical reactions used for the coupling: cycloaddition, nucleophilic addition and radical reaction [START_REF] Burley | Fullerene Amino Acids and Related Derivatives[END_REF][START_REF] Pochkaeva | Fullerene derivatives with amino acids, peptides and proteins: From synthesis to biomedical application[END_REF].

For the synthesis of the first fullerene-peptide conjugate [START_REF] Prato | Synthesis and Characterization of the First Fuilerene-Peptide Italy methanofullerene which contains a covalently linked[END_REF] in 1993, it was employed a [2+1] cycloaddition reaction[53] by using a diazomethane derivative 1 that once deprotected to a carboxylic acid 3 was coupled to a pentapeptide to form the first fulleryl derivative 6 (Scheme 3).

SCHEME 3: First peptide conjugation

Then more efficient attempts followed, by using directly diazoacetates and diazoamides to obtain peptide-fullerenes derivatives by reducing the number of steps [START_REF] Skiebe | A facile method for the synthesis of amino acid and amido derivatives of C60[END_REF][START_REF] Ouihia | A new diazoacylating reagent: preparation, structure, and use of succinimidyl diazoacetate[END_REF].

Diazoacetate 7 once linked on C60 ( 8) is deprotected to carboxylic acid 9 and then coupled to amino acids by using standard condition (DCC and TEA). Instead by using a diazoamide 12 is enough a single step, a direct addition, to obtain compound 11 (Scheme 4).

SCHEME 4: [2+1] cycloaddition by diazoacetates and diazoamides

Another cycloaddition reaction largely employed for the preparation of these conjugated compounds is the Diels-Alder ([4+2] cycloaddition). A functionalized diene 13 reacts with the electron-deficient double bond presents on C60 to provide compound 14. Different diene derivatives were used in order to synthesized fullerene derivatives with amino acids or peptides [START_REF] An | Synthesis of a-Amino Acid Derivatives of C60 from 1,9-(4-Hydroxycyclohexano)-buckminsterfullerene[END_REF][START_REF] Kotha | A Diels-Alder approach for the synthesis of highly functionalized benzo-annulated indane-based α-amino acid derivatives via a sultine intermediate[END_REF][START_REF] Kotha | Synthesis of novel quinone-amino acid hybrids via cross-enyne metathesis and Diels-Alder reaction as key steps[END_REF] (Scheme 5). Alternatively, organic azides (the linker group) can be used to yield fulleroaziridines derivatives [START_REF] Prato | Addition of Azides to c60: synthesis of azafulleroids[END_REF][START_REF] Yan | Photochemical and Thermal Reactions of C60 with N-Succinimidyl 4-Azido-2,3,5,6-tetrafluorobenzoate: A New Method for Functionalization of C60[END_REF][START_REF] Strom | A simple quick route to fullerene amino acid derivatives[END_REF] (Scheme 7B); finally, azomethine ylides (via Prato reaction) [START_REF] Watanabe | Synthesis of novel fullerene amino acids and their multifullerene peptides[END_REF][START_REF] Bianco | Synthesis, chiroptical properties, and configurational assignment of fulleroproline derivatives and peptides[END_REF][START_REF] Milic | Fullerene unsymmetrical bis-adducts as models for novel peptidomimetics[END_REF] can be used for the synthesis of fullerenoprolines (fpr) and its derivatives [START_REF] Prato | Synthesis and applications of fulleropyrrolidines[END_REF] (Scheme 7C) and other peptide-fullerene derivates such as conjugated products presenting GABA [START_REF] Bjelaković | An approach to nanobioparticles -Synthesis and characterization of fulleropeptides[END_REF], or porphyrin moieties [START_REF] Kobata | Synthesis and characterization of dendritic poly(l-lysine) containing porphyrin-fullerene moieties[END_REF], as well as proline-rich fullerene [START_REF] Sofou | Synthesis of a proline-rich [60]fullerene peptide with potential biological activity[END_REF] and intermediates used in solidphase synthesis of peptides [START_REF] Aroua | C60 pyrrolidine bis-carboxylic acid derivative as a versatile precursor for biocompatible fullerenes[END_REF][START_REF] Pellarini | A novel [60]fullerene amino acid for use in solid-phase peptide synthesis[END_REF].

SCHEME 7: Different type of [3+2] cycloaddition

Cycloaddition is not the only way to obtain amino acids or peptides derivatives, indeed another approach involves nucleophilic addictions.Bingel belongs to this class of reactions, Ruiz-Santaquiteria et al. [START_REF] Ruiz-Santaquiteria | Multivalent Tryptophan-and Tyrosine-Containing [60]Fullerene Hexa-Adducts as Dual HIV and Enterovirus A71 Entry Inhibitors[END_REF] exploited it to obtain a hexa-adduct fullerene containing tryptophane or tyrosine (Scheme 8A).

Instead, Burley et al. [74] and Ball et al. [START_REF] Ball | Structural reassignment of the mono-and bis-addition products from the addition reactions of N-(diphenylmethylene)glycinate esters to [60]fullerene under bingel conditions[END_REF] have reported a different mechanism of this famous reaction, using N-diphenylmethyleneglycinate esters as reagents. Indeed the 60fullerenyldihydropyrroles (Scheme 8B) are the products of these type of reaction. Also, primary and secondary amines seem able to undergo nucleophilic addition with C60 double bond. Romanova et al. linked on fullerene amino groups belonging to different amino acids (Scheme 8C) by means of this approach [START_REF] Romanova | pin, Addition of amino acids and dipeptides to fullerene C60 giving rise to monoadducts[END_REF][START_REF] Belik | Nanoparticles of water-soluble dyads based on amino acid fullerene C60 derivatives and pyropheophorbide: Synthesis, photophysical properties, and photodynamic activity[END_REF]. Photolysis reaction is another method used for the synthesis of peptide-fullerenes derivatives [START_REF] Gan | Synthesis of fullerene amino acid derivatives by direct interaction of amino acid ester with C60[END_REF]. The mechanism is reported in Scheme 9; the photoreaction starts with the formation of -carbon radical that reacts with fullerene. Moreover, some reactions as [3+2] cycloaddition mediated by azide could be activated by photochemical or thermal stimuli [START_REF] Yan | Photochemical and Thermal Reactions of C60 with N-Succinimidyl 4-Azido-2,3,5,6-tetrafluorobenzoate: A New Method for Functionalization of C60[END_REF].

