Protocols

Synthetic Peptides

Peptides were obtained by solid-phase synthesis using the Fmoc strategy. Cleavable TentaGel® R RAM from Rapp Polymers was used for synthesis. Deprotection of the Fmoc-protected amino group on the surface of the TentaGel®-Resin was performed with 20% piperidine in dimethylformamide (2x15 min). Cysteine was activated with HBTU (0.97 eq.) 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 (0.97 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 was removed by washing the resin with 1 % trifluoroacetic acid (TFA) in DCM with gravity flow. Carboxyfluorescein was activated with HBTU(0.97 eq.) and coupled twice to the free ε-amino group of the lysine, whereas the second coupling occurred overnight. After extensive washing and deprotection of the α-amino group, the synthesis of the tag-specific region was performed automatically using an Invatis AG ResPep System. 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. N-methyl-2-pyrrolidone (NMP) was added to the solvents increasing 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 performed by high-performance liquid chromatography (HPLC) and the resulting product was checked by mass spectroscopy using electrospray ionization (ESI).