Prelude automated peptide synthesizer

Fully protected linear octapeptide was prepared via solid phase peptide synthesis (SPPS) on a Prelude Automated Peptide Synthesizer™ (Protein Technologies Inc.) using 2-chlorotrityl chloride resin as the solid support. The first Fmoc-protected amino acid was coupled to the resin using DIPEA (4 eq.) in DMF, followed by capping of unreacted resin sites using a solution of MeOH : DIPEA : DCM (7 : 1 : 2, v/v/v). Deprotection of the Fmoc group of the amino acids was done using 20% piperidine in DMF. Subsequent amino acids were coupled using Fmoc-amino acids (5 eq.), HCTU (5 eq.) and NMM (10 eq.) in DMF. In the last step, the linear octapeptide was cleaved from the resin (while keeping protecting groups on) by a solution of 20 vol% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM. Yield quantitative.

Overlapping 15-mer synthetic peptides corresponding to the matured sequence of sNTX (accession: P60770) and CTX A3 (accession: P60301) were synthesized by the solid-phase method using a Prelude automated peptide synthesizer (Protein Technologies, Inc., Tucson, AZ, USA). In the process, α-amino groups were protected by the fluorenylmethoxycarbonyl group and the final de-blocking step was carried out with a mixture of TFA/triisopropylsilane/phenol/water/1,2-ethanedithiol. The crude peptide was precipitated with ether and further purified by reverse-phase HPLC. Purity was ensured to be higher than 90% by optical detector (UV 214 nm) performed on an Agilent 1100 Series LC/MSD high-performance ion-trap mass spectrometer.