Lisa Tanzi
email: lisa.tanzi01@universitadipavia.it

Davide Rubes
email: davide.rubes01@universitadipavia.it

Teodora Bavaro
email: teodora.bavaro@unipv.it

Matthieu Sollogoub
email: matthieu.sollogoub@sorbonne-universite.fr

Massimo Serra
email: massimo.serra@unipv.it

Yongmin Zhang
email: yongmin.zhang@upmc.fr

Marco Terreni
email: marco.terreni@unipv.it

Controlled Decoration of [60]Fullerene with Polymannan Analogues and Amino Acid Derivatives through Malondiamide-Based Linkers

Keywords: bis-functionalized fullerenes, amino acid conjugates, glycofullerenes, mannose-based oligosaccharides, cyclopropanation

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Buckminster fullerene (C60), the smallest stable fullerene (diameter 10.34 Å), is characterized by high symmetry and two different types of carbon-carbon bonds. The 5,6 bond is a single bond of 1.45 Å length between 5-member and 6-member rings, whilst the 6,6 bond of 1.38 Å between two 6-member rings has a double bond character and is usually involved in fullerene reactivity. In the last years, fullerenes have attracted attention for their possible therapeutic applications in humans. Pure C60 is soluble only in organic solvent and can present potentially high toxicity, but these limitations could be overcome by submitting fullerene to chemical modifications with polar biomolecules. There are three main synthetic approaches to gain these functionalized derivatives, i.e., by exploiting cyclopropanation, hydroxylation and cycloaddition reactions [START_REF] Tanzi | Synthesis and biological application of glyco-and peptide derivatives of fullerene C60[END_REF].

Depending on the conjugated molecule, fullerene derivatives acquire different therapeutic properties [START_REF] Riley | Recent advances in carbon nanomaterials for biomedical applications: A review[END_REF][START_REF] Sharoyko | Biologically active water-soluble fullerene adducts: Das Glasperlenspiel (by H. Hesse)?[END_REF]. Conjugation of fullerene with carbohydrates for biological application has been largely investigated [4]. The most explored reaction to decorate C60 with sugar derivatives is cyclopropanation. Exploiting a Bingel cyclopropanation, up to six malonate ester chains carrying azido or propargyl terminal groups were one-pot linked to fullerene [START_REF] Cecioni | Synthesis of dodecavalent fullerene-based glycoclusters and evaluation of their binding properties towards a bacterial lectin[END_REF]. However, other conjugation methods were deeply investigated [START_REF] Ramos-Soriano | Maleimide and Cyclooctyne-Based Hexakis-Adducts of Fullerene: Multivalent Scaffolds for Copper-Free Click Chemistry on Fullerenes[END_REF][START_REF] Enes | Synthesis and solvent dependence of the photophysical properties of [60]fullerene-sugar conjugates[END_REF][START_REF] Uhlmann | Glycosylidene Carbenes. Part 20. Synthesis of deprotected, p -k -g y of C60[END_REF][9][START_REF] Yashiro | Fullerene glycoconjugates: A general synthetic approach via cycloaddition of per-O-acetyl glycosyl azides to [60]fullerene[END_REF].

T ff f g y y g y f f f " " f f sugars is to improve fullerene solubility in water or if they are involved in targeted drug delivery. Glycofullerene derivatives have been investigated in photodynamic therapy [START_REF] Serda | Development of photoactive Sweet-C 60 for pancreatic cancer stellate cell therapy[END_REF][START_REF] Serda | Developing[END_REF][START_REF] B Ń K E.; W Ć-Cudak | Interactions of a water-soluble glycofullerene with glucose transporter 1. Analysis of the cellular effects on a pancreatic tumor model[END_REF], in HIV-1 protease inhibition [START_REF] Strom | Fullerene-based inhibitors of HIV-1 protease[END_REF][START_REF] Tanimoto | Chemical methods for degradation of target proteins using designed lightactivatable 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] or against neurodegenerative diseases [START_REF] Ishida | Photo-degradation of amyloid β by a designed fullerene-sugar hybrid[END_REF][START_REF] Horie | Antioxidant action of sugar-pendant C60 fullerenes[END_REF].

On the contrary, the role of the sugars " " f y have a defined therapeutic activity. In this case, t " y" p p y y g g activity of sugar-modified fullerene, e.g., these derivatives have been investigated as potential ligands for receptors and enzymes used by viruses and bacteria (e.g., E. coli, Ebola or Dengue viruses) during the infection process [START_REF] Muñoz | Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection[END_REF][START_REF] Ramos-Soriano | Glycodendritic structures as DC-SIGN binders to inhibit viral infections[END_REF][START_REF] Nierengarten | Giant Glycosidase Inhibitors: First-and Second-Generation Fullerodendrimers with a Dense Iminosugar Shell[END_REF][START_REF] Durka | The inhibition of liposaccharide heptosyltransferase WaaC with multivalent glycosylated fullerenes: A new mode of glycosyltransferase inhibition[END_REF].

Similarly, the conjugation of amino acids or peptides with fullerene [START_REF] Tanzi | Synthesis and biological application of glyco-and peptide derivatives of fullerene C60[END_REF][START_REF] Jennepalli | Fullerenyl amino acids and peptides: A review of their synthesis and applications[END_REF][START_REF] Burley | Fullerene Amino Acids and Related Derivatives[END_REF] provides structural diversity and specific recognition properties [25]. Amino acid derivatives of fullerenes have been mainly studied as anti-infective agents against viruses such as cytomegalovirus and HIV [START_REF] Poletaeva | Antioxidant properties of water-soluble amino acid derivatives of fullerenes and their role in the inhibition of herpes virus infection[END_REF][START_REF] Friedman | Inhibition of the HIV-1 Protease by Fullerene Derivatives: Model Building Studies and Experimental Verification[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]. Fullerene conjugated with amino acids or peptides were also tested in the treatment of different pathologies that still require efficient therapeutic approaches, such as Alzheimer [START_REF] Xie | The molecular mechanism of fullerene-inhibited aggregation of A z ' β-amyloid peptide fragment[END_REF], lupus [START_REF] Sofou | Synthesis of a proline-rich [60]fullerene peptide with potential biological activity[END_REF] and in cancer diagnosis [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].

In the last few years, thanks to their intrinsic properties, such as size, biocompatibility, and the possibility to undergo chemical modification, nanomaterials have attracted increasing attention as promising tools for the development of new generation vaccines [START_REF] Donnelly | Vaccine delivery systems[END_REF][START_REF] Wendorf | Molecular Nanomedicine Towards Cancer[END_REF]. Some studies reported the efficiency of C60 derivatives as vaccine adjuvants [START_REF] Liu | The adjuvant effect of C60 (OH)22 nanoparticles promoting both humoral and cellular immune responses to HCV recombinant proteins[END_REF][START_REF] Masalova | Immunostimulation Effect of Water-Soluble Fullerene Derivatives ; Potential Adjuvants for the New Generation of Vaccines[END_REF], suggesting the idea that fullerene could be exploited as a carrier for covalent conjugation of immunogenic or antigenic sugars and/or peptides.

The high expression of Mannose Receptors (MR) on Antigen Presenting Cells (APCs) indicates that MR play a key role in antigen recognition. Indeed, mannosylated peptides and proteins stimulate MHC class II specific T cells with 200 to 10,000-fold higher efficiency compared to peptides or proteins that are not mannosylated [START_REF] Tan | Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells[END_REF]. Fairbanks et al. [START_REF] Mcintosh | Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells[END_REF] reported a stronger immune response obtained after the mannosylation of a peptide-epitope of a cytomegalovirus antigen compared to the non-glycosylated peptide.

