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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2387

S1

Supplementary Information

Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection

Antonio Muñoz,§ David Sigwalt,¥,£ Beatriz M. Illescas,§ Joanna Luczkowiak,‡ Laura

Rodríguez,§ Iwona Nierengarten,¥ Michel Holler,¥ Jean‐Serge Remy,£ Kevin Buffet,¶

Stéphane P. Vincent,¶ Javier Rojo,†,* Rafael Delgado,‡,* Jean‐François Nierengarten,¥,*

Nazario Martín§,,*

§Departamento de Química Orgánica, Facultad de Química, Universidad Complutense, 28040

Madrid, Spain, e‐mail: [email protected]; † Glycosystems Laboratory, Instituto de

Investigaciones Químicas (IIQ), CSIC – Universidad de Sevilla, Av. Américo Vespucio 49, Seville

41092 Spain. Tel: + 34 954489568; FAX +34 954460165; e‐mail: [email protected]; £ Laboratory V‐SAT (CAMB UMR 7199, CNRS), Labex Medalis, Université de Strasbourg, 74

Route du Rhin, 67401 Illkirch‐Graffenstaden, France; ¶ University of Namur (FUNDP),

Département de Chimie, Laboratoire de Chimie Bio‐Organique, rue de Bruxelles 61, B‐5000

Namur, Belgium; ‡Laboratorio de Microbiología Molecular, Instituto de Investigación Hospital

12 de Octubre (imas12) 28041 Madrid, Spain, e‐mail: [email protected]; ¥ Laboratoire de Chimie des Matériaux Moléculaires, Université de Strasbourg et CNRS (UMR

7509), Ecole Européenne de Chimie, Polymères et Matériaux, 25 rue Becquerel, 67087

Strasbourg, France, e‐mail: [email protected]; IMDEA‐Nanoscience, Campus

Cantoblanco, 28049 Madrid, Spain.

Table of Contents ........................................................................................................................ S1 Synthesis and Characterization .................................................................................................. S2 DLS Analysis .............................................................................................................................. S44 TEM .......................................................................................................................................... S47 XPS ............................................................................................................................................ S48 Biological assays ....................................................................................................................... S50 Cytotoxicity studies .................................................................................................................. S51

Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection

© 2015 Macmillan Publishers Limited. All rights reserved



S2

Synthesis and Characterization General Reagents and solvents were purchased as reagent grade and used without further purification. Compounds 4,1 6,2 8,3 10,4 11,5 13a,6 13b,7 and 168 were prepared according to previously reported procedures. All reactions were performed in standard glassware under an inert Ar atmosphere. Evaporation and concentration were done at water aspirator pressure and drying in vacuum at 10‐2 Torr. Column chromatography: silica gel 60 (230‐400 mesh, 0.040‐0.063 mm) was purchased from E. Merck. Thin Layer Chromatography (TLC) was performed on glass sheets coated with silica gel 60 F254 purchased from E. Merck, visualization by UV light. IR spectra (cm‐1) were measured on a Perkin Elmer Spectrum One. NMR spectra were recorded on a Bruker AC 300, AC 400, AMX‐500 or AMX‐700 equipped with cryoprobe, with solvent peaks as reference. MALDI‐TOF‐mass spectra were carried out on a Bruker BIFLEX

TM or a

Bruker ULTRAFLEX III matrix‐assisted laser desorption time‐of‐flight mass spectrometer. Copper analysis was carried out using a Varian ICP‐MS.

1 J. Iehl, R. Pereira de Freitas, B. Delavaux‐Nicot, J.‐F. Nierengarten, Chem. Commun. 2008, 2450‐2452. 2 J.‐F. Nierengarten, J. Iehl, V. Oerthel, M. Holler, B. M. Illescas, A. Munoz, N. Martin, J. Rojo, M. Sanchez‐Navarro, S. Cecioni, S. Vidal, K. Buffet, M. Durka, S. P. Vincent, Chem. Commun. 2010, 46, 3860‐3862. 3 H. Sekiguchi, K. Muranaka, A. Osada, S. Ichikawa, A. Matsuda, Bioorganic & Medicinal Chemistry 2010, 18, 5732‐5737. 4 F. Wessendorf, J.‐F. Gnichwitz, G. H. Sarova, K. Hager, U. Hartnagel, D. M. Guldi, A. Hirsch J. Am. Chem. Soc. 2007, 129, 16057‐16071. 5 J. Iehl, R. Pereira de Freitas, J.‐F. Nierengarten Tetrahedron Lett. 2008, 49, 4063. 6 a) E. Arce, P. M. Nieto, V. Díaz, R. García‐Castro, A. Bernad, J. Rojo, Bioconjug. Chem. 2003, 14, 817; b) Lindhorst, T. K.; Kötter, S.; Krallmann‐Wenzel U.; Ehlers, S. J. Chem. Soc., Perkin Trans. 1 2001, 823. 7 Davis, B.G.; Maughan, M.A.T.; Ullman, A.; Jones, J.B. Tetrahedron: Asymm. 2000, 11, 245. 8 M. Sánchez‐Navarro, A. Muñoz, B. M. Illescas, J. Rojo, N. Martín Chem. Eur. J. 2011, 17, 766‐769.




