Enhancing Membrane Permeability by Fatty Acylation of Oligoarginine Peptides

The improved bioavailability of drugs in the treatment of diseases produces a prolonged therapeutic effect and, as a consequence, reduced toxicity and cost. Most oligonucleotides, peptides, or proteins are poorly taken up by cells due to their insufficient association with the lipid bilayer of the plasma membrane. In many cases, therapeutic agents need to possess lipophilic properties in order to achieve the desired pharmacokinetic profile. Thus, the lipophilicity of the molecules assists in the penetration of cytoplasmic and intracellular membranes. Traditionally, the incorporation of homologous series of alkyl groups into a drug of interest produced increases in pharmacological effects. More recently, a short peptide (RKKRRQRRR), derived from HIV Tat-protein, as well as other arginine-containing peptides have attracted attention due to their high cellular uptake efficiency. These membrane-penetrating peptides have been applied as delivery vectors for various biological and medical applications. A systematic study of Tat-peptide indicated that the positively charged arginine residues are crucial to its membrane-penetrating ability, a property that has also been mimicked by a hepta-arginine peptide. It has thus been concluded that the cationic guanidine moiety on the arginine side chain provides the exceptional translocation properties that are not observed in peptides containing similar amino acids such as ornithine, lysine, histidine or citrulline. Although this membrane-translocalization phenomenon has been reported for more than a decade, the detailed mechanism still has to be determined. Here we described the systematic exploration of the changes in cell-localizing ability brought about by the modification of oligoarginine peptides with fatty acid analogues. We originally hypothesized that such modifications would lead to enhanced association with lipid membranes and, potentially, improved transmembrane delivery. The 7-mer oligoarginine Tat-peptide mimetic, originally reported by Wender et al. , was selected as the peptide template on which to study the effect of the length of the acyl chain. A series of fatty acid groups, including hexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, and palmitoyl, were attached to the N terminus of the amidated peptide via a b-alanine spacer on solid support by using a modified Schotten±Baumann reaction. Thereafter, the lipopeptides were labeled with fluorescein isothiocyanate