Supporting Information

Supporting Information Wiley-VCH 2008 69451 Weinheim, Germany

High Catalytic Activity of Dendritic C 60 Monoadducts in Metal-Free Superoxide Dismutation** Gao-Feng Liu, Miloš Filipović, Ivana Ivanović-Burmazović* Department of Chemistry and Pharmacy, University of Erlangen-ürnberg, Egerlandstr. 1, 91058 Erlangen, Germany. Florian Beuerle, Patrick Witte, Andreas Hirsch* Department of Chemistry and Pharmacy & Interdisciplinary Center for Molecular Materials (ICMM), University of Erlangen-ürnberg, Henkestrasse 42, 91054 Erlangen, Germany Supporting Information Experimental Section General Methods Chemicals: C 60 was obtained from Hoechst AG/Aventis and separated from higher fullerenes by a plug filtration. [1] All chemicals were purchased by chemical suppliers and used without further purification. All analytical reagent-grade solvents were purified by distillation. Dry solvents were prepared using customary literature procedures. [2] Thin layer chromatography (TLC): Riedel-de-Haën silica gel F254 and Merck silica gel 60 F254. Detection: UV lamp and iodine chamber. Flash column chromatography (FC): Merck 30 silica gel 60 (230 400 mesh, 4 63 nm). Analytical high performance liquid chromatography (HPLC): Shimadzu Liquid Chromatograph LC-10 with Busmodule CBM-10A, auto injector SIL-10A, two pumps LC-10AT, diode array detector. The HPLC-grade solvents were purchased from SDS or Acros rganics; analytical column ucleosil 5 μm, 200 4 mm, Macherey agel, Düren. UV/Vis spectroscopy: Shimadzu UV-3102 PC UV/Vis/IR scanning spectrophotometer; absorption maxima λ max are given in nm. Mass spectrometry: Micromass Zabspec, FAB 1

(LSIMS) mode, matrix 3-nitrobenzyl alcohol. MR spectroscopy: JEL JM EX 400 and JEL JM GX 400 and uker Avance 300. The chemical shifts are given in ppm relative to TMS. The resonance multiplicities are indicated as s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet) and m (multiplet), non-resolved and broad resonances as br. Elemental analysis (C, H, ): Succeeded by combustion and gas chromatographical analysis with an EA 1110 CHS analyser (CE Instruments). The precursor compound S1 was synthesized according to literature procedure. [3] H R 1 S1 a) R 1 H H S2 H H b) R 1 H c) R 1 H S3 S4 d) H R 1 3 3 a) formic acid, rt, 48 h; b) 2-bromoethanol, DCC, DMAP, 1-HBt, THF, 0 C rt, 24 h; c) C 60, C 4, DBU, toluene, rt, 6 h; d) pyridine, 60 C, two days. 2