SCHEME 9: Photoreaction for coupling amino acids and peptides with fullerene

Resuming, the strategies of functionalization of C60 reported in literature involve different part of the amino acid structure in the conjugation [START_REF] Jennepalli | Fullerenyl amino acids and peptides: A review of their synthesis and applications[END_REF]. Thus, amino acids can be directly linked via the -carbon as well as involving its amino or carboxyl group. In this context, fullerenyl amino acids conjugates, mainly synthesized by Diels-alder reactions or [3+2] cycloaddictions, were used in the peptide synthesis as a non-natural amino acid and therefore fullerene was introduced in a peptide sequence.

Terapeutic application of C60 derivatives

Functionalised derivatives of fullerene can be considered for biological application because of its therapeutic characteristics and physical and chemical properties that make fullerene derivatives perfect candidates as nanomaterial for drug delivery and as bioactive molecule for specific diseases. First of all it's interesting to evaluate the fullerene characteristics that make C60 effective for biological and therapeutic applications.

The mains properties that should be considered for drug delivery application are the following: size (it is considered a nanomaterial since its dimension is around 0.7-1 nm), high surface area that is also regular and reproducible, solubility in water when it is properly derivatized, reversible aggregation and possibility of covalent conjugation.

Moreover we can't forget also the chemical properties that make fullerene a molecule of relevant therapeutic prospective: C60 has the ability to be either a reactive oxygen species (ROS) generator and a radical scavenger depending on the presence or absence of light [START_REF] Zhu | Biological applications of hydrophilic C60 derivatives (hC60s)-a structural perspective[END_REF].

Under light exposition C60 is able to generate ROS by two pathways: via energy transfer reaction (from the triplet excited state of C60) and via electron transfer reaction (from the C60 radical anion) [START_REF] Aroua | C60 pyrrolidine bis-carboxylic acid derivative as a versatile precursor for biocompatible fullerenes[END_REF]. Conversely in the dark, it has an exceptional capacity for radical scavenging, indeed it's also called "radical sponge" [START_REF] Acquah | Review-The Beautiful Molecule: 30 Years of C 60 and Its Derivatives[END_REF].

Such features make fullerene a very good candidate for photodynamic therapy (PTD) [START_REF] Otake | Effect and mechanism of a new photodynamic therapy with glycoconjugated fullerene[END_REF] , for antioxidant therapy or in diagnosis technics [START_REF] Watson | Use of fullerenes In diagnostic and/or therapeutic agents[END_REF].

Therapeutic applications of sugar conjugated of C60

Focusing on applications of glycoconjugated fullerene, we can discern two groups based on the role of sugars. There are applications where carbohydrates have an indirect action because they make fullerene water soluble as required for pharmacological applications and, in some cases, also mediate target delivery of fullerene (the active pharmaceutical agent).

On the other hand, there are applications where sugars have a biological role by mediating the interaction with specific therapeutical targets (Direct action).

Indirect action

As previously discussed, one of the main possible fullerene employments is in PDT. In this context, one important application of PDT is in cancer therapy. An improved selectivity of the sugar-fullerene derivatives towards cancer cells can lead a great efficiency and a low frequency of side effects.

Accordingly, Mikata et al.[84] in 2003 demonstrated that sugar-fullerene derivatives exhibit phototoxicity against HeLa cells (std cancer cells) as confirmed in 2010 by Otake et. al. [START_REF] Otake | Effect and mechanism of a new photodynamic therapy with glycoconjugated fullerene[END_REF].

The glycoconjugated derivatives of C60 (with one or more glucoses linked on fullerene) are more specific against cancer than healthy cells. So, these compounds showed a tumourselective cytotoxicity both in vivo and vitro tests, that may be ascribed to an increased presence of GLUT (glucose transporters) in cancer cells.

Later, other scientists designed glycoconjugated derivatives that resulted even more active towards cancer cells. Yano et al. [START_REF] Yano | Efficient singlet oxygen generation from sugar pendant C60 derivatives for photodynamic therapy[END_REF] discovered that the type of conjugation influences the activity, Glucose-C60 conjugate (19, Figure 4) exhibits higher toxicity than Glc-azafulleroid (20, Figure 4). Instead Serda et al. [START_REF] Serda | Development of photoactive Sweet-C 60 for pancreatic cancer stellate cell therapy[END_REF], based on the fact that the selectivity depends on the high presence of GLUT, designed a compound with six linked sugars, the hexakis-glucosamine C60 derivative 21 (sweet-C60) in Figure 4, that showed high selectivity and strong cytotoxic activity once illuminated by light, above all in pancreatic stellate cells in which 21 is predominately accumulated. The same research group [START_REF] Serda | Glycofullerenes as non-receptor tyrosine kinase inhibitors-towards better nanotherapeutics for pancreatic cancer treatment[END_REF] in 2020 discovered also that these kind of glycofullerenes can be considered inhibitors of non-receptor tyrosine kinase (cytosolic enzymes able to regulate cell growth, proliferation, differentiation, adhesion, migration and apoptosis). Baranska et. al. [START_REF] Barańska | Interactions of a water-soluble glycofullerene with glucose transporter 1. Analysis of the cellular effects on a pancreatic tumor model[END_REF] in 2021, always based on the selectivity due to high GLUT presence and the non-toxicity of these carbon carrier in absence of light, confirmed the potentiality of Sweet-C60 as vehicle for the treatment of pancreatic cancer.

The effect of fullerene glycosylation was also confirmed when Narumi et Al. [START_REF] Narumi | C60 Fullerene with Tetraethylene Glycols as a Well-defined Soluble Building Block and Saccharide-conjugation Producing PDT Photosensitizer[END_REF] carried out a study showing that a glycoconjugated TEG-C60 (22, Figure 4, a fullerene conjugated with a malonamide chain bearing two glycosylated tetraethylene glycol, TEG) could be a promising approach to increase the photodynamic activity of C60. Moreover, it seems that it acts in aqueous medium forming micelles by hydrophobic interaction between C60 units that exposes on the surface the glycosylated TEG chains (the portion responsible of the tumour-targeting and the biocompatibility). About improving PDT activity of fullerene derivatives as photosensitiser, it has been shown that the use of specific oligosaccharides, such as cyclodextrins, helps this function in addition of making fullerene water soluble [START_REF] Zhu | Secondary-Rim γ-Cyclodextrin Functionalization to Conjugate with C60: Improved Efficacy as a Photosensitizer[END_REF].