Since our research activity is focused on the design of new glycovaccines, we considered the use of fullerene as a potential carrier for both immunogenic/antigenic sugars and peptides for the development of new generation nano-vaccines.

In this work we reported the decoration of C60 core by using malondiamide-based linkers endowed with natural polymannan analogues and natural/unnatural amino acid derivatives. The derivatization strategy is based on the conversion of fullerene into methanofullerenes through a Bingel cyclopropanation reaction, which usually takes place between fullerene and a halide-activated malonic acid derivative in the presence of a base [START_REF] Biglova | Nucleophilic cyclopropanation of [60]fullerene by the addition-elimination mechanism[END_REF].

Malondiamide-based linkers were chosen due to their higher chemical stability with respect to the corresponding ester derivatives. As a title of example, amide bond is stable under the basic reaction conditions required for the removal of acetyl protecting groups from sugar derivatives obtained through chemoenzymatic approaches, thus allowing the obtainment of fully deprotected sugar bioconjugates. Nevertheless, C60 functionalization with diamide-based linkers is a less used approach due to their low reactivity if compared with ester-based analogues. Among diamide-based linkers, those characterized by an aromatic amide moiety turned out to be more reactive than aliphatic ones. For instance, by using simple diarylamide derivatives, Li et al. succeeded in functionalizing fullerene in 20-35% yields (calculated on consumed fullerene) [START_REF] Li | Synthesis and stabilization mechanism of novel stabilizers for fullerene-malonamide derivatives in nitrocellulose-based propellants[END_REF]. Wang and co-workers exploited arylamide-derived chains carrying a porphyrin moiety to functionalize the C60 core in yields up to 44% [START_REF] Wang | Hydrogen bonding-mediated foldamerbridged zinc porphyrin-C60 dyads: Ideal face-to-face orientation and tunable donor-acceptor interacion[END_REF]. Similarly, a diarylamide-based linker was employed in a modified Bingel-type reaction to afford, in 77% yield, the first fullerene-based X-ray contrast agent [START_REF] Wharton | Highly-iodinated fullerene as a contrast agent for X-ray imaging[END_REF]. Only one example of direct functionalization of C60 with a sugar-modified malondiamide-based linker has been previously reported [START_REF] Kato | Sugar balls: Synthesis and supramolecular assembly of [60]fullerene glycoconjugates[END_REF]. By exploiting an aliphatic bis-malonamide derivative, Hirsch and coworkers decorated fullerene with fully acetyl-protected α-D-mannopyranose.

Since this last reaction proceeded in a quite low 6% yield, a further goal of our study was to improve the yield of fullerene functionalization with sugar-modified aliphatic diamide chains.

In order to better control the derivatization process, our idea was to assemble the sugar/amino acidmodified malondiamide chains exploiting a copper-mediated Huisgen 1,3-dipolar cycloaddition and then proceed to the C60 functionalization.

In the last part of this work, we focused our attention on the synthesis of heterosubstituted C60 scaffolds. Actually, a few studies of hetero-functionalization of C60 are described in literature [START_REF] Gulumkar | Synthesis of an Azide-and Tetrazine-Functionalized [60]Fullerene and Its Controlled Decoration with Biomolecules[END_REF][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], and there are no examples of C60 functionalization with both sugar and amino acid derivatives through the use of malondiamide-based linkers.

Results

Synthesis of Propargyl-Mannose Glycans

The first step involved the synthesis of natural polymannan analogues. For this purpose mannose-based di-w α(1 2) α(1 6) f w g y z . T w obtainment of sugar derivatives simpler than natural polymannans (composed by 10 or more mannose units) and bearing a propargyl group in anomeric position.

The synthesis of the desired di-and trimannans was planned by means of a chemoenzymatic approach. Acetylated building blocks 1a-c, characterized by a free hydroxyl group in position C2 or C6 suitable as acceptors for the synthesis of the corresponding disaccharides 4-6, were prepared by exploiting immobilized enzymes such as Candida rugosa lipase (CRL) and Bacillus pumilus acetyl xylan esterase (AXE) (Scheme 1) [START_REF] Tanzi | Developing a Library of Mannose-Based Mono-and Disaccharides : A General Chemoenzymatic Approach to Monohydroxylated Building Blocks[END_REF].

By adopting slightly different reaction conditions (time and temperature), the monodeprotected building blocks (1a-c) were conjugated by Schmidt glycosylation reaction with man-trichloroacetimidate 2 [START_REF] Schmidt | Facile Synthesis of α-and β-O-G y y Imidates; Preparation of Glycosides and Disaccharides[END_REF] to give disaccharides 4-6 (Scheme 2).

Man(α1,6)man disaccharides 4 and 5 were obtained in 86% and 90% yield after 2.5 and 4 h, respectively, working at 0 °C. The synthesis of man(α1,2)man disaccharide (6) required w p (-50/-70 ° ) to afford 6 in 80% yield.

The disaccharide 4 was converted into the corresponding trichloroacetimidate 4b through anomeric deprotection/activation in 52% overall yield. The subsequent coupling reaction with acceptor 1b afforded, after 4 h at 0 °C, the desired trisaccharide 7 in 56% yield. Scheme 1. Synthesis of propargyl-modified sugar derivatives. Experimental condition: (i) BF3 Et2O, dry DCM, 0 °C, 2.5 h (86%); (ii) BF3 Et2O, dry DCM, 0 °C to rt, 4 h (90%); (iii) BF3 Et2O y D -50 ° 18 (80%); ( ) y z D F 50 °C, 5 h (92%); (v) trichloroacetonitrile, DBU, dry DCM, 0 °C, 4 h (57%); (vi) BF3 Et2O, dry DCM, 0 °C, 4 h (56%).

Preparation of Glycosylated C60 Derivatives

As mentioned above, our approach to fullerene decoration consisted of the use of malondiamide-based linkers previously modified with the desired sugar derivatives through 1,3 Huisgen dipolar cycloaddition. Thus, the synthesis of a malondiamide chain endowed with two terminal azide groups was accomplished (Scheme 2). 1-Amino-3-chloropropane hydrochloride was reacted with sodium azide to afford 3-azidopropan-1-amine 8 in a 91% yield. This intermediate was then coupled with malonyl dichloride to give the bis-azido malondiamide 9 in a 44% yield. The click reaction was initially performed between the malondiamide chain 9 and the acetylated monosaccharide 3 to obtain product 10. Then, the optimized conditions were applied to conjugate disaccharides 5 and 6 and trisaccharide 7. Scheme 2. Synthesis of sugar-modified malondiamide linkers. Experimental conditions: (i) 3-chloro propylamine, NaN3, H2O, 80 °C, 6 h (91%); (ii) malonyl chloride, aqueous NaOH, CH3Cl, 0 °C, 20 min (44%); (iii) CuSO4, Na ascorbate, THF/ H2O, overnight.

The procedure of the Huisgen 1,3-dipolar cycloaddition consisted of dissolving the malondiamide chain in the presence of the proper sugar derivative working in a tetrahydrofuran/H2O (1:1) mixture. Then, copper sulphate and sodium ascorbate were added. The desired products were obtained in good yields (78-83%) after flash chromatography purification.