S3

Compound 2

Ethylmalonyl chloride (1.70 mL, 13 mmol) was added dropwise to a solution of 1 (1.7 mL, 20 mmol) and pyridine (1.3 mL, 20 mmol) in dry CH2Cl2 (100 mL) at 0° C under Argon. After 1 h, the mixture was allowed to slowly warm to room temperature (within 1 h), then stirred for 18 h and evaporated. Column chromatography (SiO2, CH2Cl2/cyclohexane, 4:1) gave 2 (3.69 g, 99%). Colorless oil. IR (neat): 2175 (C≡C), 1733 (C=O). 1H NMR (CDCl3, 300 MHz), δ: 4.23 (q, J = 7 Hz, 2H), 4.20 (t, J = 7 Hz, 2H), 3.37 (s, 2H), 2.32 (t, J = 7 Hz, 2H), 1.87 (m, 2H), 1.29 (t, J = 7 Hz, 2H), 0.14 (s, 9H). 13C NMR (CDCl3, 75 MHz), δ: 165.7, 165.6, 105.4, 84.6, 63.2, 60.7, 40.7, 26.7, 16.4, 15.6, ‐0.8.

1H NMR spectrum of compound 2 (CDCl3, 300 MHz, 298 K)




S4

13C NMR spectrum of compound 2 (CDCl3, 75 MHz, 298 K)

DEPT 135 spectrum of compound 2 (CDCl3, 75 MHz, 298 K)




S5

Compound 3

DBU (0.6 ml, 3.5 mmol) was added to a stirred solution of C60 (998 mg, 1.4 mmol), 2 (375 mg, 1.4 mmol) and I2 (528 mg, 2.0 mmol) in dry toluene (1 L) under argon. The resulting solution was stirred for 16 h, then filtered through a short plug of SiO2 (CH2Cl2) and evaporated. Column chromatography (SiO2, CH2Cl2/cyclohexane, 3:2) gave 3 (675 mg, 49%). Brown solid. IR (neat): 2176 (C≡C), 1747 (C=O). UV‐Vis (CH2Cl2): 326 (38000), 426 (2800), 486 (1540), 688 (180). 1H NMR (CDCl3, 300 MHz), δ: 4.56 (m, 4H), 2.46 (t, J = 7 Hz, 2H), 2.07 (m, 2H), 1.51 (t, J = 7 Hz, 3H), 0.15 (s, 9H). 13C NMR (CDCl3, 75 MHz), δ: 162.65, 162.6, 145.4, 144.3, 144.25, 144.2, 143.9, 143.65, 143.6, 143.55, 143.0, 142.95, 142.2, 142.1, 142.0, 141.0, 140.9, 140.05, 140.0, 138.2, 137.9, 104.2, 85.1, 70.6, 64.8, 62.1, 51.1, 26.7, 15.7, 13.4, ‐0.7. MALDI‐MS: 988.5 ([M]+, calcd for C73H20O4Si: 988.11).





S6


DEPT 135 spectrum of compound 3 (CDCl3, 75 MHz, 298 K)




S7

MS (MALDI‐TOF) of compound 3

IR spectrum of compound 3




S8

Compound 5

OO

OO

OO

OO

O

O O

OO

O OO

OO

O

OO

OO O

SiMe3N3N3

N3

N3

N3

N3

N3 N3

N3

N3

5 DBU (0.5 ml, 3 mmol) was added to a stirred solution of 3 (140 mg, 0.14 mmol), CBr4 (4.7 g, 14

mmol) and 4 (459 mg, 1.7 mmol) in ODCB (30 mL) at room temperature. The resulting solution

was stirred for 72 h, then filtered through a short plug of SiO2 (CH2Cl2) and evaporated. Column

chromatography (SiO2, cyclohexane/AcOEt, 2:1) gave 5 (220 mg, 67%). Caution: owing to its

high number of azide residues, this compound must be handled with special care. Upon

evaporation, compound 5 has never been dried under high vacuum and the use of metallic

spatula avoided. Furthermore, this compound has been always prepared on a small scale.

Orange glassy product. IR (neat): 2176 (C≡C), 2092 (N3), 1739 (C=O). 1H NMR (CDCl3, 300 MHz),

δ: 4.35 (m, 24H), 3.41 (m, 20H), 2.34 (t, J = 7 Hz, 2H), 1.95 (m, 22H), 1.34 (t, J = 7.0 Hz, 3H), 0.15

(s, 9H). 13C NMR (CDCl3, 75 MHz), δ: 163.4, 145.7, 141.2, 105.185.7, 69.0, 65.5, 63.8, 63.1, 47.8,

45.3, 27.9, 27.4, 16.4, 14.0, 0.0.





