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

In the last few years, nanomaterials based on fullerene have begun to be considered promising tools in the development of efficient adjuvant/delivery systems for vaccination, thanks to their several advantages such as biocompatibility, size, and easy preparation and modification. In this work we reported the chemoenzymatic synthesis of natural polymannan analogues (di- and tri-mannan oligosaccharides characterized by α1,6man and/or α1,2man motifs) endowed with an anomeric propargyl group. These sugar derivatives were submitted to 1,3 Huisgen dipolar cycloaddition with a malondiamide-based chain equipped with two azido terminal groups. The obtained sugar-modified malondiamide derivatives were used to functionalize the surface of Buckminster fullerene (C60) in a highly controlled fashion, and yields (11–41%) higher than those so far reported by employing analogue linkers. The same strategy has been exploited to obtain C60 endowed with natural and unnatural amino acid derivatives. Finally, the first double functionalization of fullerene with both sugar- and amino acid-modified malondiamide chains was successfully performed, paving the way to the possible derivatization of fullerenes with immunogenic sugars and more complex antigenic peptides.


Introduction
Buckminster fullerene (C 60 ), 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 C 60 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 [1].
The effect of glycosylation on biological activity of fullerene is defined "indirect" if the role of the 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 [11][12][13], in HIV-1 protease inhibition [14][15][16] or against neurodegenerative diseases [17,18].
On the contrary, the role of the sugars can be considered "direct" if they have a defined therapeutic activity. In this case, the "multivalency" is the essential property that usually ensures a good biological 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 [19][20][21][22].
Similarly, the conjugation of amino acids or peptides with fullerene [1,23,24] 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 [26][27][28]. 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 [29], lupus [30] and in cancer diagnosis [31,32].
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 [33,34]. Some studies reported the efficiency of C 60 derivatives as vaccine adjuvants [35,36], 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 [37]. Fairbanks et al. [38] 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 C 60 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 [39].
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, C 60 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) [40]. Wang and co-workers exploited arylamide-derived chains carrying a porphyrin moiety to functionalize the C 60 core in yields up to 44% [41]. 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 [42]. Only one example of direct functionalization of C 60 with a sugarmodified malondiamide-based linker has been previously reported [43]. 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 acid-modified malondiamide chains exploiting a copper-mediated Huisgen 1,3-dipolar cycloaddition and then proceed to the C 60 functionalization.
In the last part of this work, we focused our attention on the synthesis of heterosubstituted C 60 scaffolds. Actually, a few studies of hetero-functionalization of C 60 are described in literature [44,45], and there are no examples of C 60 functionalization with both sugar and amino acid derivatives through the use of malondiamide-based linkers.

Synthesis of Propargyl-Mannose Glycans
The first step involved the synthesis of natural polymannan analogues. For this purpose mannose-based di-and trisaccharides with α(1,2) and α(1,6) motifs were designed and synthesized. The idea was the 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) [46]. 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 [47] 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 lower temperatures (−50/−70 • C) 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.

Preparation of Glycosylated C 60 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 bisazido 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.
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/H 2 O (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)(11)(12)(13) were then reacted with fullerene exploiting a Bingel-Hirsch cyclopropanation for the preparation of the corresponding glycosylated fullerenes (Scheme 3). 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.

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, fullerene-man 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 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 C 60 (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, fullerene-man 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 fullerene-man(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 C 60 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). Lowering the temperature to −20 • C did not afford any improvement in terms of yield (entries 13 and 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 variation in the reagent's ratio (0.7 eq of sugar and 1 eq of C 60 ) did not lead to any improvement.
To obtain fullerene-man(α1,6)man(α1,6)man (17) the corresponding malondiamidechain 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 C 60 with Amino Acid Derivatives
By exploiting the approach described for the functionalization of C 60 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 [48]. 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 CHCl 3 . 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 one-pot approach but replacing iodine with CBr 4 [49]. The addition of DBU to a 1:1 mixture of C 60 and 19 in toluene, in the presence of CBr 4 , using CHCl 3 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.
These results prompted us to reconsider the functionalization protocol. We decided to preserve the one-pot approach but replacing iodine with CBr4 [49]. The addition of DBU to a 1:1 mixture of C60 and 19 in toluene, in the presence of CBr4, using CHCl3 as a cosolvent, 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 C 60 : Sugar and Amino Acid Derivative
With the optimized protocol in our hands, we wondered if it was possible to functionalize C 60 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 C 60 14 was then reacted with the (Aib)-functionalized malondiamide chain 22 in the presence of CBr 4 and DBU (Scheme 5). Due to the similar solubility of 14 and 22 in CHCl 3 , 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 [50]. 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 [50][51][52]. 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 C 60 with both sugar and amino acid moieties exploiting a malondiamide-based linker.

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 [43].
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 bisfunctionalized 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.

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 C 60 , 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 [43].
Through a slight modification of the functionalization procedure, we demonstrated that natural and non-proteinogenic quaternary amino acids can be also easily linked to C 60 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 bisfunctionalized 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 C 60 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, MA, USA; Alfa Aesar Ward Hill, MA, USA; Fluorochem Limited, Hadfield, U.K.) and used without further purification. Solvents were purified according to the guidelines in the Purification of Laboratory Chemicals [53]. All solvents were freshly distilled from the appropriate drying agent. THF, and toluene were distilled from sodium/benzophenone ketyl; TEA and DCM from CaH 2 . Reactions requiring anhydrous conditions were performed under Ar or N 2 . Yields were calculated for compounds purified by flash chromatography and judged homogeneous by thin-layer 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 µm (Sigma-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 solution of 4,4 -methylenebis-N,N-dimethylaniline, ninhydrin, and KI in an aqueous ethanolic solution of AcOH, or by spraying with 5% H 2 SO 4 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 shifts (δ) are given in ppm and were referenced to the solvent signals. Signal multiplicities are abbreviated as 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 USA) equipped with the Twin Sprayer ESI probe and coupled to an ExionLC™ system (SCIEX). The 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.
The reaction was then treated with saturated KOH solution (10 mL), added dropwise at 0 • C. The aqueous layer was extracted with Et 2 O (3 × 25 mL). The organic phase was dried over MgSO 4, filtered and concentrated in vacuo at room temperature (aliquots of pentane were added and evaporated to extract any Et 2 O 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 [54]. 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 H 2 O (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 MgSO 4 , filtered and concentrated in vacuo.