Cyclic Peptoid-Peptide Hybrids as Versatile Molecular Transporters

Abstract

Addressing intracellular targets is a challenging task that requires potent molecular transporters capable to deliver various cargos. Herein, we report the synthesis of hydrophobic macrocycles composed of both amino acids and peptoid monomers. The cyclic tetramers and hexamers were assembled in a modular approach using solid as well as solution phase techniques. To monitor their intracellular localization, the macrocycles were attached to the fluorophore Rhodamine B. Most molecular transporters were efficiently internalized by HeLa cells and revealed a specific accumulation in mitochondria without the need for cationic charges. The data will serve as a starting point for the design of further cyclic peptoid-peptide hybrids presenting a new class of highly efficient, versatile molecular transporters.

Keywords: amide; cyclization; molecular transport; peptidomimetic; peptoid.

FIGURE 1

Phenylalanine (Green and Loewenstein, 1988) as an example for an amino acid as the building block of a peptide (A) and N1ph (Frankel and Pabo, 1988) as an example for an alkylated glycine monomer (B).

SCHEME 1

Synthetic approach for the assembly of linear precursors on solid support. [A]: Fmoc-protected amino acid, N,N′-diisopropylethylamine (DIPEA), N-methyl-2-pyrrolidone (NMP), 21°C, 16 h; [B]: piperidine, dimethylformamide (DMF), 21°C, 3 × 5 min; [C]: Fmoc-protected amino acid, N,N’-diisopropylcarbodiimide (DIC), hydroxybenzotriazole, NMP, 21°C, 4 h; [D]: bromoacetic acid, DIPEA, DMF, 21°C, 1 h; [E]: amine, DMF, 21°C, 1–16 h; [F]: bromoacetic acid, DIC, DMF, 21°C, 30 min; [G]: hexafluoroisopropanol, methylene chloride, 21°C, 16 h.

FIGURE 2

Alkylated glycine monomers 2 and 12–14 used for the synthesis of molecular transporters and their applied designation.

FIGURE 3

Macrocyclic hybrids 15–24 composed of alkylated glycine monomers and amino acids that were assembled via a combination of solid and solution phase methods.

SCHEME 2

Conjugation of the chromophore Rhodamine B to a cyclic hybrid exemplarily shown for macrocycle 15. [A] Rhodamine B, HATU, DIPEA, DMF, 36 h, 21°C.

FIGURE 4

Localization of the macrocyclic conjugates 15-Rhod to 24-Rhod in HeLa cells. After incubation with a 5.0 µm solution of the respective transporters for 5 h, the cellular uptake was monitored via fluorescent confocal microscopy (Rhodamine B labeled hybrids: λ _Exc = 532 nm, λ _Em = 570–620 nm; MitoTracker^® Green: λ _Exc = 488 nm, λ _Em = 490–540 nm; Hoechst 33,342: λ _Exc = 405 nm, λ _Em = 430–490 nm). Counterstaining with MitoTracker^® Green visualized mitochondria (green). Staining with 2 μg/ml Hoechst 33,342 indicated nuclei (blue). Scale bar: 10 µm.