The malondiamide-sugar chains (10-13) were then reacted with fullerene exploiting a Bingel-Hirsch cyclopropanation for the preparation of the corresponding glycosylated fullerenes (Scheme 3). Scheme 3. Preparation of glycosylated fullerenes 14-17.

The mannose-functionalized malondiamide 10 was used as a model compound to optimize the reaction conditions. In a first attempt, 10 was dissolved in dry toluene with C60 (1 eq.) and iodine (1.2 eq.), then DBU was added at 0 °C and the mixture was stirred at room temperature for 90 min. After purification, fullereneman 14 was obtained in a 17% yield in a one-pot fashion (Table 1, entry 1).

Interestingly, monitoring the reaction over time by TLC, we observed the gradual decrease in the product spot and, at the same time, the formation of various new spots ascribable to possible side-products. Assuming that the low conjugation yield could have been due to the degradation of the desired product, the reaction time was shortened. We were pleased to find that functionalized fullerene 14 was obtained in a 30% yield after 1 h (entry 2) and in 41% after 45 min at rt (entry 3).

Each glycosylated malondiamide chain required the application of different reaction conditions to achieve satisfactory yields (Table 1), highlighting a structure-reactivity correlation which characterizes these sugar derivatives. As shown in Table 1, their reactivity decreased as the length of the sugar chain increased. This could be due, probably, to the steric hindrance of the different glycosidic residues, affecting the reactivity of the malondiamide moiety. The mannosylated chain 11 was first reacted at room temperature for 60 and 90 min to afford fullereneman(α1,6)man 15 in 12% and 10% yields, respectively (entries 4 and 5). By decreasing the reaction times, the yields increased, and the desired product was isolated in 16% after 30 min and 24 % yields after only 7 min at rt (entries 6 and 8). These results, suggesting a lower stability of 15 at the applied reaction conditions with respect to 14, prompted us to decrease the reaction temperature. Indeed, by performing the C60 functionalization at 0 °C, the yield raised to 31% (entry 11). The same yield was obtained by shortening the reaction time to 1 h (entry 10).

When the reaction conditions adopted for the preparation of fullerene-man(α1,6)man 15 (1 h, 0 °C) were applied to man(α1,2)man malondiamide 12, the desired product 16 was obtained in a low 8% yield (entry 12). L w g p -20° ff y p f y ( 13 14). On the contrary, when the reaction was performed at rt for three hours, the product was obtained in a 11% yield (entry 15). Longer reaction times resulted again in lower yields (entries 17 and 18). Likewise, the g ' (0.7 q f g 1 q f 60) did not lead to any improvement. To obtain fullerene-man(α1,6)man(α1,6)man ( 17) the corresponding malondiamide-chain 13 was initially reacted in the same reaction conditions adopted for the conjugation of man(α1,6)man chain 11. The desired product 17 was obtained in a 12% yield (entry 19). Attempts to improve the reaction yield by changing reaction time and temperature turned out to be unsuccessful.

Conjugation of C60 with Amino Acid Derivatives

By exploiting the approach described for the functionalization of C60 with sugar derivatives, the conjugation of a model amino acid such as L-alanine was attempted. The natural amino acid was first converted into the corresponding methyl ester, then its amino group was reacted with 5-hexynoic acid in the presence of EDC*HCl and DMAP to introduce the alkyne moiety (Scheme 4). The obtained amino acid derivative 18 was then submitted to azide-alkyne Huisgen cycloaddition with the malondiamide chain by using copper sulphate and sodium ascorbate in a mixture of tetrahydrofuran/water as the solvent [START_REF] Van Berkel | A macrocyclic coumarin-containing tripeptide via CuAAC chemistry[END_REF]. The desired bis-functionalized compound 19 was obtained in a 75% yield after flash chromatography purification (scheme 4).

The first conjugation experiments were performed by following the previously reported protocol based on a Bingel-Hirsch reaction. Given the very poor solubility of the amino acid-chain in toluene, we explored the replacement of toluene with 1,2-dichlorobenzene and the use of various co-solvents, such as DMSO, DMF, DCM, 1,2-dichlorobenzene and CHCl3. However, only traces of the desired functionalized product (structure confirmed by HRMS analysis) were obtained. Attempts to improve the yield by prolonging the reaction time, working in the presence of molecular sieves, under different inert atmospheres (argon, nitrogen), or performing the reaction at various temperatures (from -20 to 40 °C) proved to be fruitless; the only difference being, at higher temperatures, the increasing formation of side-products of undefined structures.

These results prompted us to reconsider the functionalization protocol. We decided to preserve the onepot approach but replacing iodine with CBr4 [START_REF] Camps | Efficient cyclopropanation of C60 starting from malonates[END_REF]. The addition of DBU to a 1:1 mixture of C60 and 19 in toluene, in the presence of CBr4, using CHCl3 as a co-solvent, allowed the obtainment of the functionalized product 20 in a satisfactory 20% yield.

To prove the applicability of this protocol to different amino acid derivatives such as non-proteinogenic or quaternary amino acids, we decided to prepare the 2-aminoisobutyric (Aib) functionalized malondiamide chain 22. The preparation and the coupling of the Aib moiety to azide 9 was performed, by following the previously reported protocol, with very similar reaction yields. The same can be said for the conjugation reaction, which afforded the Aib-functionalized fullerene 23 in a 25% yield.

Double Conjugation of C60: Sugar and Amino Acid Derivative

With the optimized protocol in our hands, we wondered if it was possible to functionalize C60 with both a malondiamide-sugar chain and a malondiamide-amino acid chain in two subsequent steps. Indeed, if proved to be feasible, this protocol would not only be adaptable to the coupling of different derivatives but would also allow controlling the stoichiometry of the reaction in a more accurate fashion.

The mannosylated C60 14 was then reacted with the (Aib)-functionalized malondiamide chain 22 in the presence of CBr4 and DBU (Scheme 5). Due to the similar solubility of 14 and 22 in CHCl3, the reaction was performed in the latter solvent, avoiding the use of toluene. To our delight, TLC monitoring of the reaction revealed the exclusive formation of a new product. After 24 h the reaction mixture was submitted to chromatographic purification, affording the bis-functionalized fullerene 24 in a 37% yield, as a single regioisomer. This result is particularly worthy of note, as, for two different addends, nine regioisomeric bisadducts are in principle possible. Each isomeric bis-addition pattern displayed a typical absorption feature in the visible light region, almost independent of the type of addends and the substituents [START_REF] Djojo | Regiochemistry of Twofold Additions to [6,6] Bonds in C60: Influence of the Addend-Independent Cage Distortion in 1,2-Monoadducts[END_REF]. Regarding the structural assignment of 24, the UV-Vis spectrum is in accordance with that of a trans-3 bisadduct (see supplementary materials), which usually is the preferred regioisomer in the case of hetero functionalization with sterically demanding addends [START_REF] Djojo | Regiochemistry of Twofold Additions to [6,6] Bonds in C60: Influence of the Addend-Independent Cage Distortion in 1,2-Monoadducts[END_REF][START_REF] Trinh | Regioselective Preparation of Fullerene Bis-adducts from Cleavable Macrocyclic Bis-malonates[END_REF][START_REF] Djojo | Synthesis and chiroptical properties of enantiomericaily pure bis-and trisadducts of C60 with an inherent chiral addition pattern[END_REF]. However, a further confirmation of this data through NMR experiments was hampered due to the broad 1H and 13C signals generated by the presence of rotamers.

