Chemical and Biological Investigations of β‐Oligoarginines
Authors: Seebach Dieter , Namoto Kenji , Mahajan Yogesh R. , Bindschädler Pascal , Sustmann Reiner , Kirsch Michael , Ryder Neil S. , Weiss Markus , Sauer Markus , Roth Christian , Werner Sabine , Beer Hans‐Dietmar , Munding Christine , Walde Peter , Voser Matthias 
Published: February 2004

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 β‐homoarginines are central in our investigations of β‐peptides (Fig. 1). The preparation of β2‐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‐β3hArg(Boc)2‐OH is used for manual solid‐phase synthesis of β3‐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, β3‐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‐β2hArg‐OH and (S)‐H‐β3hArg‐OH were tested for their ability to function as substrates for NO synthase (iNOS); the β3‐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 β‐homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 μM; Table 1), and no significant antibiotic activity (MIC≥64 μg/ml; Table 2). Cell‐penetration studies with the fluorescence‐labelled, peptidase‐resistant β3‐oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer‐chain β‐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° and 4° with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non‐endocytotic and non‐energy‐dependent uptake mechanism; ii) the β‐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‐μM concentration of the fluorescence‐labelled β‐octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the β‐octaarginine derivative show migration of the β‐peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence‐labelled β‐octa‐ and β‐decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the β‐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 β‐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 β‐oligoarginines are discussed.