S2: Compound S1 (1.37 g, 1.58 mmol) was dissolved in formic acid (15 ml). The reaction mixture was stirred for 48 h at room temperature and the progress of the reaction was monitored by TLC. The reaction mixture was concentrated and dried in vacuum to afford S2 as a white solid. (1.09 g, 1.58 mmol, 99 %). 1 H-MR (300 MHz, RT, THF-d 8 ): δ = 10.33 (s, br, 3H, CH), 6.21 (s, br, 1H, CH), 4.17 (t, 3 J = 6.6 Hz, 2H, CH 2 ), 4.14 (t, 3 J = 6.8 Hz, 2H, CH 2 ), 3.32 (s, 2H, CCH 2 C), 2.22 (t, 3 J = 7.7 Hz, 6H, CH 2 CH), 2.11 (t, 3 J = 7.4 Hz, 2H, CCH 2 ), 1.98 (t, 3 J = 7.8 Hz, 6H, HC(CH 2 ) 3 ), 1.67 (m, 6H, CH 2 ), 1.25 (m, 32H, CH 2 ), 0.91 (t, 3 J = 6.7 Hz, 3H, CH 3 ) ppm. 13 C-MR (75 MHz, RT, THF-d 8 ): δ = 174.88 (3C, CH), 172.12 (1C, CH), 166.87 (1C, C), 166.79 (1C, C), 65.66 (1C, CH 2 ), 65.31 (1C, CH 2 ), 57.19 (1C, HC(CH 2 ) 3 ), 41.44 (1C, CCH 2 C), 37.19 (1C, CH 2 C), 31.86 (1C, CH 2 ), 29.89 (3C, HC(CH 2 ) 3 ), 29.68, 29.64, 29.61, 29.49, 29.48, 29.37, 29.19 (10C, CH 2 ), 28.71 (3C, CH 2 CH), 28.39, 28.18, 25.71 (5C, CH 2 ), 25.48 (1C, CH 2 CH 2 C), 25.18, 22.66 (2C, CH 2 ), 14.12 (1C, CH 3 ) ppm. IR(ATR): ν ~ = 3402, 2963, 1871, 1648, 1507, 1482, 1467, 1457, 1432, 1324, 1266, 1117, 950, 791, 686, 624 cm -1. MS (FAB, BA): m/z = 699 [M] +. C 37 H 65 11 C 2 HF 3 2 : calcd. C 57.55, H 8.17, F 7.00, 1.72, 25.55; found: C 57.99, H 8.52, 2.01. S3: A solution of S2 (1.09 g, 1.56 mmol) and 2-bromoethanol (0.79 g, 6.32 mmol) in dry THF (150 ml) was cooled to 0 C under nitrogen atmosphere. DMAP (193 mg, 1.58 mmol), 1-HBt (747 mg, 5.53 mmol) and DCC (1.14 g, 5.53 mmol) were added subsequently. After stirring the solution under 2 for 2 h at 0 C, it was left at room temperature for another 24 h. Progress of the reaction was monitored by TLC. The solution was filtered and after evaporation of the solvent the residue was dissolved in ethyl acetate and filtered again for several times to remove the remaining DCU. Purification was obtained by flash column chromatography (Si 2, dichloromethane/ethyl acetate, 15:1 to 5:1). The purified material was dried in vacuum affording S3 as a light yellow oil. (1.22 g, 1.20 mmol, 76 %). 1 H-MR (400 MHz, RT, CDCl 3 ): δ = 5.68 (s, br, 1H, CH), 4.39 (t, 3 J = 6.2 Hz, 6H, CH 2 CH 2 ), 4.15 (t, 3 J = 6.7 Hz, 2H, CH 2 ), 4.13 (t, 3 J = 6.7 Hz, 2H, CH 2 ), 3.52 (t, 3 J = 6.1 Hz, 6H, CH 2 ), 3.37 (s, 2H, CCH 2 C), 2.36 (t, 3 J = 7.8 Hz, 6H, CH 2 C), 2.14 (t, 3 J = 7.6 Hz, 2H, CCH 2 ), 2.06 (t, 3 J = 7.2 3

