Team:Tuebingen/Notebook/Protocols/SyntheticPeptides

Synthetic Peptides

Peptides were obtained by solid-phase synthesis using the Fmoc strategy.
Cleavable TentaGel® R RAM beads from Rapp Polymers was used for synthesis. Deprotection of the Fmoc-protected amino group on the surface of the TentaGel®-Resin occurred twice with 1 % piperidine in dimethylformamide (DMF) for 30 min. Cysteine was activated with 1.2 equivalents of HBTU and added twice in 5-fold excess. The product was washed 5 times in DMF and 2 times in dichloromethane (DCM). Deprotection of the Fmoc-protected amino group of the coupled cysteine was performed like the deprotection of the TentaGel®-Resin. HBTU (1.2 eq.) activated lysine (Fmoc-Lys(Mtt)-OH) was coupled twice in 3-fold excess, with the second coupling taking place overnight. The 4-methyltrityl (Mtt) protection group of the washed product was removed by usage of 1 % trifluoroacetic acid (TFA) in DCM and flow-through method. The now unprotected ε-amino group of the lysine was coupled twice with 5-fold excess to HBTU activated carboxyfluorescein, whereas the second coupling occurred overnight. After extensive washing and deprotection of the α-amino group, the resin was transferred into a peptide synthesis automat and processed over two days. Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Ahx-OH were used for this purpose. Additionally added N-methyl-2-pyrrolidone (NMP) to the solvents increased the solubility of the amino acids and prevented precipitation in the machine.

The processed product was cleaved from the resin and unprotected from their side chain protection groups using a cleavage solution consisting of 670 mg of phenol, 450 μl of water, 450 μl of triisopropylsilyl (TIPS) and 9 ml of TFA. The resulting peptides were precipitated in cold diethyl ether and lyophilized overnight. Purification was executed by high-performance liquid chromatography (HPLC) and the resulting product was checked by mass spectroscopy using electron electrospray ionization (ESI).