In conclusion, the described protocol allows a highly controlled hetero functionalization of Buckminster fullerene, on which the two addends are present in a 1:1 ratio, as confirmed by NMR and HRMS analysis. To the best of our knowledge, this is the first example of the contemporary functionalization of C60 with both sugar and amino acid moieties exploiting a malondiamide-based linker. Scheme 5. Synthesis of bis-functionalized fullerenes.

Conclusions

We have developed an efficient synthetic protocol for the functionalization of Buckminster fullerene with both polymannan analogues and natural/non-proteinogenic amino acid derivatives.

Simpler analogues of natural polymannan endowed with a propargyl group at the anomeric position were synthesized in their acetylated form exploiting a chemoenzymatic approach. These compounds were submitted to azide-alkyne 1,3 dipolar cycloaddition with a malondiamide-based linker carrying two azido terminal groups. The obtained sugar-modified malondiamide chains were then reacted in the presence of C60, affording in a one-pot the corresponding functionalized Buckminster fullerenes with yields ranging from 11% to 41%, higher than those reported in the literature with similar derivatives [START_REF] Kato | Sugar balls: Synthesis and supramolecular assembly of [60]fullerene glycoconjugates[END_REF].

Through a slight modification of the functionalization procedure, we demonstrated that natural and non-proteinogenic quaternary amino acids can be also easily linked to C60 in a satisfactory 20-26% overall yields.

Finally, the first hetero functionalization of fullerene with both amino acid-and sugar-modified malondiamide linkers has been successfully performed affording the bis-functionalized derivative as a single regioisomer in a 37% yield.

Noteworthy, this conjugation strategy allowed the obtainment of properly decorated fullerenes in a highly controlled fashion.

The derivatization protocol showed to be reliable and, in principle, suitable for the conjugation of more complex oligopeptide derivatives. The synthesis of mannosylated C60 endowed with antigenic peptides, which could find future potential application as new generation nanovaccines, is currently under investigation in our group.

Materials and Methods

Reactants and chemicals were purchased from commercial sources (Sigma-Aldrich, Burlington, Massachusetts, U.S. (Merck KgaA group), Alfa Aesar Ward Hill, Massachusetts, U.S. (Thermo Fisher Scientific group), Fluorochem Limited, Hadfield, U.K., and used without further purification. Solvents were purified according to the guidelines in the Purification of Laboratory Chemicals [START_REF] Armarego | Purification Of Laboratory Chemicals[END_REF]. All solvents were freshly distilled from the appropriate drying agent. THF, and toluene were distilled from sodium/benzophenone ketyl; TEA and DCM from CaH2. Reactions requiring anhydrous conditions were performed under Ar or N2. Yields were calculated for compounds purified by flash chromatography and judged homogeneous by thinlayer chromatography, NMR and mass spectrometry.

Compound purification was performed by flash chromatography using Silica Gel high-purity grade, pore size 60 Å 70-230 mesh, 63-200 μ ( g -Aldrich). Analytical thin layer chromatography (TLC) was performed on silica gel F254 precoated aluminium sheets (0.2 mm layer, Merck, Darmstadt, Germany), visualized by a 254 nm UV lamp, and stained with aqueous ceric molybdate solution or iodine and a f 4 4′-methylenebis-N,N-dimethylaniline, ninhydrin, and KI in an aqueous ethanolic solution of AcOH, or by spraying with 5% H2SO4 in ethanol, followed by heating to 150 °C. Characterization of purified compounds was performed by NMR spectroscopy. NMR spectra were recorded on a Bruker Advance III 400 MHz/600 MHz spectrometer (Bruker Corporation, Billerica, MA, USA). All 1D and 2D NMR spectra were acquired using the standard pulse sequences available with Bruker Topspin 3.6 software package. Chemical f (δ) g pp w f g . g p follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; td, triplet of doublets; m, multiplet. Structures assignment was performed by means of 2D-COSY and HSQC. Infrared spectra were recorded on a Perkin-Elmer ATR-FTIR 1600 series spectrometer using neat samples. UV-Vis spectra were recorded on a Shimadzu UV-1900 spectrophotometer.

High-resolution mass HRMS data of compounds 7, 10-17, were acquired using a Bruker Micro-TOF spectrometer in electrospray ionization (ESI) mode, using Tuning-Mix as reference. High-resolution mass HRMS data of compounds 9, 18-24 were acquired using a X500B QTOF System (SCIEX, Framingham, MA 01701 A) q pp w Tw p y E p p Ex L ™ y ( EX). T SCIEX OS software 2.1.6 was used as an operating platform. For MS detection the following parameters were applied: Curtain gas 30 psi, Ion source gas 1 45 psi, Ion source gas 2 55 psi, Temperature 450 °C, Polarity positive, Ionspray voltage 5500 V, TOF mass range 50-2800 Da, declustering potential 60 V and collision energy 10 V.

Sugar Synthesis

The chemoenzymatic synthesis of compounds 1a-c, 2, 3, 4, 4a, 4b, 5, and 6 was performed by following previously described procedures, and their spectroscopic data were matched with literature values [START_REF] Tanzi | Developing a Library of Mannose-Based Mono-and Disaccharides : A General Chemoenzymatic Approach to Monohydroxylated Building Blocks[END_REF]. Propargyl-2" 3" 4" 6"-tetra-O-acetyl-α-D-mannopyranosyl-(1→6)-2′ 3′ 4′-tri-O-acetyl-α-Dmannopyranosyl-(1→6)-2,3,4-tri-O-acetyl-α-D-mannopyranoside [START_REF] Enes | Synthesis and solvent dependence of the photophysical properties of [60]fullerene-sugar conjugates[END_REF]. 2′ 3′ 4′ 6′-tetra-O-acetyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-acetyl-α-D-mannopyranosyl trichloracetimidate (4b) (250 mg, 0.658 mmol, 2 eq.) and propargyl 2,3,4-tri-O-acetyl-α-D-mannopyranoside (1b) (113 mg, 0.329 mmol, 1 eq.) were dissolved in dry dichloromethane (10 mL) in the presence of activated molecular sieves and cooled to 0 °C under nitrogen atmosphere. BF3 Et2O (40.6 µL, 0.329 mmol, 1 eq.) was added and the mixture was stirred at 0 °C for 4 h. The reaction was quenched with triethylamine (45.644 µL, 0.329 mmol, 1 eq.), stirred for 5 min, filtered and concentrated in vacuo.

The reaction mixture was monitored by TLC (DCM/acetone 9:1, Rf = 0.52 or EtOAc/n-hexane 5:5, Rf = 0.19) and purified by flash chromatography (DCM/acetone 9:1 and EtOAc/n-hexane 5:5). The desired product (181 mg, 56%) was obtain as a white solid.

IR (neat, cm

Functionalization of C60 with Sugar Derivatives

3-azidopropan-1-amine (8) [START_REF] Hodgson | Reproducible Dendronized PEG Hydrogels via SPAAC Cross-Linking[END_REF].

3-chloropropylamine hydrochloride (6 g, 46.147 mmol, 1 eq.) and NaN3 (11.4 g, 175.358 mmol, 3.8 eq.) was dissolved in H2O (20 mL) and the mixture was stirred at 80 °C for 6 h.