Hz, 6H, HC(CH 2 ) 3 ), 1.64 (m, 6H, CH 2 ), 1.25 (m, 32H, CH 2 ), 0.88 (t, 3 J = 6.9 Hz, 3H, CH 3 ) ppm. 13 C-MR (100 MHz, RT, CDCl 3 ): δ = 172.73 (3C, C), 172.35 (1C, CH), 166.69 (1C, C), 166.62 (1C, C), 65.69 (1C, CH 2 ), 65.21 (1C, CH 2 ), 64.01 (3C, CH 2 CH 2 ), 57.24 (1C, HC(CH 2 ) 3 ), 41.54 (1C, CCH 2 C), 37.11 (1C, CH 2 C), 31.86 (1C, CH 2 ), 29.81 (7C, HC(CH 2 ) 3, CH 2 ), 29.64, 29.47, 29.34 (6C, CH 2 ), 28.77 (3C, CH 2 C), 28.58 (3C, CH 2 ), 28.53, 28.29 (4C, CH 2 ), 25.91 (1C, CH 2 CH 2 C), 25.63, 25.29, 22.80 (3C, CH 2 ), 14.22 (1C, CH 3 ) ppm. IR(ATR): ν ~ = 3379, 2972, 2922, 1832, 1501, 1455, 1432, 1281, 1268, 1102, 950, 799, 791, 697, 686, 624 cm -1. MS (FAB, BA): m/z = 1020 [M] +. C 43 H 74 3 11 : calcd. C 50.60, H 7.31, 23.48, 1.37, 17.24; found: C 59.87, H 7.48, 1.52. S4: C 60 (741 mg, 1.03 mmol) was dissolved in dry toluene (ca. 0.5 ml toluene per mg C 60 ) under a nitrogen atmosphere. S3 (750 mg, 0.73 mmol) and C 4 (342 mg, 1.03 mmol) were added subsequently. DBU (153 μl, 1.03 mmol) in 20 ml toluene was added dropwise over a period of 1 h to the stirred solution at room temperature. The reaction mixture was stirred at room temperature for additional 6 h and the progress of the reaction was monitored by TLC. The product was isolated by flash chromatography (Si 2, toluene/ethyl acetate, 80:5 to 80:25) and dried in vacuum affording S4 as a red brownish solid. (406 mg, 0.28 mmol, 32 %). 1 H-MR (400 MHz, RT, CDCl 3 ): δ = 5.59 (s, br, 1H, CH), 4.47 (t, 3 J = 6.6 Hz, 4H, CH 2 ), 4.36 (t, 3 J = 6.1 Hz, 2H, CH 2 CH 2 ), 3.49 (t, 3 J = 6.1 Hz, 6H, CH 2 ), 2.33 (t, 3 J = 7.6 Hz, 6H, CH 2 C), 2.12 (t, 3 J = 7.5 Hz, 2H, CCH 2 ), 2.03 (t, 3 J = 7.3 Hz, 6H, HC(CH 2 ) 3 ), 1.83 (m, 4H, CH 2 ), 1.67 (m, 2H, CH 2 ), 1.45 (m, 2H, CH 2 ), 1.25 (m, 30H, CH 2 ), 0.85 (t, 3 J = 7.0 Hz, 3H, CH 3 ) ppm. 13 C-MR (100 MHz, RT, CDCl 3 ): δ = 173.08 (3C, C), 172.48 (1C, CH), 164.00 (1C, C), 163.92 (1C, C), 145.65, 145.64, 145.55, 145.48, 145.47, 145.42, 145.17, 144.99, 144.96, 144.93, 144.89, 144.16, 143.40, 143.38, 143.31, 143.28, 143.26, 142.49, 142.15, 141.25, 141.22, 139.24, 138.14 (58C, C 60 -sp 2 ), 71.84 (2C, C 60 -sp 3 ), 67.74 (1C, CH 2 ), 67.28 (1C, CH 2 ), 64.25 (3C, CH 2 CH 2 ), 57.46 (1C, HC(CH 2 ) 3 ), 52.61 (1C, CCC), 37.32 (1C, CH 2 C), 32.08 (1C, CH 2 ), 29.87 (5C, HC(CH 2 ) 3, CH 2 ), 29.82, 29.78, 29.77, 29.52, 29.38 (7C, CH 2 ), 28.85 (3C, CH 2 ), 28.75 (2C, CH 2 ), 28.56 (3C, CH 2 C), 28.47 (2C, CH 2 ), 26.15 (1C, CH 2 CH 2 C), 25.81, 25.27, 22.84, 21.59 (4C, CH 2 ), 14.28 (1C, CH 3 ) ppm. IR(ATR): ν ~ = 3332, 3062, 2992, 2634, 1832, 1766, 1703, 4

