Synthesis of potential cell-penetrating peptide nucleic acid conjugates

Abstract

In this work, polycationic peptides and a lipophilic phosphonium, namely (KFF)₃K R₁₀, and phosphonium cation (P⁺) has been evaluated for their cellular deliver of PNA to E. coli ATCC 25922 cells. Three model PNA conjugates, Flu-O-T₃-O-(KFF)₃K-NH₂, Flu-O-T₃-O-R₁₀-NH₂ and Flu-O-T₃-O-Lys-(P⁺)-NH₂ have been successfully prepared by sequential coupling using standard solid phase synthesis. Fluorescence techniques were used to determine the cell penetration of the PNA conjugates into the cells. It was demonstrated that (KFF)₃K was a better carrier than R₁₀ and photphonium cation. In addition, the cell growth rates showed that Flu-O-T₃-O-(KFF)₃K-NH₂ and Flu-O-T₃-O-Lys-(P⁺)-NH₂ had no cytotoxicity. By contrast, Flu-T₃-R₁₀ conjugate should be considered as somewhat toxic because a reduced rate of cell growth was observed. The PNA length requirement has also determined using a longer PNA conjugate namely Flu-O-T₉-O-(KFF)₃K-NH₂. The results demonstrated that despite the increased length of the PNA part, the uptaking ability of Flu-O-T₉-O-(KFF)₃K-NH₂ was better than Flu-O-T₃-O-(KFF)₃K-NH₂. To determine the importance of the position of attachment of (KFF)₃K, H-(KFF)₃K-T₉-Lys(Flu)-NH₂ were also prepared. No conclusion can be made with H-(KFF)₃K-O-T₉-Lys(Flu)-NH₂ possibly due to precipitation or degradation under the assay conditions. The stability of a model PNA: Ac-T₉-Lys-NH₂ towards a model proteolytic enzyme: proteinase K has been evaluated. The control: ACTH 4-10 was completely digested under condition 0.15 units/mL of proteinase K at 37℃ whereas the PNA remained stable over 18.5 h at 15 units/mL protenase K at 37℃