The reaction was then treated with saturated KOH solution (10 mL), added dropwise at 0 °C. The aqueous layer was extracted with Et2O (3 × 25 mL). The organic phase was dried over MgSO4, filtered and concentrated in vacuo at room temperature (aliquots of pentane were added and evaporated to extract any Et2O residue). The desired product was obtained as a yellow oil (4.18 g, 91%). 1 H and 13 C NMR were in agreement with previously reported data [START_REF] Hodgson | Reproducible Dendronized PEG Hydrogels via SPAAC Cross-Linking[END_REF].

N 1 ,N 3 -bis(3-azidopropyl)malondiamide (9) 3-azidopropan-1-amine (8) (2.5 g, 0.025 mol, 1 eq.) was dissolved in chloroform (20 mL) and a solution of NaOH (2 g) in H2O (4 mL) was added. The mixture was cooled to 0 °C and a solution of malonyl chloride (1.21 mL, 0.0125 mol, 0.5 eq.) in chloroform (10 mL) was added dropwise, then was stirred for 20 min at 0 °C. The mixture was extracted with chloroform, the organic phase was dried over MgSO4, filtered and concentrated in vacuo.

The reaction mixture was monitored by TLC (ethyl acetate 100%. Rf = 0.45). Column chromatography (SiO2, ethyl acetate 100%) gave the desired product (1.5 g, 44%) as a white powder.

IR (neat, cm Man malondiamide chain (10) Propargyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (3) (86.38 mg, 0.224 mmol, 3 eq.) was dissolved in THF (8 mL) and H2O (8 mL). N 1 ,N 3 -bis(3-azidopropyl)malondiamide (9) (20 mg, 0.0746 mmol, 1 eq.) then copper sulphate pentahydrate (186.2 mg, 0.746 mmol, 10 eq.) and sodium ascorbate (252 mg, 1.268 mmol, 17 eq.) were added and the mixture was stirred overnight at rt. The reaction was extracted with dichloromethane, and the organic layer was washed with saturated NaHCO3 solution, then dried over MgSO4, filtered and concentrated in vacuo.

The reaction mixture was analysed by TLC (DCM/MeOH 95:5, Rf = 0.31). Column chromatography (SiO2, dichloromethane/MeOH 95:5) gave the desired product (62 mg, 80%) as white solid.

IR (neat, cm 13 C{ 1 H}-NMR (100 MHz, CDCl3): δ = 20.6-20.8 (8C, CH3COO), 29.9 (NHCH2CH2CH2N), 36.5 (NHCH2CH2CH2N), 42.8 (COCH2CO), 47.9 (NHCH2CH2CH2N), 60.9 (triazole-CH2O), 62.4 (C-6a, C-6b), 66.0 (C-3), 68.7 (C-5), 69.1 (C-2), 69.4 (C-4), 96.8 (C-1), 123.6 (NCH=C), 143.5, 167.8 (COCH2CO), 169.7-170.7 (8C, CH3COO); HRMS (ESI) calculated for C43H60N8O22Na [M + Na] + 1063.3714, found 1063.3716 (Δ = -0.2 ppm).

Man(α1 6)man malondiamide chain [START_REF] Serda | Development of photoactive Sweet-C 60 for pancreatic cancer stellate cell therapy[END_REF] Propargyl-2′ 3′ 4′ 6′-tetra-O-acetyl-α-D-mannopyranosyl-(16)-2,3,4-tri-O-acetyl-α-D-mannopyranoside (5) (37.6 mg, 0.0558 mmol, 3 eq.) was dissolved in THF (2.5 mL) and H2O (2.5 mL). N 1 ,N 3 -bis(3-azidopropyl) malondiamide (9) (5 mg, 0.0186 mmol, 1 eq.) then copper sulphate pentahydrate (46.4 mg, 0.186 mmol, 10 eq.) and sodium ascorbate (63 mg, 0.316 mmol, 17 eq.) were added and the mixture was stirred overnight at rt. The reaction was extracted with dichloromethane, and the organic layer was washed with saturated NaHCO3 solution, then dried over MgSO4, filtered and concentrated in vacuo. The reaction mixture was analysed by TLC (DCM/MeOH 95:5, Rf = 0.28). Column chromatography (SiO2, dichloromethane/MeOH 95:5) gave the desired product (25 mg, 83%) as a white solid.

IR (neat, cm (α1 2) (12) Propargyl-2′ 3′ 4′ 6′-tetra-O-acetyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranoside (6) (71 mg, 0.105 mmol, 3 eq) was dissolved in THF (3 mL) and H2O (3 mL). N 1 ,N 3 -bis(3-azidopropyl) malondiamide (9) (9.4 mg, 0.0351 mmol, 1 eq.) then copper sulphate pentahydrate (118.8 mg, 0.351 mmol, 10 eq.) and sodium ascorbate (87.64 mg, 0.596 mmol, 17 eq.) were added and the mixture was stirred overnight at rt. The reaction was extracted with dichloromethane, and the organic layer was washed with saturated NaHCO3 solution, then dried over MgSO4, filtered and concentrated in vacuo.

The reaction mixture was monitored by TLC (dichloromethane/MeOH 95:5, Rf = 0.28). Column chromatography (SiO2, dichloromethane/MeOH 95:5) gave the desired product (43.7 mg, 77%) as white solid.

IR (neat, cm (α1 6) (α1 6) (13) Propargyl-2" 3" 4" 6"-tetra-O-acetyl-α-D-mannopyranosyl-(1→6)-2′ 3′ 4′-tri-O-acetyl-α-Dmannopyranosyl-(1→6)-2,3,4-tri-O-acetyl-α-D-mannopyranoside (7) (32 mg, 0.0336 mmol, 3 eq.) was dissolved in THF (2 mL) and H2O (2 mL). N 1 ,N 3 -bis(3-azidopropyl) malondiamide (9) (3 mg, 0.0112 mmol, 1 eq.) then copper sulphate pentahydrate (27.9 mg, 0.112 mmol, 10 eq.) and sodium ascorbate (37.9 mg, 0.190 mmol, 17 eq.) were added and the mixture was stirred overnight at rt. The reaction was extracted with dichloromethane, and the organic layer was washed with saturated NaHCO3 solution, then dried over MgSO4, filtered and concentrated in vacuo. The reaction mixture was monitored by TLC (DCM/MeOH 95:5, Rf = 0.30). Column chromatography (SiO2, DCM/MeOH 95:5) gave the desired product (18.5 mg, 75%) as white solid.

IR (neat, cm Fullerene-man ( 14) C60 (7 mg, 0.0096 mmol, 1 eq.), compound 10 (10 mg, 0.0096 mmol, 1 eq.) and I2 (3 mg, 0.0117 mmol, 1.22 q.) w y (4.5 L). DB (3 μL 0.0216 2.25 q.) was added to the mixture under argon at 0 °C and the reaction was stirred at rt for 45 min. The reaction mixture was monitored by TLC (DCM/acetone 1:1. Rf = 0.77). The solvent was removed under reduced pressure and a column chromatography (SiO2, DCM/acetone 1:1) gave the desired product (7 mg, 41%) as a black-brown solid.