1654, 1603, 1533, 1478, 1403, 1281, 1235, 1107, 1033, 799, 787, 653 cm -1. MS (FAB, BA): m/z = 1739 [M] +. UV/Vis (CH 2 Cl 2 ): λ max = 325.5, 425.5, 492 nm. 3: A solution of S4 (150 mg, 86 mmol) in 10 ml of dry pyridine was stirred for two days at 60 C. After the addition of 10 ml of toluene, the reaction mixture was filtrated and the residue was suspended in toluene and distilled under vacuum for several times to remove traces of pyridine. Reprecipitation from methanol/diethyl ether gave 3 as red brownish solid. (156 mg, 79 mmol, 92 %). 1 H-MR (400 MHz, RT, DMS-d 6 ): δ = 9.13 (d, 3 J = 5.6 Hz, 6H, o-pyrh), 8.65 (t, 3 J = 7.8 Hz, 3H, p-pyrh), 8.20 (dd, 3 J = 6.0, 7.7 Hz, 6H, m-pyrh), 7.20 (s, br, 1H, CH), 4.91 (m, 6H, CH 2 -Pyr), 4.51 (m, 10H, CH 2 CH 2 -Pyr, CH 2 ), 2.13 (m, 6H, CH 2 C), 2.05 (m, 2H, CCH 2 ), 1.73 (m, 6H, HC(CH 2 ) 3 ), 1.48 (m, 2H, CH 2 ), 1.35 (m, 4H, CH 2 ), 1.17 (m, 32H, CH 2 ), 0.84 (t, 3 J = 6.2 Hz, 3H, CH 3 ) ppm. 13 C-MR (100 MHz, RT, CDCl 3 ): δ = 172.59 (3C, C), 172.56 (1C, CH), 162.92 (1C, C), 162.90 (1C, C), 146.33 (3C, p-pyrc), 145.56 (6C, o- PyrC), 145.51, 145.16, 144.95, 144.94, 144.89, 144.86, 144.80, 144.73, 144.52, 144.36, 144.34, 144.30, 144.25, 143.54, 143.51, 142.81, 142.74, 142.73, 141.85, 141.84, 141.53, 141.46, 140.67, 138.74, 138.22 (58C, C 60 -sp 2 ), 128.21 (6C, m-pyrc), 71.55 (2C, C 60 -sp 3 ), 67.37 (2C, CH 2 ), 62.56 (3C, CH 2 CH 2 -Pyr), 59.76 (3C, CH 2 -Pyr), 56.29 (1C, HC(CH 2 ) 3 ), 52.70 (1C, CCC), 35.84 (1C, CH 2 C), 31.42 (6C, HC(CH 2 ) 3 ), 29.18, 29.11, 29.01 (4C, CH 2 ), 28.84 (3C, CH 2 C), 28.65, 28.57, 28.13, 27.80, 27.67 (7C, CH 2 ), 25.69 (1C, CH 2 CH 2 C), 25.08, 24.97, 22.22 (4C, CH 2 ), 14.06 (1C, CH 3 ). IR(ATR): ν ~ = 3368, 3020, 3001, 2651, 1799, 1765, 1654, 1613, 1546, 1434, 1406, 1256, 1249, 1237, 1111, 1023, 807, 753, 653, 603 cm -1. MS (FAB, BA): m/z = 1897 [M] +, 908 [M] 2+. UV/Vis (DMS/H 2 ): λ max = 258, 326 nm. 5