S9






S10

Compound 7

A mixture of CuSO4.5H2O (15 mg, 94 mol), sodium ascorbate (30 mg, 152 mol), 5 (367 mg, 157 mol) and 6 (412 mg, 1.89 mmol) in THF/H2O (7 mL, 1:1) was heated 2 h under microwave irradiation (100°C). The product was precipitated by addition of MeOH (20 mL). The precipitate was washed with MeOH and dried under reduced pressure. Gel permeation chromatography (Sephadex G‐50, H2O/MeOH; 90:10) gave 7 (647 mg, 91%). Orange powder. IR (neat): 3308 (OH), 1738 (C=O). 1H NMR (400 MHz, D2O), δ: 8.05 (m, 10H), 4.80 (m), 4.43 (m), 3.75 (m), 2.25 (m), 1.16 (m), 0.15 (s, 9H, TMS). 13C NMR (100 MHz, D2O), δ: 163.8 (broad), 145.6 (broad), 141.2 (broad), 125.1 (broad), 99.6, 73.0, 70.7, 70.1, 66.7, 64.6, 60.9, 58.3, 47.6, 45.6, 38.9, 31.8, 28.7, 13.7, 0.1. MALDI‐MS: 4465.6 (8%, M‐TMS+Na+, calcd for C205H216O84N30Na: 4465.3), 4120.5 (8%, M‐1/2 malonate+Na+, calcd for C192H197O76N27Na: 4119.2), 3759.4 (34%, M‐1 malonate+Na+, calcd for C178H177O68N24Na: 3761.1), 3414.2 (26%, M‐1 malonate‐1/2malonate+Na+, calcd for C165H157O60N21Na: 3415.0), 3054.2 (36%, M‐2 malonates+Na+, calcd for C151H136O52N18Na: 3056.8), 2708.9 (21%, M‐2 malonates‐1/2malonate+Na+, calcd for C138H117O44N15Na: 2710.7), 2349.7 (17%, M‐TMS‐3 malonates+Na+, calcd for C124H97O36N12Na: 2352.6), 2003.6 (8%, M‐3 malonates‐1/2malonate+Na+, calcd for C111H77O28N9Na: 2006.5), 1644.4 (6%, M‐4 malonates+Na+, calcd for C97H57O20N6Na: 1648.3).




S11

1H NMR spectrum of compound 7 (D2O, 400 MHz, 298 K). The broadening of the spectrum is due to aggregation.

13C NMR spectrum of compound 7 (D2O, 100 MHz, 298 K)




S12

DEPT 135 spectrum of compound 7 (D2O, 100 MHz, 298 K)

MS (MALDI‐TOF) of compound 7. A series of typical fragments resulting from successive retro‐Bingel reactions are observed. Further fragments resulting from the cleavage of a malonic ester unit followed by a decarboxylation are also observed.




S13





S14

Compound 9

A 1 M solution of TBAF in THF (0.1 mL, 1 mmol) was added to a mixture of CuSO4.5H2O (3 mg, 19 mol), sodium ascorbate (12 mg, 60 mol), 7 (273 mg, 60 mol) and 8 (481 mg, 1.14 mmol) in THF/H2O (3.5 mL, 0.5:3). The resulting mixture was heated for 1.5 h at 80°C under microwave irradiation. THF and the major part of water were evaporated. Then the crude product was precipitated by addition of a mixture of acetone/CH2Cl2 and extensively washed with CH2Cl2 and acetone. This procedure was repeated three times. A gel permeation chromatography (Sephadex G‐50, H2O/MeOH; 90:10) gave 9 as a brown‐orange glassy solid (254 mg, 87 %). IR (neat) 3324 (OH), 2107 (N3), 1738 (C=O) cm‐1; 1H NMR (400 MHz, D2O), : 8.05 (m, 11H; triazole), 4.80(m), 4.43 (m), 3.66 (m), 2.25 (m), 1.24 (m). 13C NMR (100 MHz, D2O), δ: 163.6 (broad), 146.6 (broad), 143.8 (broad), 125.0 (broad), 99.5, 97.8 73.5, 73.0, 71.5, 70.7, 70.2, 70.1, 69.7, 69.3 66.7, 65.6, 64.2, 60.9, 59.8, 50.3, 49.0, 47.2, 39.2, 28.8, 31.8, 28.7, 21.2, 13.6.

1H NMR spectrum of compound 9 (D2O, 400 MHz, 298 K)




S15

13C NMR spectrum of compound 9 (D2O, 100 MHz, 298 K)

DEPT 135 spectrum of compound 9 (D2O, 100 MHz, 298 K)




S16





S17

Compound 12

OO

OO

OO

O

O

O

O O

OO

O OO

OO

O

O

O

OO O

12

Br

DBU (1.02 g, 8 mmol) was added to a solution of 10 (400 mg, 0.4 mmol), 11 (0.94 g, 4 mmol) and CBr4 (7 g, 21.3 mmol) in dry toluene (400 mL) under argon atmosphere. The mixture was kept with stirring and under argon atmosphere for 72 h, after which a Na2S2O3 solution (150 mL) was added. The organic solution was washed with HCl 1M (2 x 150 mL), water (2 x 150 mL) and brine (150 mL), dried (Na2SO4) and evaporated. Column chromatography (SiO2, CH2Cl2/ AcOEt 100:5) yielded 12 (428 mg, 49 %) as a red solid. FTIR (KBr): 3292 (≡CH), 2117 (C≡C) 1742 (C=O) cm‐1; 1H NMR (300 MHz, CDCl3), δ: 4.55 – 4.10 (m, 24H), 3.44 – 3.23 (m, 2H), 2.39 – 2.14 (m, 19H), 2.05 – 1.69 (m, 30H), 1.48 – 1.15 (m, 12H). 13C NMR (75 MHz, CDCl3), δ: 163.8, 146.1, 140.8, 82.2, 70.1, 69.1, 65.6, 63.0, 33.7, 32.50, 28.4, 27.2, 24.8, 17.1, 16.5, 15.1, 14.2. MS (MALDI‐TOF) calculated for [M]+ C136H87O24Br= 2185.04; found: 2184.5.

