Click-Free Synthesis of a Multivalent Tricyclic Peptide as a Molecular Transporter

Abstract

The cellular delivery of cell-impermeable and water-insoluble molecules remains an ongoing challenge to overcome. Previously, we reported amphipathic cyclic peptides c[WR]₄ and c[WR]₅ consisting of alternate arginine and tryptophan residues as nuclear-targeting molecular transporters. These peptides contain an optimal balance of positive charge and hydrophobicity, which is required for interactions with the phospholipid bilayer to facilitate their application as a drug delivery system. To further optimize them, we synthesized and evaluated a multivalent tricyclic peptide as an efficient molecular transporter. The monomeric cyclic peptide building blocks were synthesized using Fmoc/tBu solid-phase chemistry and cyclization in the solution and conjugated with each other through an amide bond to afford the tricyclic peptide, which demonstrated modest antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli (E. coli) with a minimum inhibitory concentration (MIC) of 64-128 µg/mL. The tricyclic peptide was found to be nontoxic up to 30 µM in the breast cancer cell lines (MDA-MB-231). The presence of tricyclic peptide enhanced cellular uptakes of fluorescently-labeled phosphopeptide (F'-GpYEEI, 18-fold), anti-HIV drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), and stavudine (F'-d4T), 1.7-12-fold), and siRNA (3.3-fold) in the MDA-MB-231 cell lines.

Keywords: MDA-MB-231; antibacterial activity; cell-penetrating peptide; cellular uptake; cyclic peptide; cytotoxicity; drug delivery; phosphopeptide; siRNA; tricyclic.

Figure 1

The chemical structures of previously reported mono- and bicyclic peptides, c[WR]₅, c[WR]₄ [16], c[R₄W₄] [23], and (c[W₅G]-(triazole)-c[KR₅], and c[W₅E]-(β-Ala)-c[KR₅]) [22].

Scheme 1

Synthesis of monocyclic peptides containing free amino or free carboxylic acid or both.

Scheme 2

Synthesis of cyclic peptides containing two free amino or two free carboxylic acids.

Scheme 3

Synthesis of cyclic peptides containing one free amino group (16) and one free carboxylic acid (17 and 18).

Scheme 4

Synthesis of tricyclic peptide 20 from monomeric cyclic peptides 9 and 17.

Figure 2

Cytotoxicity of tricyclic peptide 20 in MDA-MB-231 cells (if p < 0.01 then **). DMSO (35% v/v) was used as the positive control. The compound was used in the aqueous solution.

Figure 3

The cellular uptake of fluorescently labeled G-(pTyr)-Glu-Glu-Ile (F′-GpYEEI) using tricyclic peptide 20 in the MDA-MB-231 cells (if p < 0.0001 then ****). Untreated cells are shown on the left.

Figure 4

The cellular uptake of fluorescently labeled anti-HIV drugs (F′-FTC, F′-3TC, and F′-d4T) in the presence of tricyclic peptide 20 in MDA-MB-231 cells (if p < 0.01 then **, if p < 0.001 then ***).

Figure 5

The cellular uptake of fluorescently-labeled siRNA in the presence of tricyclic peptide 20 in MDA-MB-231 cells (if p < 0.01 then **, if p < 0.0001 then ****).