Considering the biological effect of fullerene under light irradiation, other possible applications may be envisaged. First of all, as antiviral agent as demonstrated by Zhu et al. [START_REF] Zhu | Design, synthesis and biological evaluation of water-soluble per-O-methylated cyclodextrin-C60 conjugates as antiinfluenza virus agents[END_REF] that investigated the potential use of C60 as anti-influenza virus agent (with mechanism still unknown), once conjugated with cyclodextrins (thanks to the increase in water solubility). In particular, two types of cyclodextrins were employed : -CD, composed by six glucose subunits, and -CD by eight glucoses. It was observed that only -CD C60 conjugates gave antiviral activity hypnotizing that this could be due to some -CD specific properties (different from alpha type): a) it has higher water solubility than -CD, making C60 even more hydrophilic and b) it allows less aggregation between C60 conjugates and therefore generates more singlet oxygen [START_REF] Quaranta | Photophysical studies of six amphiphilic 2:1 cyclodextrin:[60]fullerene derivatives[END_REF]. Although the IC50 values of C60 conjugates with -CD are half compared with Oseltamivir (that was considered as positive control), they exhibit an enough anti-influenza virus activity to consider this class of compounds for further optimizations.

Another antiviral application concerned fullerene alone or linked with some amino acids is the inhibition of HIV-1 protease by photoirradiation [START_REF] Grösser | Ring Expansion of the Fullerene Core by Highly Regioselective Formation of Diazafulleroids[END_REF][START_REF] Strom | Fullerenebased inhibitors of HIV-1 protease[END_REF], but when the conjugation is with sugars a better activity is obtained. Because of the simultaneous presence of a hydrophobic centre and hydrophilic chains, the C60-glycoconjugate interaction with the HIV enzyme (HIV-1 protease) is reinforced by different hydrogen bonds.

HIV-1 protease is an important target since it is essential for the replication of the virus.

Tanimoto et al. [START_REF] Tanimoto | Chemical methods for degradation of target proteins using designed light-activatable organic molecules[END_REF][START_REF] Tanimoto | Target-selective photodegradation of HIV-1 protease and inhibition of HIV-1 replication in living cells by designed fullerene-sugar hybrids[END_REF] discovered that fullerene could degrade HIV-1 protease under photo irradiation, thanks to the production of 1 O2. Indeed, the cleavage of this enzyme protein backbone is carried out by ROS or radical species.

The use of sugar-fullerene derivatives improves this effect: 1) sugars increase water solubility and 2) it was demonstrated that fullerene-sugar hybrid compounds such as 23 (Figure 5) have high affinity for this enzyme and its inhibitory activity increased of 6.7 fold under light irradiation.

The same research groups evaluated the activity of conjugate 24 (Figure 5) against Aβ42 peptide (amyloid β) that is involved in Alzheimer disease (AD) [START_REF] Yano | Efficient singlet oxygen generation from sugar pendant C60 derivatives for photodynamic therapy[END_REF][START_REF] Ishida | Photo-degradation of amyloid β by a designed fullerene-sugar hybrid[END_REF]. This molecule has affinity with the amyloid peptide and is able to degraded it, always by photo-irradiation and the production of ROS species. Also, oligomers of the Aβ42 peptide can be degraded under long-wavelength UV radiation, without any additives (such as metals or reducing agents as norally used). Once in the dark, fullerene photosensitizer changes its properties and becomes an antioxidant agent. Always talking about neurodegenerative diseases, antioxidants could be used in some pathologies where the triggering effect is the oxidative stress. Horie et al. [START_REF] Horie | Antioxidant action of sugar-pendant C60 fullerenes[END_REF] measured the capability of two type of glycoconjugated fullerene derivatives (the first with one sugar-chain and the other with two sugar-chains) to inhibit the free radicals coming from lipid peroxidation remarking that these compounds act as radical scavenger with an antioxidant activity comparable to phenols.

Moreover in order to increase this activity, Kop et al. [START_REF] Kop | Polysaccharide-fullerene supramolecular hybrids: Synthesis, characterization and antioxidant activity[END_REF] suggested to formulate these polysaccharides-fullerene products with hydrophobic materials (such as cholesterol) in order to form nanoparticles and observed that antioxidant activity increases improving the hydrophobicity of the formulation.

An unusual application always related to antioxidant properties of fullerene is in the treatment of shock. Shock is a syndrome characterized by an insufficient tissue perfusion.

Since it seems that ROS is one of the causes involved in shock parthenogenesis, fullerene, used as a radical sponge, can reduce the irreversible cellular damage, but only the conjugation with polar molecules as sugars enables the use in-vivo [99].

A recent study [START_REF] Lee | Glycofullerenes inhibit particulate matter induced inflammation and loss of barrier proteins in HaCaT human keratinocytes[END_REF] investigated fullerene, according to its good radical scavenger properties, in the treatment of particulate matter (PM) that induces skin diseases and pulmonary and cardiovascular disfunctions. Indeed, PM is involved in skin inflammation and in the activation of ROS. Hexa-adduct fullerenes presenting twelve sugars (to increase C60 water solubility) resulted protective against ROS productions and so valid candidates for PM therapy. It seems that all inflammatory consequences due to PM could be reduced by pre-treatment with this kind of glycofullerenes.

Direct action

We are used to thinking of a monovalent "lock and kay" type of binding between a biological target and its ligand, but this kind of interaction is typically weak when the ligand is an oligosaccharide.

For this reason, some kind of protein (for example lectins) benefits from multivalency for their biological activity. The multivalency, multiple epitopes that interact with multiple cells receptors, is a mode of molecular recognition involved in important biological processes mediated by oligosaccharides, above all in the cell-cell adhesion such as the interaction of host cells with pathogenic microorganisms [START_REF] Kiessling | Multivalency in Protein -Carbohydrate Recognition[END_REF]. The multivalent presentation of carbohydrates enhances the affinity and selectivity by an effect also known as " cluster glycoside effect" [START_REF] Lundquist | The cluster glycoside effect[END_REF].