1H NMR spectrum of 12 (300 MHz, CDCl3, 298 K)




S18

13C NMR spectrum of 12 (75 MHz, CDCl3, 298 K)

MS (MALDI‐TOF) of compound 12




S19

IR spectrum of 12




S20

Compound 14a

O

HO

HO O

HO

HO

OO

OO

OO

OO

O

O O

OO

O OO

OO

O

OO

OO O

O

HO

HO

OHOHO

OHO

OH

O

HO OH OHO

OH

O

HOOH

O

OH

HO

O

HO

HO

O

OHHO

O

OH

HO

OOH

HO

O

OHHO

O OH

HO

O

OHHO

O

OH

OH

O OH

HO

NNN

NNN

NNN

NNN

NNN

NNN

NN N

NN N

N NN

N NN

Br

O

OH

OHO

OH

OH

14a

A mixture of 12 (35 mg, 0.016 mmol), 13a (73 mg, 0.29 mmol), CuBr∙S(CH3)2 (16 mg, 0.08 mmol), sodium ascorbate (24 mg, 0.12 mmol) and a piece of copper metal wire in DMSO (2 mL) was deoxygenated and kept under argon with vigorous stirring for 48 h. The crude reaction was filtered through a QuadraSilTM Mercaptopropyl column. Et2O was added to the mixture and the precipitate was centrifuged and extensively washed with AcOEt, then dried under high vacuum to give compound 14a (64 mg, 86 %) as a dark red solid. FTIR (KBr): 3386 (OH), 1741 (C=O) cm‐1; 1H NMR (700 MHz, DMSO‐d6), δ: 7.84 (s, 10H), 4.61 (m, 12H), , 4.52 – 4.26 (m, 44H), 3.93 (s, 8H), 3.77 (s, 8H), 3.58 (m, 8H), 3.53 – 3.12 (m, 12H), 3.03 – 2.98 (m, 96H), 2.06 (s, 24H), 1.41 – 1.05 (m, 32H); 13C NMR (175 MHz, DMSO‐d6), δ: 163.3, 146.1, 145.5, 141.2, 122.8, 100.2, 74.5, 71.3, 70.4, 69.2, 67.2, 65.3, 61.6, 55.1, 49.6, 28.2, 21.8, 14.3. MS (MALDI‐TOF) calculated for [M]+ C216H237BrN30O84= 4673,44; found: 4697 [M+Na]+




S21

1H NMR spectrum of 14a (700 MHz, DMSO‐d6, 298 K)

13C NMR spectrum of 14a (175 MHz, DMSO‐d6, 298 K)




S22

DEPT 135 spectrum of 14a (175 MHz, DMSO‐d6, 298 K)

MS (MALDI‐TOF) of compound 14a




S23

IR spectrum of 14a




S24

Compound 15a

A mixture of compound 14a (140 mg, 0.03 mmol) and sodium azide (8 mg, 0.120 mmol) in DMSO (2 mL) was deoxygenated with argon for 5 min and heated 3 h under microwave irradiation (70°C). AcOEt was added and the precipitate was centrifuged, washed with MeOH and MeOH/AcOEt 3:1, then dried under high vacuum to give compound 15a (117 mg, 84%) as a red solid. FTIR (KBr): 3385 (OH), 2099 (N3), 1740 (C=O) cm‐1; 1H NMR (700 MHz, DMSO‐d6), δ: 7.85 (s, 10H), 4.68 (m, 12H), , 4.59 – 4.09 (m, 44H), 3.91 (s, 8H), 3.76 (s, 8H), 3.61 (m, 8H), 3.53 (s, 8H), 3.46 – 3.25 (m, 96H), 3.12 (m, 4H), 2.04 (m, 24H), 1.36 – 0.98 (m, 32H); 13C NMR (175 MHz, , DMSO‐d6), δ: 163.3, 146.1, 145.8, 141.4, 122.7, 100.2, 78.6, 74.5, 71.2, 70.5, 67.2, 65.3, 61.6, 52.5, 52.0, 49.6, 28.2, 21.8, 14.4; MS (MALDI‐TOF) calculated for [M]+ C216H237N33O84= 4636,53; found: 4659 [M+Na]+.




