Peptide Synthesis
Starting from the Fmoc-Lys(Boc)-OCH2-Wang resin (0.175 g, 0.1 mmol, 0.57 mmol/g), the peptide was elongated by CEM Liberty Blue using a 0.1 mmol scale protocol to give an Fmoc-AAGGGAAAAK(Boc)-resin. The resulting resin was divided into two portions and one was used for the further synthesis to obtain the Fmoc-AAAGGGAAAAK-resin (P1 peptide). For the other, Fmoc-C(Trt)-OH (0.25 mmol act by HBTU 0.25 mmol, DIEA 0.5 mmol) was reacted for 30 min at 50 ºC to give the Fmoc-C(Trt)AAGGGAAAAK(Boc)-resin (P2 peptide).
Starting from the Fmoc-Arg(Pdf)-OCH2-PEG-resin (0.23 g, 0.05 mmol, 0.22 mmol/g), the peptide chain was elongated by CEM to obtain Ac-GAN(Trt)APK(Boc)E(OBut)PQ(Trt)R(Pbf)-OCH2-PEG-resin (P4 peptide). Starting from the Fmoc-Lys(Boc)-OCH2-Wang resin (0.0875 g, 0.05 mmol, 0.57 mmol/g), the peptide chain was elongated by CEM to obtain NH2-N(Trt)C(Trt)AGH(Trt)K(Boc)-OCH2-Wang-resin (P3 peptide).
The deprotected P1 peptide (AAAGGGAAAAK) anchored to the dried resin was stirred with a 20 % piperidine solution at room temperature for 30 minutes, washed with DMF and dried under a vacuum. The peptide-resin was treated with an excess of the non-cleavable heterobifunctional protein cross-linker BMPS ((N-β-maleimidopropyloxy)succinimide ester) from MERCK dissolved in DMF spiked with traces of trimethylamine and the reaction proceeded during a period of 30 minutes. The resin was washed with DMF and dried under a vacuum. In the case of P4, it was dissolved in 100 mM Tris/HCl buffer, pH=6.8 and treated with an excess of a solution of BMPS in DMF. The reaction proceeded for 30 minutes and the P4 peptide modified with a maleimide group at the ε-amino group of Lysine residue was purified by rp-HPLC.
All peptides anchored to the resin were deprotected and cleaved from the resin by treatment with a mixture containing TFA:H2O:TMS in a ratio of 95:2.5:2.5 (v/v). The peptides were precipitated with diethyl ether, the excess diethyl ether was removed by centrifugation and the peptide pellet was dried under a stream of nitrogen. These peptides were dissolved in H2O/TFA (0.1 % v/v) and purified by rp-HPLC.
As a result, four peptides were synthesized: P1 (*AAAGGGAAAAK), P2 (CAAGGGAAAAK), P3 (NCAGHK), and P4 (Ac-GANAPK*EPQR). Asterisks in the sequences of P1 and P4 peptides mean that their primary amino groups were modified with an N -propionyl maleimide group. Ac- in the P4 peptide sequence indicates that an acetyl group is located at theN -terminal end.
Cross-linked peptides were synthesized by the maleimide-thiol chemistry1,2. Approximately equal amounts of the free Cys containing peptides (P2 and P3) and the maleimide N -propionyl maleimide-containing peptides (P1 and P4) were dissolved at equal concentrations in 100 mM Tris/HCl, pH=6.8 and mixed in the correct combinations to synthesize four cross-linked peptides with the thiosuccinimide linker. These compounds are referred to hereafter as (P1-P2), (P1-P3), (P3-P4), and (P2-P4). The reactions proceeded during 20 minutes at room temperature with stirring. The final products of the reactions were analyzed by rp-HPLC and the completion was confirmed by the fact that one of the starting peptides had been completely consumed. The new generated peak fractions that potentially contained the desired product were collected and directly analyzed by MALDI-MS.