4.4. Peptide Cleavage

Cleavage was performed on half or all of the resin with a TFA:H₂O:TIS (95:2.5:2.5) solution for 2 h. The cleavage solution was collected and concentrated with a gentle stream of N₂, down to about 300 µL. The peptide was precipitated and washed with 4 mL cold (−20 °C) diethyl ether, 3 times. The residual ether was left overnight to evaporate. Next, the crude product was dissolved in 90% water and 10% ACN and freeze-dried, ready for analysis and purification. Peptides were identified using Matrix Assisted Laser Desorption Ionization time of flight mass spectroscopy (MALDI–ToF MS). To this end, 1 µL peptide solution and 1 µL α-cyano4-hydroxycinnamic acid matrix (10 mg/ mL in ACN:H₂O:TFA, 50:47.5:2.5) were spotted onto a target plate, followed by detection using a MicroflexTM. FlexControl software was used to obtain the spectra and the data were processed using flexAnalysis [Bruker Daltonik GmbH]. RP–HPLC was used to assess peptide purity. The system consisted of a WatersTM In-Line Degasser AF, a WatersTM 600 pump, a WatersTM 2996 Photodiode Array Detector, and a WatersTM Symmetry C18, 4.6 × 250 mm, 5 mm column. The data were processed using Empower3 software. Purity was ≥95% for all tested peptide. Peptide purification was done by dissolving the peptides in minimal H₂O/can, and 300–500 µL was injected into the HPLC system to achieve separation.