S25






S26

DEPT 135 spectrum of 15a (175 MHz, DMSO‐d6, 298 K)

MS (MALDI‐TOF) of compound 15a




S27

IR spectrum of 15a




S28

Compound 14b

A mixture of 12 (35 mg, 0.016 mmol), 13b (72 mg, 0.29 mmol), CuBr∙S(CH3)2 (16 mg, 0.08 mmol), sodium ascorbate (25 mg, 0.12 mmol) and a piece of copper metal wire in DMSO (2 mL) was deoxygenated and kept under argon with vigorous stirring for 48 h. The crude reaction was filtered through a QuadraSilTM Mercaptopropyl column. Et2O was added to the mixture and the precipitate was centrifuged and extensively washed with AcOEt, then dried under high vacuum to give compound 14b (64 mg, 86 %) as a dark red solid. FTIR (KBr): 3422 (OH), 1740 (C=O) cm‐1; 1H NMR (700 MHz, DMSO‐d6), δ: 7.93 (s, 10H), 5.01–4.72 (m, 12H), , 4.64–4.18 (m, 44H), 3.86 (s, 8H), 3.78 (s, 8H), 3.61–3.53 (m, 16H), 3.41–3.18 (m, 96H), 3.09 (m, 4H), 2.10 (s, 24H), 1.40–0.95 (m, 32H); 13C NMR (175 MHz, DMSO‐d6), δ: 177.1, 167.8, 163.1, 147, 146.1, 145, 140.8, 124.7, 123.2, 103.9, 97.8, 82.7, 75.7, 73.9, 70.8, 69.1, 68.8, 68.5, 67.3, 63.5, 61, 60.2, 49.9, 28.1, 21.9, 15.5; MS (MALDI‐TOF) calculated for [M]+ C216H237BrN30O84= 4673.44; found: 4674.

O

HO

HOO

HO OH

OO

OO

OO

OO

O

O O

OO

O OO

OO

O

OO

OO O

O

HO

OHOHO

OH

OHO

OH

O

HO

HO

OHO

OH

O

OH

HO

O

OH

HO

O

HO

OH

O

OHHO

O

HO

OH

OOH

HO

O

HO

OH O OH

HO

O

OH

OH

O

OH

OHO OH

HO

NNN

NNN

NNN

NNN

NNN

NNN

NN N

NN N

N NN

N NN

Br

O

OH

OHO

OHHO




S29

1H NMR spectrum of 14b (700 MHz, DMSO‐d6, 298 K)

13C NMR spectrum of 14b (175 MHz, DMSO‐d6, 298 K)




S30

MS (MALDI‐TOF) of compound 14b

IR spectrum of 14b




S31

Compound 15b

A mixture of compound 14b (30 mg, 0.007 mmol) and sodium azide (5 mg, 0.077 mmol) in DMSO (1.5 mL) was deoxygenated with argon for 5 min and heated 3 h under microwave irradiation (70°C). AcOEt was added and the precipitate was centrifuged, washed with MeOH and MeOH/AcOEt 3:1, then dried under high vacuum to give compound 15a (26 mg, 81%) as a red solid. FTIR (KBr): 3123 (OH), 2097 (N3), 1740 (C=O) cm‐1; 1H NMR (700 MHz, DMSO‐d6), δ: 7.83 (s, 10H), 4.84– 4.72(m, 12H), , 4.64 – 4.16 (m, 44H), 3.94 (s, 8H), 3.74 (s, 8H), 3.63–3.52 (m, 16H), 3.41–3.18 (m, 96H), 2.10 (s, 24H), 1.40–0.95 (m, 32H); 13C NMR (175 MHz, DMSO‐d6), δ: 163.4, 146.1, 145.5, 141.2, 123.1, 100.2, 78.2, 77.9, 74.5, 71.3, 70.5, 67.2, 65.1, 61.4, 52.4, 49.4, 28.3, 25.4, 21.9, 14.2; MS (MALDI‐TOF) calculated for [M]+ C216H237N33O84= 4636,53; found: 4638.

