title: Chemical and biological investigations of beta-oligoarginines.
abstract: In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-beta3 hArg(Boc)2-OH is used for manual solid-phase synthesis of beta3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, beta3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2-4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-beta2 hArg-OH and (S)-H-beta3 hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the beta3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free beta-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 microM; Table 1), and no significant antibiotic activity (MIC > or = 64 microg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant beta3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain beta-oligoarginine amides (8 and 10 residues; Figs. 4-6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37 degrees and 4 degrees with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the beta-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-microM concentration of the fluorescence-labelled beta-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the beta-octaarginine derivative show migration of the beta-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled beta-octa- and beta-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the beta-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a beta-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the 'protein transduction' by beta-oligoarginines are discussed.
authors.affiliation: Laboratorium für Organische Chemie, ETH-Zürich, ETH-Hönggerberg, HCI, CH-8093 Zürich.
authors.forename: Dieter
authors.initials: D
authors.lastname: Seebach
authors.forename: Kenji
authors.initials: K
authors.lastname: Namoto
authors.forename: Yogesh R
authors.initials: YR
authors.lastname: Mahajan
authors.forename: Pascal
authors.initials: P
authors.lastname: Bindschädler
authors.forename: Reiner
authors.initials: R
authors.lastname: Sustmann
authors.forename: Michael
authors.initials: M
authors.lastname: Kirsch
authors.forename: Neil S
authors.initials: NS
authors.lastname: Ryder
authors.forename: Markus
authors.initials: M
authors.lastname: Weiss
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
authors.forename: Christian
authors.initials: C
authors.lastname: Roth
authors.forename: Sabine
authors.initials: S
authors.lastname: Werner
authors.forename: Hans-Dietmar
authors.initials: HD
authors.lastname: Beer
authors.forename: Christine
authors.initials: C
authors.lastname: Munding
authors.forename: Peter
authors.initials: P
authors.lastname: Walde
authors.forename: Matthias
authors.initials: M
authors.lastname: Voser
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000494:pharmacology; D002478:Cells, Cultured; D006367:HeLa Cells; D006709:Homoarginine / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D008297:Male; D051379:Mice; D051381:Rats
chemical_list: D006709:Homoarginine; D001120:Arginine