Figure S1 Cyclic voltammogramms of 2-7 in DMS purged with nitrogen. Conditions: [Fullerene] = 5 x 10-4 M, [Bu 4 BF 4 ] = 0.1 M, T = 298 K, scan rates = 0.2 V/s. -1.5x10-5 -1.5x10-5 -1.2x10-5 -1.2x10-5 -9.0x10-6 -6.0x10-6 -3.0x10-6 3.0x10-6 6.0x10-6 -9.0x10-6 -6.0x10-6 -3.0x10-6 3.0x10-6 6.0x10-6 9.0x10-6 0.2-0.2-0.4-0.6-0.8-1.0-0.2-0.4-0.6-0.8-1.0 2 3-4.0x10-5 -4.0x10-5 -3.0x10-5 -3.0x10-5 -2.0x10-5 -2.0x10-5 -1.0x10-5 -1.0x10-5 1.0x10-5 0.2-0.2-0.4-0.6-0.8-1.0 1.0x10-5 2.0x10-5 0.2-0.2-0.4-0.6-0.8-1.0 4 5-1.2x10-4 -2.5x10-5 -2.0x10-5 -1.0x10-4 -8.0x10-5 -6.0x10-5 -1.5x10-5 -1.0x10-5 -4.0x10-5 -2.0x10-5 -5.0x10-6 2.0x10-5 4.0x10-5 6.0x10-5 5.0x10-6 -0.2-0.4-0.6-0.8-1.0 8.0x10-5 -0.5-1.0-1.5-2.0-2.5-3.0 6 7 6

Figure S2 The time resolved UV/vis spectra for the reaction between a) 6.25 x 10-5 M malonate of 7 and b) 5 x 10-5 M 7 and 1 mm K 2 in DMS at room temperature. a) 1,50 Absorbance 1,00 0,50 65,0 45,0 0,0 325 375 425 475 525 575 625 675 5,0 25,0 Time (sec) Wavelength (nm) b 1,60 1,40 1,20 Absorbance 1,00 0,80 0,60 0,40 90 0,20 0,0 300 350 400 450 500 550 600 650 700 Wavelength (nm) 10 50 Time (sec) 7

Figure S3 The time resolved UV/Vis spectra for the reaction between electrochemically reduced cytochrome c and 6 under an argon atmosphere (50 mm potassium phosphate buffer, ph = 7.8; 25 C; [cyt c] = 10 μm and [6] =20 μm). Inset: corresponding absorbance change with time at 550 nm. Cyt C II with 7 absorbance [a.u.] 2.5 2.0 1.5 1.0 Absorbance 0.58 0.56 0.54 0.52 0.50 0.48 0.46 0.44 0.42 0.40 550 nm, 0.38 0 200 400 600 800 10001200140016001800 time, s 0.5 350 400 450 500 550 600 650 700 wavelength [nm] Determination of H 2 2 To detect the formation of hydrogen peroxide, product of superoxide dismutation, solid K 2 (to the final concentration of 250 μm) was added into a 25 μm solution of the fullerenes in 50 mm potassium phosphate buffer. The solutions were incubated for 5 min at 37 C and the concentration of formed H 2 2 was determined. H 2 2 was quantified by measuring spectrophotometrically the coloured product, formed by peroxidase-catalyzed oxidation of 4-aminoantipyrine.(I. Ioannidis, H. de Groot, Biochem. J. 1993, 296(Pt 2), 341-345) As a control, H 2 2 formation after K 2 addition was measured in buffer without the addition of fullerenes. For comparison the quantity of H 2 2 was also followed in samples containing native MnSD (E. coli) (500 U/ml). 8

[1] a) U. Reuther, Ph. D. Dissertation, University of Erlangen-uremberg, Germany, 2002; b) L. Isaacs, A. Wehrsig, F. Diederich, Helv. Chim. Acta 1993, 76, 1231 1250. [2] D. D. Perrin. W. L. F. Amarego, Purification of Laboratory Chemicals, 3rd ed., Pergamon Press, xford, 1988. [3] P. Witte, F. Beuerle, U. Hartnagel, R. Lebovitz, A. Savouchkina, S. Sali, D. Guldi,. Chronakis, A. Hirsch, rg. Biomol. Chem., 2007, 5(22), 3599-3613. 9