4.3. Protocol of peptide synthesis

Synthesis of the protected linear peptide was conducted on a Protein Technologies Prelude automated peptide synthesizer using standard Fmoc solid-phase peptide chemistry. Synthesis was carried out using TCP-Resin pre-loaded with Fmoc-Thr(tBu)-OH (0.1 mmol scale). Coupling of all Fmoc-amino acids was performed using the same standard protocol 3 molar equivalents (relative to resin loading) of the Fmoc amino acid and the coupling reagent HCTU in DMF, with activation in situ using 6 molar equivalents of DIPEA. The reaction was left to proceed for 50 min at room temperature which was followed by washing of the resin with DMF. Fmoc deprotection was conducted using the protocol 20% piperidine in DMF (1 × 5 min, 1 × 10 min), followed by washing of the resin with DMF at room temperature. Removal of the ivDde protecting group was achieved with 3% hydrazine in DMF (4 × 15 min), followed by washing of the resin with DMF. The protected linear peptide was then cleaved from the resin by treating the resin with 10% hexafluoroisopropanol (HFIP) in DCM (1 × 30 min, 1 × 5 min). This solution was concentrated in vacuo to give the crude protected linear peptide. The protected linear peptide was dissolved in DMF (5 mL) to which DIPEA 0.6 mmol, 104 μL and DPPA, 0.3 mmol, 0.65 μL were added, and the resulting solution stirred for 6 h at room temperature. The cyclisation solution was concentrated under vacuum overnight and the resulting residue taken up in a solution of 2.5% EDT and 5% TIPS in TFA and stirred for 2 h at room temperature. To this solution, 40 mL of diethyl ether was added to precipitate the crude peptide. The resulting precipitate was collected by centrifugation, washed twice with diethyl ether (40 mL), then dried to give the crude cyclic peptide product as a white solid. The crude peptide was dissolved in Milli-Q water (5 mL), de-salted using a Vari-Pure IPE SAX column, then purified by RP-HPLC on a Waters Prep LC system with a Waters 486 tuneable absorbance detector (214 nm) and a Phenomenex Axia Luna C8(2), 250 × 21.2 mm i.d., 100 Å, 10 μm column. Peptides were eluted from the column with a gradient of 0–60% buffer B over 60 min at a flow rate of 15 mL min⁻¹; buffer A was 0.1% TFA/water, and buffer B was 0.1% TFA/acetonitrile.