Cell penetrating peptides (CPPs) containing unnatural α,α-disubstituted amino acid form the stable amphiphilic helical structures and show high cell penetrating ability. In the present study, we synthesized CPPs containing lysine and α-aminoisobutylic acid (Aib) by using chemoenzymatic polymerization and examined the secondary structures and the cell penetrating activities. The chemoenzymatic reaction of tripeptides (Lys-Aib-Xxx, Xxx = Glysine, Alanine, Leucine, Lysine, Phenylalanine) was performed using papain or protenase K in a buffer at 40-60 °C for 1 h. We analyzed the properties of the obtained peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and circular dichroism (CD) spectroscopy. The peaks assigned to the dimer and trimer of the Lys-Aib-Xxx unit were observed, indicating the successful polymerization of Lys-Aib-Xxx esters using papain and proteanse K. We measured the CD spectroscopy of the obtained peptides in various buffer solutions. The CD spectra did not show any specific secondary structures in phosphate buffer (pH 8.0), whereas these peptides form the helical-rich secondary structures in 0.1% sodium dodecyl sulfate solution. This result indicates that these peptides can form the suitable helical-rich secondary structures to penetrate the cell membrane in a physiological condition. Finally, we evaluated the cell penetrating activity of the obtained peptides by confocal microscopic observation. Cell penetrating activity of the novel peptides was confirmed by the internalization of fluorescence dye, which is connected to the N-terminal of the peptides (Figure), in human embryonic kidney 293 (HEK) cells.

Synthesis of cell penetrating peptide containing α-aminoisobutylic acid using chemoenzymatic polymerization

256th National Meeting (2018)

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