IR (neat, cm Fullerene-(α1 6) (15) C60 (6.24 mg, 0.00866 mmol, 1 eq.), Compound 11 (14 mg, 0.00866 mmol, 1 eq.) and I2 (2.68 mg, 0.0105 1.22 q.) w y (4.5 ). DB (2.9 μL 0.0195 2.25 q.) w solution under argon at 0 °C and the reaction was stirred at 0 °C for 1 h. The reaction mixture was monitored by TLC (DCM/Acetone 6:4, Rf = 0.75). The solvent was removed under reduced pressure and a column chromatography (SiO2, DCM/acetone 6:4) gave the desired product (6 mg, 30 %) as a black-brown solid.

IR (neat, cm Fullerene-(α1 2) (16) C60 (4.4 mg, 0.00618 mmol, 1 eq.), Compound 12 (10 mg, 0.00618 mmol, 1 eq.) and I2 (1.9 mg, 0.00754 1.22 q.) w y (4 L). DB (2.1 μL 0.0139 2.25 q.) w added to the solution under argon at 0 °C and the reaction was stirred at rt for 1.5 h. The reaction mixture was monitored by TLC (DCM/acetone 6:4, Rf = 0.73). The solvent was removed under reduced pressure and a column chromatography (SiO2, DCM/acetone 6:4) gave the desired product (1.4 Fullerene-(α1 6) (α1 6) (17) C60 (5 mg, 0.007 mmol, 1 eq.), Compound 13 (15.5 mg, 0.007 mmol, 1 eq.) and I2 (2.18 mg, 0.00854 mmol, 1.22 q.) w y (4 L). DB (2.4 μL 0.0157 2.25 q.) w x under argon at 0 °C and the reaction was stirred at rt for 1.5 h. The reaction mixture was monitored by TLC (DCM/acetone 6:4, Rf = 0.74). The solvent was removed and a column chromatography (SiO2, DCM/acetone 6:4) gave the desired product (3 mg, 10%) as a black-brown solid.

IR (neat, cm

Functionalization of C60 with Amino Acid Derivatives

Methyl hex-5-ynoyl-L-alaninate (18) A suspension of L-alanine methyl ester hydrochloride salt (125 mg, 0.896 mmol, 1 eq.) in DCM (0.1 M) was added with 5-hexynoic acid (95 , 0.896 mmol, 1 eq.) and DMAP (196 mg, 1.60 mmol, 1.8 eq.) at rt under nitrogen atmosphere. The mixture was cooled at 0 °C and EDC HCl (172 mg, 0.896 mmol, 1 eq.) was added. After 30 min the reaction was allowed to warm to rt and maintained under stirring overnight.

The reaction was added with 15 mL of 1 M HCl and extracted with DCM. The organic phases were collected and washed with saturated NaHCO3 solution, then dried over Na2SO4, filtered, and concentrated in vacuo to give 18 (155 mg, 89%) as a colourless oil without the need of further purifications.

The reaction was monitored by TLC analysis (EtOAc/Hex 6:4, Rf = 0.33). IR (neat, cm Compound 18 (100 mg, 0.581 mmol, 2.1 eq.) and N 1 ,N 3 -bis(3-azidopropyl)malondiamide 9 (75 mg, 0.227 mmol, 1 eq.) were dissolved in 27 mL of THF/H2O (2:1) mixture and added with copper sulphate pentahydrate (48 mg, 0.193 mmol, 0.7 eq.) and sodium ascorbate (61 mg, 0.304 mmol, 1.1 eq.). Reaction completion was monitored by TLC analysis (EtOAc/Hex 6:4 to evaluate starting material consumption; DCM/MeOH 8:2 to monitor product formation) and, after 4 h, the solvent was evaporated at reduced pressure. The crude mixture was purified by flash chromatography (DCM/MeOH 85:15) to obtain compound 19 as a white solid (135 mg, 73%) Rf = 0.29 IR (neat, cm -1 ) 3298, 3120, 3066, 2952, 2850, 1736, 1638, 1625, 1539, 1211, 1166, 1055; 1 H-NMR (CDCl3, 400 MHz,) δ 1.41 (d, J = 7.2 Hz, 6H, CHCH3), 2.01 (quintuplet, J = 7.1 Hz, 4H, COCH2CH2CH2), 2.16 (m, 4H, NHCH2CH2CH2N), 2.27 (t, J = 7.3 Hz, 4H, COCH2CH2CH2), 2.79 (t, J =7.0, 4H, COCH2CH2CH2), 3.17 (s, 2H, COCH2CO), 3.27 (q, J = 6.2 Hz, 4H, NHCH2CH2CH2N), 3.76 (s, 6H, CO2Me), 4.41 (t, J = 6.5, 4H, NHCH2CH2CH2), 4.60 (q, J = 7.2, 2H, CHCH3), [START_REF] Ramos-Soriano | Maleimide and Cyclooctyne-Based Hexakis-Adducts of Fullerene: Multivalent Scaffolds for Copper-Free Click Chemistry on Fullerenes[END_REF] Fullerene-L-alanine (20) C60 (55 mg, 0.075 mmol, 1 eq.), compound 19 (50 mg, 0.075 mmol, 1 eq.) and CBr4 (28 mg, 0.083 mmol, 1.1 eq.) were dissolved in 28 mL of toluene/CHCl3 (3:1). DB (25 μL 0.166 2.2 q.) w mixture under nitrogen atmosphere at 0 °C and the reaction was allowed to warm to rt. After 48 h the solvent was evaporated at reduced pression and the crude mixture was purified by flash chromatography (toluene to recover unreacted C60, then DCM/MeOH 9:1) to obtain compound 20 as a red-brown solid (21mg, 20%) Rf = 0.38.

IR (neat, cm A suspension of 2-aminoisobutyric acid methyl ester hydrochloride salt (145 mg, 0.945 mmol, 1 eq.) in DCM (0.1 M) was added with 5-hexynoic acid (100 , 0.945 mmol, 1 eq.) and DMAP (208 mg, 1.70 mmol, 1.8 eq.) at rt under nitrogen atmosphere. The mixture was cooled at 0 °C and EDC HCl (181 mg, 0.945 mmol, 1 eq.) was added. After 30 min, the reaction was allowed to warm to rt and maintained under stirring overnight.

The reaction was added with 15 mL of 1 M HCl and extracted with DCM. The organic phases were collected and washed with saturated NaHCO3 solution, then dried over Na2SO4, filtered, and concentrated in vacuo to give 21 (161 mg, 81%) as a white solid without the need of further purifications.

The reaction was monitored by TLC analysis (EtOAc/Hex 6:4, Rf = 0.39). IR (neat, cm Compound 21 (101 mg, 0.478 mmol, 2.1 eq.) and N 1 ,N 3 -bis(3-azidopropyl)malondiamide 9 (61 mg, 0.227 mmol, 1 eq.) were dissolved in 21 mL of THF/H2O (2:1) mixture and added with copper sulphate pentahydrate (39 mg, 0.158 mmol, 0.7 eq.) and sodium ascorbate (49 mg, 0.248 mmol, 1.1 eq.). Reaction completion was monitored by TLC analysis (EtOAc/Hex 6:4 to evaluate starting material consumption; DCM/MeOH 8:2 to monitor product formation) and, after 3 h, the solvent was evaporated at reduced pressure. The crude mixture was purified by flash chromatography (DCM/MeOH 80:20) to obtain compound 22 as a colourless oil (152 mg, 98%) Rf = 0.37.