The main classes of receptors involved in this type of recognition are lectins. In the last years, since this category of protein is important for the adhesion of virus, bacteria and pathogens to host cells, specific molecules have been investigated to block the infection by inhibiting the sugar receptors interaction using multivalent compounds [START_REF] Bojarová | Sugared biomaterial binding lectins: achievements and perspectives[END_REF][START_REF] Da Ros | Carbon nanostructures for biomedical applications[END_REF].

FimH (a bacterial adhesin belongs to E. Coli) [START_REF] Durka | The functional valency of dodecamannosylated fullerenes with Escherichia coli FimH-towards novel bacterial antiadhesives[END_REF], and PA-IL (a bacterial lectin from Pseudomonas aeruginosa) [START_REF] Cecioni | Synthesis of dodecavalent fullerene-based glycoclusters and evaluation of their binding properties towards a bacterial lectin[END_REF] were the first examples of lectins that were inhibited by this multivalent approach and above all by using dodecavalent-adducts of fullerene presenting 12 sugar units linked through six malonate chains (called also hexa-adduct with regards to number of linker chains).

Indeed, it has been demonstrated through assays such as SPR (surface plasmon resonance), ELLA (enzyme-linked lectin assay), HIA (hemagglutination inhibition assay) and ITC (isothermal titration calorimetry) that the affinity of these dodecavalent glycoconjugate of C60 is 12000-fold higher compared to the free monosaccharides, and they can be considered as potential competitors in order to avoid the pathogen binding that leads to the beginning of the infection [START_REF] Bhatia | Multivalent glycoconjugates as vaccines and potential drug candidates[END_REF]. Further studies demonstrated that a good inhibition is promoted by a right spatial distribution of carbohydrates involved in the interaction with lectins [START_REF] Timmer | Spatially well-defined carbohydrate nanoplatforms: Synthesis, characterization and lectin interaction study[END_REF].

Another group of lectins involved in cell-cell adhesion is DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin). Some viruses target DC-SIGN (a Ctype lectin receptor) to enter to the host cell and start the infection, so antagonists of this receptor can stop the infection process.

One of the first applications was against Ebola virus. Luczkowiak et al. were the first to prove the efficiency of dodecavalent-adducts of fullerene (compound 25, figure 6) presenting 12 mannoses linked on C60 through six malonate chains, as anti-Ebola agent, highlighting that compounds with same valency but longer spacers could be more active [START_REF] Luczkowiak | Glycofullerenes inhibit viral infection[END_REF]. This was confirmed by Engstrom et al. [START_REF] Engström | Investigation of glycofullerene dynamics by NMR spectroscopy[END_REF] that demonstrated as a spacer extension (as for compound 26 figure 6) makes glycofullerene derivatives more dynamic and flexible, favouring the binding [START_REF] Weber | Quantifying the rebinding effect in multivalent chemical ligand-receptor systems[END_REF]. This work has certainly inspired subsequent research in the same topic. In 2015, Munoz et al. [START_REF] Muñoz | Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection[END_REF], found a methods to synthesize giant globular multivalent glycolfullerenes acting as potent infection-inhibitors against Ebola virus. First of all, it necessary to emphasize the great synthetic challenge to build these fullerene sugar balls. This research group was able to synthesize a fullerene derivative presenting 120 mannoses, consisting in fullerene-based dendritic structure (with C60 at the branching points as well as at the core) functionalized with carbohydrates. They assembled 12 sugar-coated fullerenes (each one with 10 carbohydrate groups) to a central fullerene scaffold in only one synthetic step based on the click chemistry (Scheme 10).

The combination of Bingel reaction and click chemistry resulted a successfully approach [START_REF] Vidal | Glycofullerenes: Sweet fullerenes vanquish viruses[END_REF]. The glycoconjugates obtained from C60 by linked only 12 or 36 sugars (compounds 25 and 26 in figure 6) were also investigated but showed lower activity compared with tridecafullerenes 27. The first one [START_REF] Benito | Synthesis and Characterisation of the methanofullerenes, C60(CHCN) and C60(CBr2)[END_REF] have a good inhibitor activity of 2 µM (micromolar range) meanwhile compound 26 reaches an IC50 of only 68 µM [START_REF] Luczkowiak | Glycofullerenes inhibit viral infection[END_REF]; thus, the activity dramatically improves with compound 27 that expose 120 mannoses-units [START_REF] Illescas | Multivalent Nanostructures To Inhibit Ebola Virus Infection[END_REF]. Other viruses also target the same lectin to start the infection. Ramos-Soriano et al. [START_REF] Ramos-Soriano | Synthesis of Highly Efficient Multivalent Disaccharide/[60]Fullerene Nanoballs for Emergent Viruses[END_REF][START_REF] Ramos-Soriano | Glycodendritic structures as DC-SIGN binders to inhibit viral infections[END_REF] further increased the number of sugars linked to a fullerene based scaffold through a new synthetic strategy, the SPAAC (Strain-promoted azide-alkyne cycloaddition) that avoids the Lectins usually show multiple carbohydrate-binding pockets able to establish multiple simultaneous interactions promoting cross-linking between host cell and pathogen. So, it was logical to think about the use of multivalent compounds as anti-viral or -bacterial agents.

On the contrary, it would not be intuitive to adopt the same approach for enzymes such as glycosidases and glycosyltransferases, which generally only bind one oligosaccharide substrate with high selectivity and affinity as they have a single substrate-binding site.

However, contrary to what expected, even these important enzymes involved in viruses and bacteria pathogenesis, are subjected to a counterintuitive effect called "multivalent inhibition effect" [START_REF] García-Moreno | The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study[END_REF].

Glycosidases are ubiquitous enzymes that cleave glycoside anomeric bonds in glycoconjugates and oligosaccharides. They have a main role in many biological processes including pathogenic processes. For this, its inhibitors could be attractive as anti-viral, anticancer and anti-HIV agents but also for the therapy of diabetes [START_REF] Derosa | α-Glucosidase inhibitors and their use in clinical practice[END_REF] and Alzheimer disease [START_REF] Yuzwa | O-GlcNAc and neurodegeneration: Biochemical mechanisms and potential roles in Alzheimer's disease and beyond[END_REF]. Again, multivalent (multiligand) compounds proved to be highly active.

