Cell-Penetrating cis-γ-Amino-L-Proline-Derived Peptides Josep Farrera-Sinfreu, ²,‡ Ernest Giralt, ², | Susanna Castel, § Fernando Albericio,* ,², | and Miriam Royo* ,‡ Contribution from the Barcelona Biomedical Research Institute, Combinatorial Chemistry Unit, and Confocal Microscopy and Cellular Micromanipulation Facility, Barcelona Science Park, UniVersity of Barcelona, Josep Samitier 1, 08028-Barcelona, Spain, and Department of Organic Chemistry, UniVersity of Barcelona, Martı ´ i Franque ´ s 1, 08028-Barcelona, Spain Received March 15, 2005; E-mail: mroyo@pcb.ub.es; albericio@pcb.ub.es Abstract: The synthesis of cis-γ-amino-L-proline oligomers functionalized at the proline R-amine with several groups that mimic the side chains of natural amino acids, including alanine, leucine, and phenylalanine, is herein described. These γ-peptides enter into different cell lines (COS-1 and HeLa) via an endocytic mechanism. The ability of these compounds to be taken up into cells was studied at 37 °C and 4 °C by plate fluorimetry, flow cytometry, and confocal microscopy. In addition to their capacity for cellular uptake, these unnatural short length oligomers offer advantages over the well-known penetrating TAT peptide, such as being less toxic than TAT and protease resistance. Introduction In the past few years several peptides capable of crossing the cell membrane, namely cell-penetrating peptides (CPPs), 1 have been described in the literature. 2 This capacity suggests their potential application as new agents for cellular delivery of biomolecules. CPPs offer several advantages over other known cellular delivery systems, 3,4 including low toxicity, high efficiency toward different cell lines, and even inherent thera- peutic potential. Peptides and proteins are nevertheless limited by low protease resistance and, sometimes, low membrane permeability. 5,6 Hence, compounds with greater proteolytic resistance, such as loligomers, 7,8 as well as biomolecular mimetics, such as -peptides 9-12 (nonnatural peptides, formed by -amino acids, that can adopt discrete and predictable well- defined secondary structures), have been evaluated as drug delivery agents. Peptides capable of translocating the cell membrane can be classified into two groups: (i) cationic peptides with at least six charged amino acids (Lys or Arg) such as HIV-1, TAT peptide, 13 penetratin, 14 and chimeric transportan, 15 and (ii) hydrophobic peptides, such as those based on the H-region of signal-sequence proteins. 16 Bactericidal peptides are unusual in that they are both charged and contain hydrophobic regions in their primary or secondary structure. 17-21 As these features are implicated in both membrane permeabilization and pore- forming, functions which lead to microbicidal mechanisms, the ² Barcelona Biomedical Research Institute. ‡ Combinatorial Chemistry Unit. § Confocal Microscopy and Cellular Micromanipulation Facility. | Department of Organic Chemistry. (1) Abbreviations: Amp, cis-4-amino-L-proline or (2S,4S)-4-amino-pyrrolidine- 2-carboxylic acid; Boc, tert-butoxycarbonyl; (2S,4S)-Boc-Amp(Fmoc)-OH, (2S,4S)-Fmoc-4-amino-1-Boc-pyrrolidine-2-carboxylic acid; CF, 5(6)-car- boxyfluorescein; CPPs, cell-penetrating peptides; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DIC, N,N′-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; Et 3N, triethylamine; Fmoc, 9-fluorenyl- methoxycarbonyl; HF, fluorhidric acid; HIV, human immunodeficiency virus; HOAc, acetic acid; HOBt, 1-hydroxy-1,2,3-benzotriazole; MALDI-TOF, matrix-assisted laser desorption ionization, time-of-flight; MBHA, p-methylbenzhydrylamine resin; MeCN, acetonitrile; RP-HPLC, reversed-phase high performance liquid chromatography; TBME, tert- butylmethyl ether; TFA, trifluoroacetic acid; TR-DX, Texas Red-Dextran. (2) (a) Lindgren, M.; Ha ¨llbrink, M.; Prochiantz, A.; Langel, U Ä . TiPS 2000, 21, 99-103. (b) Lundberg, P., Langel, U ¨ . J. Mol. Recognit. 2003, 16, 227- 233. 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