IR (neat, cm -1 ) 3278, 3065, 2948, 1737, 1642, 1534, 1149; 1 H-NMR (CDCl3, 400 MHz,) δ 1.52 (s, 12H, C(CH3)2), 1.97 (quintuplet, J = 7.1 Hz, 4H, COCH2CH2CH2), 2.15 (quintuplet, J = 6.5 Hz, 4H, NHCH2CH2CH2N), 2.20 (t, J = 7.1 Hz, 4H, COCH2CH2CH2), 2.77 (t, J = 7.0, 4H, COCH2CH2CH2), 3.13 (s, 2H, COCH2CO), 3.28 (q, J = 6.3 Hz, 4H, NHCH2CH2CH2N), 3.73 (s, 6H, CO2Me), 4.40 (t, J = 6.5, 4H, NHCH2CH2CH2), 6.77 (s, 2H, CONHC(CH3)2), 7.27 (br s, 2H, CONHCH2), 7.45 (s, 2H, Ar CH triazole); 13 Fullerene-Aib ( 23) C60 (51 mg, 0.071 mmol, 1 eq.), compound 22 (49 mg, 0.071 mmol, 1 eq.) and CBr4 (26 mg, 0.083 mmol, 1.1 eq.) were dissolved in 25 mL of toluene/CHCl3 (4:1). DB (23 μL 0.156 2.2 q.) w mixture under nitrogen atmosphere at 0 °C and the reaction was allowed to warm to rt. After 72 h, the solvent was evaporated at reduced pression and the crude mixture was purified by flash chromatography (toluene to recover unreacted C60, then DCM/MeOH 92:8) to obtain compound 23 as a red-brown solid (26 mg, 26%) Rf = 0.30.

IR (neat, cm -1 ) 3278, 3068, 1737, 1652, 1532, 1433, 1365, 1216, 1147; 1 H-NMR (DMSO-d6, 400 MHz,) δ 1.33 (s, 12H, C(CH3)2), 1.79 (quintuplet, J = 7.3 Hz, 4H, COCH2CH2CH2), 2.07-2.17 (m, 8H, NHCH2CH2CH2N + COCH2CH2CH2), 2.59 (t, J = 7.3 Hz, 4H, COCH2CH2CH2), 3.39 (q, J = 6.3 Hz, 4H, NHCH2CH2CH2N), 3.55 (s, 6H, CO2Me), 4.40 (t, J = 7.1, 4H, NHCH2CH2CH2), 7.87 (s, 2H, Ar CH triazole); 8.17 (s, 2H, CONHC(CH3)2), 9.52 (t, J = 5.7 Hz, 2H, CONHCH2);

13 C{ 1 H}-NMR (DMSO-d6, 100 MHz) δ 24.9, 25.

Double Functionalization of C60

Fullerene-man-Aib [START_REF] Burley | Fullerene Amino Acids and Related Derivatives[END_REF] Fullerene-Man 14 (20 mg, 0.011 mmol, 1 eq.), compound 22 (8 mg, 0.011 mmol, 1 eq.) and CBr4 (4 mg, 0.012 mmol, 1.1 eq.) were dissolved in 4 mL of CHCl3. DB (4 μL 0.25 2.2 q.) w mixture under nitrogen atmosphere at 0 °C and the reaction was allowed to warm to rt. After 24 h the solvent was evaporated at reduced pression and the crude mixture was purified by flash chromatography (DCM/MeOH 9:1) to obtain compound 24 as a dark orange solid (10 mg, 37%) Rf = 0.33.

IR (neat, cm

Scheme 4 .

 4 Scheme 4. Synthesis of amino acid-functionalized fullerenes. Experimental conditions: (i) DMAP, EDC HCl, DCM, 0 °C to rt, 16.5 h (18 = 90%; 21 = 75%); (ii) CuSO4, Na ascorbate, THF/H2O, overnight (19 = 70%; 22 = 98%); (iii) DBU, CBr4, toluene/CHCl3 (4:1) (20 = 20%; 23 = 25%).

  07 (s, 2H, COCH2CO), 3.25 (s, 4H, NHCH2CH2CH2N), 3.54 (dd, J = 2.2 Hz, 11.1 Hz, 2H, H-6b), 3.74 (dd, J = 5.3 Hz, 11.0 Hz, 2H, H-6a), 3.96-4.01 (m, 2H, H-5), 4.04 (ddd, J = 2.4 Hz, 5.2 Hz, 9.7 Hz, 2H, H-5′) 4.08 (dd, J = 2.4 Hz, 12.2 Hz, 4H, H-6′ ) 4.21 ( J = 5.3 Hz, 12.2 Hz, 2H, H-6′ ) 4.39 ( 4H NHCH2CH2CH2N), 4.61 (s, 2H, triazole-CH2O), 4.77 (s, 2H, triazole-CH2O), 4.83 (d, J = 1.7 Hz, 2H, H-1′) 4.87 (s, 2H, H-1), 5.15-5.29 (m, 12H, H-2, H-2′ H-3, H-3′ H-4 H4′) 7.23 ( 2H CONHCH2), 7.74 (s, 2H, Ar CH triazole); 13 C{ 1 H}-NMR (150 MHz, CDCl3): δ = 20.6-20.8 (14C, CH3COO), 29.7 (NHCH2CH2CH2N), 36.6 (NHCH2CH2CH2N), 42.6 (COCH2CO), 47.9 (NHCH2CH2CH2N), 60.6 (triazole-CH2O), 62.4, 65.9 (C-6′ -6′ ) 66.5 (C-6a, C-6b), 66.6, 68.7, 69.1, 69.3, 69.4, 69.5, 96.6 (C-1), 97.5 (C-1′) 167.6 (COCH2CO), 169.7-170.6 (14C, CH3COO); HRMS (ESI) calculated for C67H92N8O38Na [M + Na] + 1639.5405, found 1639.5405 (Δ = 0.0 ppm).

Table 1 .

 1 Decoration of fullerene with sugar-modified malondiamide linkers: screening of the optimal reaction conditions.

	Compound	Entry	Ratio (C60:Sugar)	Temperature	Time	Yield
	14	1	1:1	rt	1.5 h	17%
		2	1:1	rt	1 h	30%
		3	1:1	rt	45 min	41%
	15	4	1:1	rt	1.5 h	10%
		5	1:1	rt	1 h	12%
		6	1:1	rt	30 min	16%
		7	1:1	rt	15 min	22%
		8	1:1	rt	7 min	24%
		9	1:1	0 °C	20 min	26%
		10	1:1	0 °C	1 h	30%
		11	1:1	0 °C	2 h	31%
	16	12	1:1	0 °C	1 h	8%
		13	1:1	-20 °C	3 h	8%
		14	1:1	-20 °C	1.5 h	<5%
		15	1:1	rt	3 h	11%
		16	1:1	rt	1.5 h	10%
		17	1:1	rt	6 h	5%
		18	1:0.7	rt	1 h	5%
	17	19	1:1	0 °C	1 h	12%
		20	1:1	0 °C	20 min	15%
		21	1:1	0 °C	10 min	13%
		22	1:1	rt	15 min	12%

