Waters 2545

Following the Fmoc method and employing HATU-collidine as coupling reagents, peptides were synthesized on solid-phase on Rink-4-methylbenzhydrylamine (MBHA) resin (loading 0.4–0.8 mmol/g) [32 (link)]. For peptide cleavage, a solution of TFA, TIS, and water (95:2.5:2.5, v/v/v) were stirred for three hours at room temperature. The peptides were then precipitated using cold diethyl ether, the pellets were reconstituted in a 75:25 v/v solution of H₂O and CH₃CN and lyophilized. Reverse-phase HPLC (RP-HPLC) was used to purify the crude peptides using a WATERS 2545 preparative system (Waters, Milan, Italy) outfitted with a WATERS 2489 UV/Vis detector. Using a Jupiter C18 (5 μm, 150 × 21.2 mm ID) column, the purification step was carried out at 15 mL/min while monitoring the absorbance at 214 nm. A linear gradient of 0.1% TFA in CH₃CN from 5% to 70% was applied for 15 min.

Crude peptides were purified by semipreparative RP-HPLC-UV-MS using a system composed by a binary gradient Waters 2545, a Waters Alliance 2767 sample manager module and an automatic fraction collector coupled to Waters 2487 dual UV-vis absorbance detector and an electrospray ion source (ESI-MS) Micromass ZQ mass spectrometer detector. The chromatographic separation of the peptides was achieved using a semipreparative column XBridge^® Prep BEH C₁₈ (19 × 100 mm, 5 µm) (Waters, Cerdanyola del Vallès, Spain) with dual solvent system formed by A: 0.1% TFA in H₂O and B: 0.1% TFA in CH₃CN at a flow rate of 25 mL/min. λ = 220 nm. Elution gradient was specific for each peptide.