15b

O

HO

HOO

HO OH

OO

OO

OO

OO

O

O O

OO

O OO

OO

O

OO

OO O

O

HO

OHOHO

OH

OHO

OH

O

HO

HO

OHO

OH

O

OH

HO

O

OH

HO

O

HO

OH

O

OHHO

O

HO

OH

OOH

HO

O

HO

OH O OH

HO

O

OH

OH

O

OH

OHO OH

HO

NNN

NNN

NNN

NNN

NNN

NNN

NN N

NN N

N NN

N NN

N3

O

OH

OHO

OHHO




S32






S33

MS (MALDI‐TOF) of compound 15b

IR spectrum of 15b




S34

Compound 17a

A mixture of hexakis‐adduct 16 (1.5 mg, 6∙10‐4 mmol), compound 15a (68 mg, 0.014 mmol), CuBr∙S(CH3)2 (20 mg, 0.08 mmol), sodium ascorbate (25 mg, 0.12 mmol) and a piece of copper metal wire in DMSO (2 mL) was deoxygenated and kept under argon with vigorous stirring for 48 h. The crude reaction was filtered through a QuadraSilTM Mercaptopropyl column. Et2O was added to the mixture and the precipitate was centrifuged and extensively washed with AcOEt, then dried under high vacuum to give compound 17a (25 mg, 73%) as a red solid. FTIR (KBr): 3419 (OH), 1740 (C=O) cm‐1; 1H NMR (700 MHz, DMSO‐d6), δ: 7.82 (s), 4.76 (m), 4.61 (m), 4.50 (m), 4.35 (m), 3.93 (s), 3.73 (m), 3.60 (m), 3.57 – 3.50 (m), 3.12 (m), 2.80 – 2.59 (m), 2.03 (m), 1.30 (m); 13C NMR (175 MHz, DMSO‐d6), δ: 163.8, 146.9, 146.4, 145.4, 141.6, 123.3, 122.9, 100.2, 78.7, 74.7, 73.4, 73, 70.2, 69.3, 68.6, 67.6, 65.6, 62.3, 61.9, 60.5, 49.8, 45.2, 42.2, 34.2, 28.2, 26.1, 22.1, 14.2. XPS: % atomic: C (284.6 eV) = 73.84, O (531.6 eV) = 20.84, N (399.63 eV) = 5.33.




S35






S36

IR spectrum of 17a




S37

Compound 17b

A mixture of hexakis‐adduct 16 (1.3 mg, 6∙10‐4 mmol), compound 15b (70 mg, 0.015 mmol), CuBr∙S(CH3)2 (18 mg, 0.08 mmol), sodium ascorbate (25 mg, 0.12 mmol) and a piece of copper metal wire in DMSO (2 mL) was deoxygenated and kept under argon with vigorous stirring for 48 h. The crude reaction was filtered through a QuadraSilTM Mercaptopropyl column. Et2O was added to the mixture and the precipitate was centrifuged and extensively washed with AcOEt, then dried under high vacuum to give compound 17b (27 mg, 79%) as a red solid. FTIR (KBr): 3423 (OH), 1738 (C=O) cm‐1; 1H NMR (700 MHz, DMSO‐d6), δ: 7.93 (s), 4.98 – 4.79 (m), 4.60 – 4.50 (m), 4.35 (m), 3.93 – 3.87 (m), 3.73 – 3.69 (m), 3.63–3.54 (m), 3.12–2.94 (m), 2.79–2.63 (m), 2.12 – 1.98 (m), 1.45 – 1.21 (m); 13C NMR (175 MHz, DMSO‐d6), δ: 163.8, 147.7, 146.4, 146.0, 141.5, 124.4, 123.0, 100.5, 74.8, 73.7, 71.7, 70.7, 69.6, 67.6, 65.6, 61.8, 61.1, 49.8, 46.3, 42.9, 33.3, 28.5, 25.7, 22.3, 14.5.

OO

OO

OO

OO

O

O O

OO

O OO

OO

O

OO

OO O

O O

RORO

RO

RO

OR

OR

ORO

O

OO

O

OOR

O

ORO

OO

O

ORO

X

NNN

OO

OROR

OR

OR

RO

RO

ROO

O

OO

O

ORO

O

ROO

OO

O

RO O

X

N NN

O

O

OR ORRO

RO

OROR

RO

O

O

O O

O

O OR

OOR

OOO

O

RO

OX

NNN

OO

OR

OR

OROR

RORO

RO

O

O

OO

O

O

ORO OR

O

OO

O

ROO

X

NNN

OO

OR

OR

OROR

RORO

RO

O

O

OO

O

O

ORO OR

O

OO

O

ROO

X

NNN

O

O

OR ORRO

RO

OROR

RO

O

O

O O

O

O OR

OOR

OOO

O

RO

O

XN

NN

OO

OROR

OR

OR

RO

RO

ROO

O

OO

O

ORO

O

ROO

OO

O

RO O

X

N NN

O O

RORO

RO

RO

OR

OR

ORO

O

OO

O

OOR

O

ORO

OO

O

ORO

X

NNN

O

O

ROROOR

OR

RORO

OR

O

O

OO

O

ORO

ORO

OO O

O

OR

O

XNNN

OO

RO

RO

RO RO

OR OR

OR

O

O

OO

O

O

ROORO

O

OO

O

ORO

X

NN N

OO

RO

RO

RO RO

OR OR

OR

O

O

OO

O

O

ROORO

O

OO

O

ORO

X

NN N

O

O

ROROOR

OR

RORO

OR

O

O

OO

O

ORO

ORO

OO O

O

OR

OX

NNN

17b -X- = -(CH2)6-, R = OHO

HO

OH

HO

O NN N




S38






S39

IR spectrum of 17b




S40

Compound 17c

A mixture of 9 (254 mg, 52 mol), 16 (10.1 mg, 3 μmol), CuSO4.5H2O (1 mg, 6 mol) and sodium ascorbate (3 mg, 15 mol) in THF/H2O (3.5 mL, 0.5:3) was heated at 80°C under microwave irradiation in a sealed tube. After 2 hours, the mixture was evaporated. Exclusion

