Solid-Phase Synthesis of Fullerene-peptides

Abstract

The solid-phase synthesis of peptides (SPPS) containing [60]fullerene-functionalized amino acids is reported. A new amino acid, fulleropyrrolidino-glutamic acid (Fgu), is used for the SPPS of a series of analogues of different length based on the natural Leu(5)-Enkephalin and on cationic antimicrobial peptides. These fullero-peptides were prepared on different solid supports to analyze the influence of the resin on the synthesis. Optimized protocols for the coupling and deprotection procedures were determined allowing the synthesis of highly pure peptides in sufficient quantities for evaluation of biological activities. In particular, to avoid side reactions of the fullerene moiety with bases and nucleophiles, the removal of the protecting groups was performed under inert conditions (nitrogen or argon in the dark). We have encountered serious problems with the recovery of the crude compounds, especially when Fgu was inserted in the proximity of the resin core as fullero-peptides tend to remain embedded inside the resin. Eventually, all of the fullero-peptides were easily purified, and the cationic peptides were tested for their antimicrobial activities. They displayed a specific activity against the Gram-positive bacterium S. aureus and also lysed erythrocytes. The availability of a fullero-amino acid easily useable in the SPPS of fullero-peptides may thus open the way to the synthesis of new types of biologically active oligomers.

Unexpected Reactions of [60]Fullerene Involving Tertiary Amines and Insight into the Reaction Mechanisms

“…All other commercial available reagents are of analytical grade. = 155.56, 153.48, 153.24, 152.37, 146.59 (2 C), 146.04, 145.95, 145.82, 145.65, 145.61, 145.50 (2 C), 145.43, 145.41, 145.29 (2 C), 145.11, 144.84 (4 C), 144.71, 144.62 (3 C), 144.57, 144.51, 144.08, 143.96, 143.71 (2 C), 142.49, 142.37, 142.02, 141.98, 141.94 = 2920, 2851, 2772, 1510, 1461, 1426, 1376, 1331, 1186, 766, 574, 526 = 155.70, 153.59, 153.23, 152.45, 146.57 (2 C), 146.12, 145.99, 145.77, 145.64, 145.57, 145.49, 145.45, 145.41, 145.36, 145.27 (2 C), 145.09, 144.91, 144.82 (2 C), 144.76, 144.70, 144.58 (3 C), 144.53, 144.47, 144.07, 143.95, 143.71, 143.68, 142.46, 142.33, 141.98, 141.95, 141.93, 141.89, 141.61, 141.51 = 2923, 2853, 2787, 1452, 1428, 1378, 1334, 1185, 736, 697, 574, 527 155.68, 153.42, 153.15, 152.23, 146.77, 146.75, 146.17, 146.04, 145.94, 145.82, 145.77, 145.67 (2 C), 145.59, 145.57, 145.45 (2 C), 145.25, 145.00 (4 C), 144.90, 144.81, 144.77 (2 C), 144.72, 144.67, 144.26, 144.10, 143.88, 143.86, 142.64, 142.52, 142.18, 142.14, 142.09 (2 C), 141.75, 141.69, 141.65 (3 C), 141.55 (2 C), 141.52 (2 C), 141.41, 141.21, 141.16, 139.75, 139.66 (2 C), 139.22, 137.12, 135.89, 135.59, 135.34, 74.67 (sp 3 -C of C 60 ), 68.74 (CHCH 3 ), 68.63 (sp 3 -C of C 60 ), 65.69 (CH 2 ), 51.24 (CH 2 COOCH 3 ), 51.10 (CH 2 COOCH 3 ), 16.14 (CHCH 3 ) ppm; FT-IR (KBr): ñ = 2921, 2851, 1736, 1510, 1461, 1428, 1378, 1188, 1169, 766, 574, 526 cm À1 ; UV/Vis (CHCl 3 ): l max = 256, 307, 430, 702 nm; 5,6]fullerene (3): A 25-mL round-bottomed flask charged with a solution of C 60 (36.0 mg, 0.05 mmol) and Et 3 N (697 mL, 5.00 mmol) in ODCB (10 mL) was wrapped with aluminum foil and heated in an oil bath preset at 220 8C for 1 h. After conventional workup, flash chromatography on a silica gel column with carbon disulfide as the eluent afforded unreacted C 60 (7.9 mg, 22 %) and adduct 3 (22.2 mg, 52 %). 5,6]fullerene (5 a 3 as relaxation reagent, all 1 C unless indicated): d = 158.26 (aryl C), 156.35, 155.81, 154.11, 153.90, 147.22, 146.88, 146.53, 146.36, 145.90, 145.57, 145.53 (2 C), 145.44 (2 C), 145.34, 145.30, 145.20, 145.14, 145.05, 144.91, 144.65, 144.55, 144.50 (3 C), 144.40 (2 C), 144.27, 143.91, 143.80, 143.71, 143.62, 142.45, 142.30, 141.93, 141.90, 141.87, 141.79, 141.70, 141.59, 141.56, 141.47, 141.44, 141.31, 141.26, 141.08, 141.07, 141.03, 140.99, 140.87, 139.34, 138.83, 138.78, 138.63, 135.29, 134.93, 134.12, 133.49, 129.58 (2 C, aryl C) = 2961, 2924, 2853, 1613, 1513, 1462, 1428, 1380, 1252, 1178, 1109, 1067,...…”

“…[4] Alternatively, amino acids and peptides can be tethered to side chains of certain fullerene derivatives. [5] Gans group has explored direct reactions between amino acid esters and C 60 induced by photoirradiation and ultrasonification. [6] We have very recently reported novel reactions of C 60 with amino acid ester hydrochlorides and CS 2 in the presence of triethylamine (Et 3 N), which afford fullerene derivatives containing biologically active amino acid, thioamide, and thiourea units.…”

Fullerene–biomolecule conjugates and their biomedicinal applications

“…Pantarotto et al 33 and Pellarini et al 34 presented a group of fullerene-peptides, called fulleropeptides, that showed great potential for bacteriostatic activity. In one study, a C 60 -functionalized amino acid was synthesized by conjugating fullerene with N-Fmoc-L-glutamic acid alpha-tert-butyl ester.…”

“…34 In another study, several similarly structured fulleropeptides were prepared through solid-phase peptide synthesis, displaying an increased potency in treating Gram-positive bacterial strains, although the molecules displayed decreased effectiveness against Gram-negative bacteria or yeasts when compared to the parent peptide. 33 Mashino et al used molecules 14 and 15 to further investigate the antimicrobial potential of fullerene derivatives. 35 Their research suggests that inhibition of bacterial growth might actually be due to interaction of fullerene molecules in the cellular energy metabolism chain rather than the physical interruption of the cellular membrane.…”

Organofullerene Materials

“…Fulleroproline was conceived as a building block for the construction of fullerene-based peptides [291]; however, the designed synthetic route resulted in the isolation of its racemic mixture, while enantiomeric resolution relied on chiral chromatography [292]. Lately, the synthesis of a protected fullerene-functionalized amino acid and its use in solid-phase synthesis for the preparation of a water-soluble peptide was achieved (Scheme 16) [293,294].…”