Solid-phase synthesis of peptide and PNA

PNA chain assembly was carried out on a rotary shaker N-500 manual peptide synthesizer (Kokusan Chemical, Tokyo, Japan) using TentaGel XV RAM resin at 5 μmol scale. Fmoc removal was carried out with a 20% piperidine solution in DMF for 5 min. Couplings were achieved using 3-fold excess of Fmoc-PNA monomer or Fmoc-L-amino acid activated with HATU (2.9 eq), DIEA (3 eq), and 2,5-lutidine (3 eq). The couplings were carried out at room temperature for 30 min. After each coupling steps, the capping of unreacted amino function was achieved using a solution of Ac₂O/lutidine/DMF (v/v/v; 5/6/89) for 5 min. Following completion of the 22-mer PNA, the resin was divided into two and one half was used to continue the synthesis of a cell penetrating peptide (ApoE: LRKLRKRLLR). The second half was acylated at the N-terminus with 3′-maleimidopropionic acid (3 eq) activated with HATU (2.9 eq) in the presence of DIEA (6 eq). Side-chain protecting groups and PNA/peptide were cleaved from the resin with a solution of TFA/TIS/H₂O (95/2.5/2.5, v/v/v) for 2 h at room temperature. After cleavage, the resin was removed by filtration, the filtrate was concentrated under a stream of nitrogen and the PNA product was precipitated in ice cold Et₂O and washed by centrifugation three times.