Cell Penetration, Herbicidal Activity, and in‐vivo‐Toxicity of Oligo‐Arginine Derivatives and of Novel Guanidinium‐Rich Compounds Derived from the Biopolymer Cyanophycin

Abstract

Oligo-arginines are thoroughly studied cell-penetrating peptides (CPPs, Figures 1 and 2). Previous in-vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti-malaria drug) have shown a 40-fold parasitaemia inhibition with P. falciparum, compared to fosmidomycin alone (Figure 3). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e. decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake (Table 1). Another pronounced difference between in-vitro and in-vivo activity was demonstrated with various cell-penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 μmol/kg (Table 2). The results show that use of CPPs for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly-aspartic acid containing argininylated side chains (Figure 4); its building block is the dipeptide H-βAsp-αArg-OH (H-Adp-OH). To test and compare the biological properties with those of octaarginines we synthesized Adp8-derivatives (Figure 5). Intravenouse injection of H-Adp8-NH2 into the tail vein of mice with doses as high as 45 μmol/kg causes no symptoms whatsoever (Table 3), but H-Adp8-NH2 is not cell penetrating (HEK293 and MCF-7 cells, Figure 6). On the other hand, the fluorescently labeled octamers FAM-(Adp(OMe))8-NH2 and FAM-(Adp(NMe2))8-NH2 with ester and amide groups in the side chains exhibit mediocre to high cell-wall permeability (Figure 6), and are toxic (Table 3). Possible reasons for this behavior are discussed (Figure 7) and corresponding NMR spectra are presented (Figure 8).

Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium‐Rich Peptides

“…The lacking cell permeability of the Adp 8 ‐derivative 2 has been interpreted as resulting from an ‘internal neutralization’ by salt formation between the guanidinum‐ and the carboxylate‐groups in the side chains under physiological conditions (pH 7.4). This would prevent the interaction of the cationic guanidinium groups with anionic phosphate groups on the cell surfaces, considered to be an important first step of cell penetration by guanidinium‐rich cell penetrating peptides (CPPs) .…”

“…This would prevent the interaction of the cationic guanidinium groups with anionic phosphate groups on the cell surfaces, considered to be an important first step of cell penetration by guanidinium‐rich cell penetrating peptides (CPPs) . Interestingly, the cell‐permeabilities of the three Adp‐peptides 2 – 4 are in line with their toxicities determined by mouse tail‐vein injection: 2 and all other peptides derived from cyanophycin with ‘free’ carboxylic acid groups in the side chains are non‐toxic, while the derivatives 3 and 4 with ester and amide groups in the side chains have toxicities comparable with those of octaarginines …”

“…These properties reverse upon esterification (→ 3 , FAM‐(AdpMe) 8 or conversion of the carboxylic‐acid group into an amide group (→ 4 , FAM‐(AdpNMe 2 ) 8 ).…”

“…We have recently studied [1] a novel type of guanidiniumrich peptides derived from the biopolymer cyanophycin, which consists of polyaspartic-acid, to the side chains of which arginine residues are attached ( Figure 1). 2 Unlike in-vitro cell-penetrating octaarginine derivatives, 3 such as 1, which turn out to be highly toxic upon intravenous administration to mice, octa-Adp derivatives with a free carboxylic-acid group in the side chains, such as the fluorescein-labeled FAM-Adp 8 (2; Figure 2), have been found to be neither cell-penetrating nor toxic.…”

“…2 Unlike in-vitro cell-penetrating octaarginine derivatives, 3 such as 1, which turn out to be highly toxic upon intravenous administration to mice, octa-Adp derivatives with a free carboxylic-acid group in the side chains, such as the fluorescein-labeled FAM-Adp 8 (2; Figure 2), have been found to be neither cell-penetrating nor toxic. [1] These properties reverse [1] upon esterification (? 3, FAM-(AdpMe) 8 4 or conversion of the carboxylic-acid group into an amide group [8] (?…”