Compain et al. [START_REF] Compain | Glycosidase Inhibition with Fullerene Iminosugar Balls: A Dramatic Multivalent Effect[END_REF] proved the inhibitory activity of a sugar-fullerene derivative linking twelve sugar units of 1-deoxynojirimycin (DNJ) by means of six malonate linkers. Thus, the fullerene conjugate 30a (figure 8) showed an enhanced activity compared to DNJ alone (a well-known glycosidases inhibitor and inhitope for several enzymes of this class). This was the first result that introduced the relevance of multivalency also for inhibition of glycosidases. In 2013 Risquez-Cuadro et al. [START_REF] Rísquez-Cuadro | Fullerene-sp2-iminosugar balls as multimodal ligands for lectins and glycosidases: A mechanistic hypothesis for the inhibitory multivalent effect[END_REF] 8) demonstrated that the matching monovalent sugar 29b has a better inhibitory potency compared with the mismatching inhitope 29a; but when these sugars are involved in the formation of multivalent compounds, they exhibit similar activity. Thus, inhitopes when used as single compounds are, as expected, not recognized by mismatching glycosidases, because their structure is different from the substrate specific for these enzymes. However, mismatching sugar derivatives become active as inhibitors towards nonspecific enzymes when involved in multivalent compounds.

It seems that two different binding modes are involved for mono and multivalent inhibitor.

For the first one, it is important the high affinity of the sugar moity with the binding site, in a typical key-lock model; instead, multivalent inhibitors activity seems also depend on interaction with non-glycone binding sites. Non-glycone binding sites with lectin-like abilities are probably implicated in this process and the presence of matching inhitopes can cooperate, but it is not fundamental for its inhibitor activity. This hypothesis was confirmed by Flos et al. [START_REF] Abellán Flos | Potent Glycosidase Inhibition with Heterovalent Fullerenes: Unveiling the Binding Modes Triggering Multivalent Inhibition[END_REF] that investigated this topic in detail, by preparing homovalent fullerene derivatives 31a-c containing 12 identical sugars and heterovalent products 32a-c linking 10 sugars and a single sp2-iminosugar inhitope moiety (figure 9) and evaluated their inhibitory potencies on 3 types of glycosidases: Jack bean αmannosidase (with an open active site and easily accessible), yeast maltase and isomaltase (with a "narrow" active site) and β-galactosidase (having an "open-to-narrow flexible" site). In this study, it was confirmed that the multivalent carbohydrate fullerene conjugates (31 and 32) exhibit dual glycosidase and lectin binding properties. This is because non-glycone regions of glycosidase with lectin-like behaviour are certainly involved in the inhibition, so if these sugar balls can interact with lectins they can also interact with these regions.

Moreover, homo and hetero-valent fullerenes show different mechanism but the different sugar moieties (hetero valency) in compounds 32a-c do not affect their inhibitory activity.

As described above the glycosidases considered in this study have a different catalytic pocket topology and accessibility which affects the type and the potency of the inhibition. Its seems that compounds 31a-c and 32a-c are not able to inhibit α-mannosidase, maybe because this enzyme requires sugars having an affinity for the "open" active site; on the contrary, 31a-c and 32a-c are potent inhibitors for the other glycosidases acting as competitive inhibitors for enzymes with "narrow" active site and uncompetitive or mixed-type for β-galactosidase (Table1).

Subsequent studies focused on finding more effective inhibitors and, as for lectin antagonists, the increase of multivalency leaded to an augmentation of the inhibitor activity. Trinh et al. [START_REF] Trinh | Construction of giant glycosidase inhibitors from iminosugar-substituted fullerene macromonomers[END_REF] demonstrated that synthesizing the giant molecule 33a composed by a fullerene core with 120 peripheral iminosugars (figure 10) similar to Munoz's compound reported in figure 6 but with sp2-iminosugars, a great inhibitory activity was obtained towards Jack bean αmannosidase (underling that a high valency is necessary for the inhibition of this "open" site enzyme). The size of these molecules seems to be the main factor allowing a good inhibitory activity also towards β-galactosidase and β-mannosidase, with a IC50 in the nanomolar range.

The inhitope density of multivalent compounds plays an important role, Nierengarten et al. [START_REF] Nierengarten | Giant Glycosidase Inhibitors: First-and Second-Generation Fullerodendrimers with a Dense Iminosugar Shell[END_REF] compared dendrimers 33b,c composed by 36 iminosugars (figure 10) and product 33d with 108 iminosugars (figure 10). Also 12-and 120-valent C60 iminosugars were included in this study. This work confirmed the relevance of the multivalency in the inhibitory activity, but added an additional factor, the density. The combination of these two aspects lead to a very potent inhibitor. The Glycosyltransferases are other enzymes subjected to multivalent inhibition. They catalyse the regio-and stereo-selective transfer of a carbohydrate from a donor to an acceptor. As glycosidases, also this class of enzyme is involved in key biological steps and could be considered as a good target for development of new therapy of several diseases (diabetes, cancer, viral infections etc..). Durka et al. [START_REF] Durka | The inhibition of liposaccharide heptosyltransferase WaaC with multivalent glycosylated fullerenes: A new mode of glycosyltransferase inhibition[END_REF] showed for the first time the efficacy of the multivalent glycofullerenes (12 sugars) as glycosyltransferases inhibitors. These compounds inhibit Heptosyltransferase WaaC (enzyme essential for biosynthesis of bacterial lypopolisaccharides) in the low micromolar range (IC50=7-45 µM) showing a much better activity compared to monomeric glycosides (IC50 above 400 µM). The mechanism is still unknown but Tikad et al. [START_REF] Tikad | Mechanistic Insight into Heptosyltransferase Inhibition by using Kdo Multivalent Glycoclusters[END_REF] provided more information. Glycosiltransferases display two recognition sites, one for the acceptor and the other for the donor. They discovered that regardless the inhitope affinity for the acceptor or donor site, the multimers interact only with the acceptor binding site (more exposed and easily accessible). Moreover, the fullerene scaffold was found to positively influence the affinity, and this leads to increase the inhibitor activity. The last possible application, for the moment at a preliminary stage, contemplates the possibility that the glycofullerenes can interfere with the action of certain toxins.