column chromatography (Econo‐Pac® 10 DG, BIO‐RAD, H2O) followed by a dialysis against milli‐Q water (Molecular Weigth CutOff: 25000) gave 17c (155 mg, 76 %). Brown‐orange glassy solid. IR (neat) 3251 (OH), 1732 (C=O) cm‐1; 1H NMR (400 MHz, DMSO‐d6), δ: 8.10 (s, 120H), 7.83 (s, 24H), 4.90‐3.18 (m, 1272H), 3.85‐3.28 (m, 744H), 2.74‐2.63 (m, 48H), 2.38‐2.11 (m, 240H), 2.08‐1.91 (m, 48H), 1.25‐1.11 (m, 36H). 13C NMR (100 MHz, DMSO‐D6), δ: 162.9, 145.5, 143.7, 140.6, 124.0, 122.3, 99.0, 74.1, 70.9, 70.2, 69.8, 69.7 (two peaks), 69.6, 69.2, 68.7, 67.0, 64.2, 61.3, 59.1, 50.0, 49.2, 46.1, 44.2, 28.6, 27.7, 21.3, 13.7.

OO

OO

OO

OO

O

O O

OO

O OO

OO

O

OO

OO O

O O

RORO

RO

RO

OR

OR

ORO

O

OO

O

OOR

O

ORO

OO

O

ORO

X

NNN

OO

OROR

OR

OR

RO

RO

ROO

O

OO

O

ORO

O

ROO

OO

O

RO O

X

N NN

O

O

OR ORRO

RO

OROR

RO

O

O

O O

O

O OR

OOR

OOO

O

RO

OX

NNN

OO

OR

OR

OROR

RORO

RO

O

O

OO

O

O

ORO OR

O

OO

O

ROO

X

NNN

OO

OR

OR

OROR

RORO

RO

O

O

OO

O

O

ORO OR

O

OO

O

ROO

X

NNN

O

O

OR ORRO

RO

OROR

RO

O

O

O O

O

O OR

OOR

OOO

O

RO

O

XN

NN

OO

OROR

OR

OR

RO

RO

ROO

O

OO

O

ORO

O

ROO

OO

O

RO O

X

N NN

O O

RORO

RO

RO

OR

OR

ORO

O

OO

O

OOR

O

ORO

OO

O

ORO

X

NNN

O

O

ROROOR

OR

RORO

OR

O

O

OO

O

ORO

ORO

OO O

O

OR

O

XNNN

OO

RO

RO

RO RO

OR OR

OR

O

O

OO

O

O

ROORO

O

OO

O

ORO

X

NN N

OO

RO

RO

RO RO

OR OR

OR

O

O

OO

O

O

ROORO

O

OO

O

ORO

X

NN N

O

O

ROROOR

OR

RORO

OR

O

O

OO

O

ORO

ORO

OO O

O

OR

OX

NNN

17c -X- = R =NNN

O O6 N

N NOHO

HO

HOHO

O




S41

1H NMR spectrum of compound 17c (DMSO‐d6, 400 MHz, 298 K)

13C NMR spectrum of compound 17c (DMSO‐d6, 100 MHz, 298 K)




S42

DEPT 135 spectrum of compound 17c (DMSO‐d6, 100 MHz, 298 K)

13C NMR spectrum of compound 17c (D2O, 100 MHz, 298 K). When compared to the spectrum recorded in DMSO‐d6, the broadening is ascribed to aggregation. This was further supported by DLS measurements in H2O.




S43

IR spectrum of compound 17c




S44

DLS analysis

For compounds 17a‐c, Dynamic Light Scattering measurements were carried out on an ALV GSC08 correlator working in a cross correlation mode with an Ar + laser operating at λ = 514.5 nm. The output signals were obtained with backscatter detection at an angle of 90o and processed with a digital correlator that computed intensity‐intensity autocorrelation of the scattered light. Measurements were made in a 1‐cm path‐length round quartz cell maintained at 298 K. Solution samples of 0.1 mg/mL were filtered through nylon Acrodisc syringe filters (Pall Life Sciences) with 0.2‐μm pore size.

17a, 0.01M 17a, 0.1M




S45

17b, 0.01M 17b, 0.1M

17c, 0.01M 17c, 0.1M

Supplementary Figure 1. Representative DLS for 17a‐c in H2O, at two different concentrations. Intensity vs. particle size (upper graphs) and volume vs. particle size (lower graphs).




S46

17a

17b

17c

Supplementary Figure 2. Representative DLS for 17a‐c in DMSO, 0.1 mg/mL. Intensity vs. particle size (left graphs) and volume vs. particle size (right graphs).




S47

TEM

For 17a‐c, TEM images were obtained using a JEOL 2100 microscope operating at 200 kV. A solution of 17a‐c in water (0.01 mg/mL or 0.1 mg/mL) was dropped onto a holey carbon copper grid (200 mesh), and the solvent was allowed to evaporate before analysis.

