Comparative Molecular Transporter Efficiency of Cyclic Peptides Containing Tryptophan and Arginine Residues

[Image: see text] Cyclic peptides containing tryptophan (W) and arginine (R) residues, [WR](5), [WR](6), [WR](7), [WR](8), and [WR](9), were synthesized through Fmoc solid-phase chemistry to compare their molecular transporter efficiency. The in vitro cytotoxicity of the peptides was evaluated using human leukemia carcinoma cell line (CCRF-CEM) and normal kidney cell line (LLC-PK1). [WR](6), [WR](7), [WR](8), and [WR](9) were not significantly cytotoxic to LLC-PK1cells at a concentration of 10 μM after 3 h incubation. Among all the peptides, [WR](9) was found to be a more efficient transporter than [WR](5), [WR](6), [WR](7), and [WR](8) in CCRF-CEM cells for delivery of a cell-impermeable fluorescence-labeled negatively charged phosphopeptide (F′-GpYEEI). [WR](9) (10 μM) improved the cellular uptake of F′-GpYEEI (2 μM) by 20-fold. The cellular uptake of a fluorescent conjugate of [WR](9), F′-[W(9)R(8)K], was increased in a concentration- and time-dependent pattern in CCRF-CEM cells. The uptake of F′-[W(9)R(8)K] was slightly reduced in CCRF-CEM cells in the presence of different endocytic inhibitors, such as nystatin, 5-(N-ethyl-N-isopropyl)amiloride, chlorpromazine, chloroquine, and methyl β-cyclodextrin. Furthermore, the uptake of F′-[W(9)R(8)K] was shown to be temperature-dependent and slightly adenosine 5′-triphosphate-dependent. The intracellular/cellular localization (in the nucleus and cytoplasm) of F′-[W(9)R(8)K] was confirmed by fluorescent microscopy in CCRF-CEM cells. These studies suggest that large cyclic peptides containing arginine and tryptophan can be used as a molecular transporter of specific compounds.