For example shiga toxin or shiga-like toxin by E. Coli, which can lead to serious consequences such as kidney damage, enters into mammalian cells through a process mediated by oligosaccharides recognition with the toxin receptor of human cells that is the glycolipid Gb3 α-D-Gal(14)β-D-Gal(14)β-D-Glc(1O-ceramide). Antagonization of this recognition could be a valid therapeutic strategy and the use of multivalent inhibitors seems to be a possible approach [START_REF] Kitov | Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands[END_REF]. One candidate could be a glycofullerene prepared by a copper-catalyzed [3 + 2] cycloaddition reaction and bearing five oligosaccharides (compound 35 in figure 12) as a Gb3-trisaccharide. The spatial orientation of these five P k trisaccharides of Gb3 leads to a tighter binding with model of shiga-like toxin (Figure 12) [START_REF] Isobe | Synthesis of fullerene glycoconjugates via a copper-catalyzed huisgen cycloaddition reaction[END_REF]. Another toxin that exploits carbohydrate-protein binding to enter in the host cell is ricin-toxin, belonging to Ricinus communis family. Also in this case, a fullerene conjugated with sugars, the bis(β-lactosyl)-fullerene, can be a tool for detecting and decontaminating from this deadly toxin [START_REF] Dohi | Bis(ß-lactosyl)-[60]fullerene as novel class of glycolipids useful for the detection and the decontamination of biological toxins of the Ricinus communis family[END_REF].

C60 peptide derivatives

The C60-peptide represents a possible example of bioconjugate that could be exploited for different biomedical applications. As for sugars, the conjugation of peptide with fullerene provides structural diversity, but also charge, flexibility and specific recognition properties [START_REF] Barron | Fullerene-peptides: bio-nano conjugates with structural and chemical diversity[END_REF].

Several medical areas were investigated for the application of these fullerene derivatives and the main applications concern the development of new anti-infective agents and immunological new tools.

Anti-infective properties

Generally, the amphiphilic nature of antimicrobial peptides (with cationic charges) is essential for their antibacterial activity; indeed, the hydrophobic residues interact with the lipidic bilayer, instead the polar and charged residues are in exposed on the membrane towards the external aqueous environment. The presence of a hydrophobic core in the C60-peptides promotes interaction with the membrane, helping the peptide to achieve its target. Pellarini et al. [START_REF] Pellarini | A novel [60]fullerene amino acid for use in solid-phase peptide synthesis[END_REF] and Pantarotto et al. [START_REF] Pantarotto | Solid-Phase Synthesis of Fullerene-peptides[END_REF] studied some peptide-fullerene derivates (36 in Figure 13) that showed remarkable effect against gram-positive bacteria (e.g. S. Aureus) but their activity was reduced in E. coli, gram-negative bacteria, maybe because of the aggregation or the large dimension of C60 that prevents the penetration of peptide to the LPS-rich membrane of gram-negative bacterium.

Some of these derivates (37 in Figure 13) have also antioxidant properties to be exploited for developing potential anti-viral products [START_REF] Pochkaeva | Fullerene derivatives with amino acids, peptides and proteins: From synthesis to biomedical application[END_REF]. Kotelnikova et al. [START_REF] Kotelńikova | Antioxidant properties of water-soluble amino acid derivatives of fullerenes and their role in the inhibition of herpes virus infection[END_REF] established a correlation between the cytomegalovirus infection (CMVI) infection and the lipid peroxidation; thus they proved that amino acids-fullerene with antioxidant activity could be a new generation of drugs for the treatment of herpes virus infections, specifically against CMVI.

The treatment HIV was also investigated with peptide derivatives of fullerene. Friedman et al.

in 1993 published a computer graphic simulation where C60 seems to be perfectly hosted by the hydrophobic cavity of HIV-1 protease [START_REF] Friedman | Inhibition of the HIV-1 Protease by Fullerene Derivatives: Model Building Studies and Experimental Verification[END_REF]. Further works [START_REF] Toniolo | A Bioactive Fullerene Peptide[END_REF][START_REF] Mashino | Human immunodeficiency virus-reverse transcriptase inhibition and hepatitis C virus RNA-dependent RNA polymerase inhibition activities of fullerene derivatives[END_REF] supported this hypothesis, indeed was demonstrated that some peptide/amino acids derivatives (38 and 39 in Figure 13) resulted inhibitors of HIV-1 protease.

Recently, Dostalova et. al [START_REF] Dostalova | Antiviral activity of fullerene C60 nanocrystals modified with derivatives of anionic antimicrobial peptide maximin H5[END_REF] studied the conjugation of an antimicrobial peptide, maximin H5 and its derivatives (isolated from Asian toad Bombina maxima) with C60, generating another example of product with antiviral activity that can be modulated by the amount of peptide linked on the fullerene surface.

Immunological properties

In the last few years, nanomaterials started to be considered as good tool in development of efficient adjuvant/delivery systems for vaccination, thanks to their several advantages as biocompatibility, size, surface properties and the possibility to introduce modification. Thus preliminary studies were made also in a possible application of fullerene for this type of therapeutic approach [START_REF] Donnelly | Vaccine delivery systems[END_REF][START_REF] Wendorf | Molecular Nanomedicine Towards Cancer[END_REF]: the potential use of fullerene derivatives nano-material in vaccination can be promising since C60 has a vast surface and a size that makes its products well recognized by APC cells and suitable to penetrate through cells walls and endothelium.

Moreover, fullerene has a regular and reproducible surface compared with other nanomaterials commonly used for pharmaceutical application (as metals or polymers).

In addition, the size and shape of possible aggregates in water are reversible and fluxional [137]. Another advantage is the possibility to link the molecule of interest by covalent conjugation to obtain stable derivatives.

However, fullerene has some limitation for its use in nanomedicine because its size is not optimal (C60 has a size around 1nm while the optimal size should be around 20 nm). This concern can be addressed using fullerenes with size higher than C60 and/or designing surface modification that improves the final size (as previously reported for the derivative obtained by conjugation with sugars).

