4.4. Solid-Phase Peptide Synthesis

The replicate was synthesised by solid-phase Fmoc chemistry using Rink amide resin in a Tribute automated peptide synthesiser (Protein Technologies, Tucson, AZ, USA) when the unequivocal primary structure of the novel peptide had been confirmed. The reaction involved deprotection of the Fmoc groups from the amino acids and coupling of peptide bonds. When the synthesis cycles were completed, the peptide was cleaved from the resin using trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (95/2.5/2.5, v/v/v) for 25 mL/g resin. The authenticity of the purified synthetic PSN-PC was verified by RP-HPLC with an analytical Jupiter C5 column (250 mm × 4.6 mm, Phenomenex, UK). The sample was eluted with a linear gradient from water/TFA (99.95/0.05, v/v) to acetonitrile/water/TFA (80/19.95/0.05; v/v/v) in 40 min at a flow rate of 1 mL/min. The degree of purity and authentication of the purified synthetic peptide was determined by RP-HPLC (Cecil, Cambridge, UK) and MALDI-TOF MS (Voyager DE, Perspective Biosystems, Foster City, CA, USA) as previously described [36].