Supplementary Figure 3. TEM images for compounds 17a‐c upon deposition of 0.01 and 0.1 mg/mL solutions in H2O. In all cases, spherical aggregates are observed, ranging from one to several molecules.

nm

160

180

200

220

240

260

280

300

0 2 4 6 8nm

nm

40

60

80

100

120

140

0 1 2 3 4 5 6nm

20 nm20 nm nm

950

1000

1050

1100

1150

1200

1250

0 1 2 3 4nm

nm

600

650

700

750

800

0 1 2 3 4 5 6 7 8nm

20 nm20 nm nm

1000

1100

1200

1300

1400

1500

0 2 4 6 8nm

20 nm20 nm

nm

6200

6400

6600

6800

7000

7200

7400

0 2 4 6 8 10 12nm

17 a ‐ 0.1 g/L

17 b ‐ 0.1 g/L

17 b ‐ 0.01 g/L

17 c ‐ 0.1 g/L

17 c ‐ 0.01 g/L

17 a ‐ 0.01 g/L




S48

XPS

XPS analyses were obtained on a SPECS GmbH (PHOIBOS 150 9 MCD) spectrometer working in the constant analyzer energy mode and a non‐monochromatic aluminium X‐ray source (1486.61 eV) powered at 200 W and a voltage of 12 eV. For recording both survey and high resolution spectra pass energies of 75 and 25 eV were applied. Survey data were acquired from kinetic energies of 1487 – 400 eV with 0.1 eV of energy step and 100 ms dwell time per point. The high resolution scans were registered around the significant emission lines with 0.1 eV steps and 100 ms dwell time per point. The spectrometer control and data handling were monitored using SpecsLab Version 2.48 software. Binding energies were calibrated relatively to the C 1s peak at 284.6 eV and the atomic ratios were computed from experimental intensity rations. The samples were introduced in the XPS apparatus as powder coated over a polycarbonate membrane.

Supplementary Table 1. Relative abundances of C, N and O determined by XPS.

Compound C O N

17a 69,7 24,3 5,9

17b 68,1 25,9 5,9

17c 66,8 25,8 7,8




S49

020040060080010000

100000

200000

300000

400000

500000

600000

700000

C 1s

O 1s

Binding Energy (eV)

N 1s400

Binding Energy (eV)

C-NN-N-N

Supplementary Figure 4. XPS survey spectrum of compound 17a (up), 17b (medium) and 17c (down) with the N 1s deconvoluted components (inset right for each compound).




S50

Biological assays

C60 (12Man) C60 (36 Man)

OO

O

O

O

O

O

O

O

O O

O

O

OO

O

O

O

O

O

O

OO O

NNN

NNN

N NN

NN

N

NN NN N

N

NNN

NN N

NNN

NNN

NNN NN

N

O O

O

O

O

O

OO

O

O

O

O

OOO

O

O

OHOH

OHOH

N NN

N NNO

N NN

O

O

O

O

OHOH

OHOH

O

OHOH

OHOH

O

O

O

O

O

OHOH

OH

OH

N NN

NNN

O

NN

N

O

O

O

O

OHOH

OH

OH

O

HOOHOH

OH

O

O

OO

O

HOHO OH

OH

NN

NNN

N

O

NNN O

OO

O

HOHO OH

OHO

HOHO OH

OH

O

O

OO

O

HOHO

OH

OH

NNN

NNN

ON

NN

OO

O

O

HOHO

OH

OH

O

HOHO

OHOH

O

O

OO

OHOHO

OHOH

NNN

NNN

ONN

N

O

O

O

OHOHO

OHOH

OHOHO

OH OH

OO

O

O

OHOHO

HO HO

NN

N

NNN

ONNN

O

O

O

O

HOHO

HO HO

O

HOHO

HO HO

OO

O

O

O

HOHO

HOHO NN

N

NNN O

NNN

O

O

O

O

HOHO

HOHO

O

HOHO

HO

HO O

O

O

OO

OHHO

HO

HO

NNN

NNN

O

NNN

O

O

OO

OHHOHO

HO

O

OHHO

HO

HO

O

O

OO

O

OHOHHO

HO

NNN

NNN

O

NN N

O

OO

O

OHOHHO

HO

O

OHOHHO

HO

O

O

O O

O

OHOH

HO

HO

NN N

NN N

ONN N

O O

O

O

OHOH

HO

HO

O

OHOH

HOHO

O

O

OO

O OHOH

HOHO

NN N

NN N

O NNN

O

O

O

O OHOH

HOHO

O OHOH

OHHO

OO

O

O

OOH

OH

OHOH

NNN

N NN

O N NN

O

O

O

O

OHOH

OHOH

O

OHOH

OHOH

OO

O

O

O

O

O

O

O

O

O

O

C60LL (36 Man)

Supplementary Figure 5. Chemical structures of multivalent glycofullerenes studied in inhibition experiments with pseudotyped Ebola virus particles.




S51

Cytotoxicity studies

1 10 100 1000 100000

20

40

60

80

100

12017a17b17cTriton X-100 (0.1%)

C+

Concentration (nM)

Viab

ility

(%)

Supplementary Figure 6. Cell proliferation assay using glycofullerenes 17a, 17b, 17c and

Positive control (Triton X‐100). The cytotoxic effect of glycofullerenes is shown as the

percentage of viability of Jurkat DC‐SIGN+ after 48h culture in the presence of different

concentrations of the glycofullerenes. The bars/plots on the graph represent the mean +/‐

SEM.


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