In addition, the design of these derivative could be performed in order to induce the formation of stable micelles with a core of fullerene that expose the antigens on the surface [START_REF] Narumi | C60 Fullerene with Tetraethylene Glycols as a Well-defined Soluble Building Block and Saccharide-conjugation Producing PDT Photosensitizer[END_REF].

Some studies showed the efficiency of different C60 derivatives as adjuvant allowing the design of a new generation of vaccines. Ramon et al. [START_REF]Surlatoxineetsur I'anatoxinediphtheriques[END_REF] defined "adjuvant" a substance that produces a more robust immune response in combination with a specific antigen respect to the antigen alone. So, the role of the adjuvants is to increase the response, allowing the use of smaller doses of vaccine and accelerating the immunization process. Adjuvants could help through one of the following mechanism to enhance the immune response: assisting the antigen delivery in the site of injection; stimulating productions of cytokines and chemokines;

promoting the cellular recruitment and promoting the antigen uptake and presentation by APC [START_REF] Awate | Mechanisms of action of adjuvants[END_REF].

According to Masalova et al. [START_REF] Masalova | Immunostimulation Effect of Water-Soluble Fullerene Derivatives ; Potential Adjuvants for the New Generation of Vaccines[END_REF], fullerene conjugated with aminocaproic acid or Nacetylglucosaminyl-N-acetylmuramyl-dipeptide (GMDP), 40 and 41 (figure 14) could be valid adjuvants thanks to their ability to increase IgG production. A group of mice was injected with pNS4 (antigenic peptide of Hepatitis C virus) without adjuvants, another group with formulations containing known adjuvants such as Freund's complete adjuvant (FCA) and aluminium hydroxide (HA) and a third group combining the antigen with the C60 derivatives 40 and 41. The results indicated the superiority of these C60 derivatives respect to the other adjuvants thanks to their higher solubility in water, the long-term effect obtained, and their ability to cause more rapid antibodies formations (a wider spectrum). Subsequent studies showed that multi-hydroxylated fullerenes (fullerenols) could be an useful component for the preparation of new vaccines for serious viral infections such as HIV [START_REF] Xu | Morphologically virus-like fullerenol nanoparticles act as the dual-functional nanoadjuvant for HIV-1 vaccine[END_REF] and hepatitis C [START_REF] Liu | The adjuvant effect of C 60 (OH) 22 nanoparticles promoting both humoral and cellular immune responses to HCV recombinant proteins[END_REF]. In addition, it was demonstrated these fullerenols have the ability to preserve the antigenic portion (peptide or DNA encoding for the antigen) from enzymatic degradation.

Another work that confirmed the possible use of fullerene as nano-carrier for designing efficient adjuvants was published by Xu et al.' [START_REF] Xu | Synthesis and immunomodulatory activity of [60]fullerene-tuftsin conjugates[END_REF]. Two fullerene-tuftsin conjugates (42 and 43 in figure 15) were compared with the non-conjugated tuftsin (a tetrapeptide, Thr-Lys-Pro-Arg, with immunostimulatory activity released by proteolysis from the IgG Fc-domain),

showing a significant enhancement of the immunostimulant activity of the peptide induced by its conjugation with fullerene(increased presence of phagocytes, major chemotaxis activity in macrophages stimulated with this conjugates). Therefore, the presence of fullerene led to two important and favourable consequences: 1) it protects the amino acids from enzyme degradation prolonging the half-life of tuftsin; 2) thanks to the nano size of fullerene, the conjugates preferentially enter in the mononuclear phagocyte system improving the bioavailability of the antigen. Therefore, all of these studies support the idea that fullerene could be a good candidate for the design of innovative nano-vaccine by using fullerene derivatives as adjuvant for the formulation of antigens and/or as a carrier for covalent conjugation of immunogenic or antigenic peptides. However, few studies have been published on this subject and it deserves further investigation.

Other applications

Several other applications have been studied for peptide-fullerenes conjugation products.

The antioxidant properties of these fullerene derivatives were exploited as anti-infective agents, but also against other pathologies such as cardiovascular disorders, neoplastic diseases, inflammations where ROS production seems to be involved in their pathogenesis [START_REF] Sharma | Analysis of fullerene based smaller chain peptidomimetics analysis of fullerene based smaller chain peptidomimetics targeting tuberculosis using invitro-antioxidant methods[END_REF]. The presence of peptide helps the solubility and decreases aggregation of C60, increasing its antioxidant potential with respect to the non-conjugated fullerene [START_REF] Bjelaković | Synthesis and characterization of highly ordered self-assembled bioactive fulleropeptides[END_REF].

On the other hand the linking of a peptide portion with fullerene could be exploited also for boosting cell penetration of peptide with pharmacological relevant activities. Yang et al. [START_REF] Yang | The use of fullerene substituted phenylalanine amino acid as a passport for peptides through cell membranes[END_REF] used a fullerene-phenylalanine derivative (Baa, compound 44 in figure 16) as building block in the synthesis of cationic peptides. These products use the strong hydrophobic properties of C60 as a passport for the intracellular delivery of the peptide into neuroblastoma cells, HEK 293 and Hep G (where the peptide alone cannot enter). Two cationic peptides were incorporated, the nuclear localization sequence (NLS) forming H-Baa-Lys(FITC)-Lys-Lys-Arg-Lys-Val-OH and a poly-lysine peptide forming H-Baa-Lys(FITC)-Lys8-OH. Also an anionic peptide (Lys(FITC)Glu4Gly3Ser-OH) was tested proving that its uptake is lower in comparison with the cationic ones. In addition, it seems that peptide derivatives of C60 could be exploited in different diseases.

For example Xiao et al. [START_REF] Xiao | A New Formyl Peptide Receptor-1 Antagonist Conjugated Fullerene Nanoparticle for Targeted Treatment of Degenerative Disc Diseases[END_REF] reported that FT-C60 (fullerene conjugated to a peptide, cFLFLF, that binds to formyl peptide receptor-1) reduces inflammation and in particular could be used against pain in treatment of the degenerative disc disease.