  -1 3270, 2956, 2923, 2118, 1741, 1368, 1211, 1038); 1 H-NMR (400 MHz, CDCl3): δ= 2.00 2.02 2.07 2.08 2.08 2.13 2.17 2.18 2.20 (9 30H O H3), 2.53 (t, J = 2.4 Hz, 1H, CH2 ≡ H) 3.55-3.65 (m, 2H), 3.81 (td, J = 5 Hz, 11.2 Hz, 2H,), 3.97-4.10 (m, 3H, H-5, H-5′ H5") 4.15 (dd, J = 2.5 Hz, 12.3 Hz, 1H), 4.30 (dd, J = 5.6 Hz, 12.3 Hz, 1H), 4.32 (d, J = 2.4 Hz, 2H, OCH2 ≡ H) 4.87 (d, J = 1.8 Hz, 1H, H-1") 4.89 ( J = 1.8 Hz, 1H, H-1′) 5.04 ( J = 1.8 Hz, 1H, H-1), 5.28-5.40 (m, 9H, H-2, H-3,

	H-4, H-2′ H-3′ H-4′ H-2" H-3" H-4");
	13 C{ 1 H}-NMR (150 MHz, CDCl3): δ= 20.6-20.8 (10C, CH3COO), 54.8 (OCH2 ≡ H) 62.4 65.9 66.3 66.4
	66.5, 68.6, 68.9, 69.0, 69.2, 69.3, 69.4, 69.9 (15C, ring carbons), 75.6 (OCH2 ≡CH), 78.2 (OCH2C≡ H) 96.0 ( -1),
	97.6, 97.7 (C-1′ -1") 169.5-170.5, (10C, CH3COO);
	HRMS (ESI) calculated for C41H54O26Na [M + Na] + 985.2796, found 985.2796 (Δ = -0.1 pp ).

  .7 20.8 20.9 29.2-29.6, 29.7, 31.9, 33.4, 37.6, 47.5, 53.8, 60.6, 62.5, 65.9, 66.5, 66.6, 68.7, 69.1, 69.2, 69.3, 69.5, 69.6, 73.7, 96.4, 97.5, 123.7, 137.7-145.3 (C-C60), 163.4, 169.7, 170.0, 170.3, 170.7; HRMS (ESI) calculated for C127H90N8O38Na [M + Na] + 2358.5281, found 2358.5283 (Δ = -1.0 pp ).

	-1 ) 3267, 3147, 3074, 2921, 2851, 1746, 1654, 1429, 1367, 1218, 1082, 1042;
	1 H-NMR (600 MHz, CDCl3): δ = 1.90 1.96 1.97 2.04 2.08 (5 42H	O H3), 2.24-2.34 (m, 4H,
	NHCH2CH2CH2N), 3.50 (t, J = 6.7 Hz, 4H, NHCH2CH2CH2N), 3.53 (dd, J = 2.6 Hz, 11.1 Hz, 2H, H-6b), 3.74
	(dd, J = 5.8 Hz, 11.1 Hz, 2H, H-6a), 3.96-4.00 (m, 2H, H-5), 4.09 (dd, J = 2.4 Hz, 12.3 Hz, 2H, H-6′ ) 4.20 (	J
	= 5.3 Hz, 12.2 Hz, 2H, H-6′ ) 4.50 ( J = 6.6 Hz, 4H, NHCH2CH2CH2N), 4.61-4.79 (m, 4H, triazole-CH2O), 4.81
	(d, J = 2.7 Hz, 2H, H-1′) 4.86 ( 2H H-1), 5.15-5.29 (m, 12 H, H-2, H-2′ H-3, H-3′ H-4, H-4′) 7.22 ( 2H A
	CH triazole), 7.88 (s, 2H, CONHCH2);

13

C{ 1 H}-NMR (150 MHz, CDCl3): δ = 20

  J = 10.5 Hz, 6H), 3.83 (ddd, J = 6.2 Hz, 11.2 Hz, 15.9 Hz, 4H,), 3.96-4.21 (m, 10H), 4.30 (dd, J = 5.2 Hz, 12.3 Hz, 4H), 4.56-5.07 (m, 12H), 5.21-5.50 (m, 18H), 8.50 (s, 2H, Ar CH triazole);

	-1 ) 3268, 3134, 3073, 2922, 2851, 1744, 1653, 1430, 1367, 1214, 1083, 1041;
	1 H-NMR (400 MHz, CDCl3): δ = 1.99 2.07 2.08 2.13 2.16 2.17 2.18 (7 60H O H3). 2.34-2.47 (m, 4H,
	NHCH2CH2CH2N), 3.61 (d, 13 C{ 1 H}-NMR (150 MHz, CDCl3): δ = 20.6-20.9 (30C), 29.2, 29.7, 31.9, 32.5, 37.6, 47.7, 53.8, 60.3, 62.4, 65.9,
	66.2, 66.3, 66.5, 68.6, 69.1, 69.3, 69.4, 69.5, 69.8, 96.6, 97.6, 97.8, 137.6-145.6 (C-C60), 163.4, 169.6-170.6 (20 C);
	HRMS (ESI) calculated for C151H122N8O54Na [M + Na] + 2934.6971, found 2934.6875 (Δ = +1.9 ppm).

  C{ 1 H}-NMR (CDCl3, 100 MHz) δ 24.3, 25.0, 25.3, 29.8, 35.0, 36.5, 42.7, 47.6, 52.5, 56.1, 122.0, 147.2, 168.0, 172.4, 175.2; HRMS (ESI) calculated for C31H51N10O8 [M + H] + 691.3886, found 691.3874 (Δ = -1.7 ppm).

  4, 25.6, 29.5, 30.2, 34.8, 37.6, 47.4, 52.2, 55.2, 122.3, 137.2-147.0 (C-C60), 148.0, 162.4, 171.9, 175.0; HRMS (ESI) calculated for C91H49N10O8 [M + H] + 1409.3729, found 1409.3705 (Δ = -1.7 ppm).

  -1 ) 3269, 3062, 2930, 1738, 1666, 1536, 1432, 1369, 1222, 1021, 1005; UV-Vis λmax (nm): 268, 281, 322, 399, 475, 636; 1 H-NMR (DMSO-d6, 400 MHz), mixture of rotamers, δ 1.32 (s, 12H, C(CH3)2), 1.70-1.84 (m, 4H, COCH2CH2CH2), 1.86-2.20 (m, 32 H in total: 24H COCH3 at 1.92, 2.02, 2.03 and 2.11 ppm, overlapped with 4H NHCH2CH2CH2N Aib + 4H COCH2CH2CH2), 2.54-2.64 (m, 8H, COCH2CH2CH2 + 4H NHCH2CH2CH2N Man), 2.90-3.32 (m, 8H, NHCH2CH2CH2N), 3.47-3.59 (m, 6H, CO2Me), 3.94-5.19 (m, 26H in total: 4H triazole-CH2O + 8H NHCH2CH2CH2-triazole, 14H sugar), 7.71-7.95 (m, 2H, CONHC(CH3)2), 8.18 (br s, 4H, Ar CH triazole), 8.76-9.82 (br m, 4H, CONHCH2); 13 C{ 1 H}-NMR (DMSO-d6, 100 MHz) δ 14.6, 20.8, 21.0, 21.2, 24.9, 25.4, 25.6, 29.5, 30.2, 34.8, 37.5, 47.5, 52.2, 55.2, 55.4, 60.2, 60.3, 62.3, 65.8, 68.5, 68.6, 69.1, 96.3, 122.3, 125.0, 137.3-147.6 (C-C60), 162.2, 170.1, 170.5, 171.8, 171.9, 175.0; HRMS (ESI) calculated for C134H107N18O30 [M + H] + 2447.7395, found 2447.7454 (Δ = +2.4 ppm).