Other pathologies considered for the treatment with peptide derivatives of fullerene are the Alzheimer disease [START_REF] Xie | The molecular mechanism of fullereneinhibited aggregation of Alzheimer's β-amyloid peptide fragment[END_REF], mixed connective tissue disease (MCTD) or systemic lupus erythematosus (SLE) [START_REF] Sofou | Synthesis of a proline-rich [60]fullerene peptide with potential biological activity[END_REF], and also in cancer therapy (e.g. as targeting molecule or in diagnostic) [START_REF] Fillmore | Conjugation of functionalized gadolinium metallofullerenes with IL-13 peptides for targeting and imaging glial tumors[END_REF][START_REF] Peng | Positron emission tomography (PET) guided glioblastoma targeting by a fullerene-based nanoplatform with fast renal clearance[END_REF].

1 2 2 4 2. 1 4 2. 2 7 2. 3 9 3

 24142739 Introduction .......................................................................................................................................... Chemical modification.......................................................................................................................... Main strategies of conjugation ..................................................................................................... Conjugation of carbohydrate ........................................................................................................ Preparation of amino acids and peptide-fullerenes conjugates ................................................... Therapeutic application of C60 derivatives .......................................................................................... 14 3.1 Therapeutic applications of sugar conjugates of C60 .................................................................. 15 3.1.1 Indirect action ...................................................................................................................... 15 3.1.2 Direct action ......................................................................................................................... 20 3.2 C60 peptide derivatives ................................................................................................................ 32 3.2.1 Anti-infective properties ....................................................................................................... 33 3.2.2 Immunological properties .................................................................................................... 34 3.2.3 Other applications ................................................................................................................ 37 1 Introduction

FIGURE 1 :

 1 FIGURE 1: Bond lengths and C60 reactivity positions

FIGURE 2 :

 2 FIGURE 2: Sugar conjugation via malonate: A,B) Multiple Bingel cyclopropanation ; C) Single cyclopropanation

FIGURE 3 :

 3 FIGURE 3: Sugar conjugation: A) Carbene addition ; B) thiolate/alkyl halide coupling reaction C) Iron salt and oxidant reaction; D,E,F) via Azide; G)sarcosine, sugar aldheydes and C60 reaction.

SCHEME 5 :SCHEME 6 :

 56 SCHEME 5: Dies-Alder reaction for preparation of peptide-fullerene derivative

SCHEME 8 :

 8 SCHEME 8: Syntheses by nucleophilic addiction

FIGURE 4 :

 4 FIGURE 4: Compound 19 (Glc-pendant C60) and Compound 20 ( Glc-pendant azafulleroid) Compound 21 (hexakis-glucosamine C60 derivative), Compound 22 (Glc-TEG-C60)

FIGURE 5 :

 5 FIGURE 5: Compound 23 (used against HIV-1 protease) and Compound 24 (used against amyloid )

SCHEME 10 :

 10 SCHEME 10: Synthetic steps for fullerene sugar ball(120 sugars)

FIGURE 6 :

 6 FIGURE 6: Hexa-adducts of C60 and its multivalent derivatives.

  catalyst, synthesized a fullerene ball of 360 disaccharides (28, in Figure7). They chose man-1,2-man disaccharides because they have higher affinity with DC-SIGN , 3-4-fold respect to monosaccharides. This compound was tested against ZIKV (Zika virus, a positive single-strand RNA virus transmitted by mosquitos) and DENV (Dengue virus) and resulted a very good inhibitor of the infection with IC50 in the picomolar range. Since there is not any therapy for ZIKV, this study opens a door on a new potential treatment of ZIKV based on inhibition of carbohydrate-protein binding.

FIGURE 7 :

 7 FIGURE 7:Potential antiviral agent against ZIKV and DENV with 1,2 binding disaccharides having higher affinity with DC-SIGN[START_REF] Ramos-Soriano | Synthesis of Highly Efficient Multivalent Disaccharide/[60]Fullerene Nanoballs for Emergent Viruses[END_REF]

  further studied the mechanism of multivalency inhibition of glycosidases. This research group used as inhitope motifs a) 1-amino-5N,6Ooxomethylydenenojirimycin (1N-ONJ) and b) its C2 epimer 1-amino-5N,6Ooxomethylydenemannnojirimycin (1N-OMJ), two sp 2 -iminosugars with ''mismatching'' (a) and ''matching''(b) structure relationships with -mannosidase. In this study, through a lectin (PNA)/glycosidase competitive assays, it was investigated the mechanism of multivalency inhibition and the relevance of the non-glycone sites for this activity; a comparison of the effect induced by simple monovalent iminosugars 29a,b and fullerene sp 2 -iminosugar balls 30b,c,d (figure

FIGURE 8 :

 8 FIGURE 8: Monovalent and multivalent inhitopes of glycosidase

FIGURE 9 :

 9 FIGURE 9: Homo and heterovalent fullerenes

FIGURE 10 :

 10 FIGURE 10: More efficient glycosidase inhibitors

  new generation of efficient inhibitors was prepared by the synthesis of some clickable keto-deoxyoctulosonate derivatives (34 a-c in figure11) that were easily conjugated with fullerene. These Kdo sugars are multivalent compounds much more active as inhibitor and affine for glycosyltransferase than the precedent glycofullerene derivatives resulting in a new potential class of glycosyltransferase inhibitors that could inspire further studies to reach therapeutic applications for cancer or HIV.

FIGURE 11 :

 11 FIGURE 11: kdo-fullerene derivatives

FIGURE 12 :

 12 FIGURE 12: Fullerene conjugated with Gb3-trisaccharides. It fit very well with a model of shiga-like toxin

FIGURE 13 :

 13 FIGURE 13: Peptide-fullerene with anti-infective properties

FIGURE 14 :

 14 FIGURE 14: Compounds developed by Masalova et al.

FIGURE 15 :

 15 FIGURE 15: C60-Tuftsin derivatives

FIGURE 16 :

 16 FIGURE 16: Fullerene-phenylalanine derivative

TABLE 1 :

 1 Values Ki (µM). Inhibition is competitive excepted 31b,c. [a] uncompetitive inhibition, [b] mixed type (both Ki from competitive and uncompetitive inhibition). N.a. (no data available). N.I. (no inhibition detected)


Vianney Le Pichon

Stéphane Bellon

Marion Desquilbet

Eve Fouilleux

Denis Lairon

Marc Tchamitchian

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