2.1. Peptide synthesis and purification

Peptides were synthesised on two different resins: Rink Amide and HMBA-AM (applied Chem-Impex). Anhydrous DMF was obtained by distillation under vacuum and stored over 4 Å molecular sieves. The peptides were purified by preparative reversed-phase HPLC. Analytical and preparative HPLC were performed on LUNA C18 preparative (10 µm, 100 × 30 mm) or analytical (5 µm, 250 × 4.6 mm) columns, both from Phenomenex, Inc. (Torrance, CA). HPLC purification was carried out with an increasing linear gradient of CH₃CN in H₂O. Mass spectra were recorded on a QToF microspectrometer, using electrospray ionisation (ESI) in the positive/negative ion mode. Data were processed using mass L-ynX ver. 4.1 calculation and de-convolution software (Waters Corp., Milford, MA). HRMS were obtained using an LTQ Orbitrap.

Coupling of the first Fmoc (9-fluorenylmethoxycarbonyl)-protected amino acid was achieved with N,N′-diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine (DMAP) in dry DMF for 4 h. The remaining Fmoc-protected amino acids were coupled with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HBTU) in DMF in combination with N,N-diisopropylethylamine (DIPEA), for a 1.5 h cycle. Fmoc deprotection was achieved with piperidine. Side-chain deprotection was achieved by treating the peptide with 5 ml of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% H₂O. Peptide cleavage from the resin was achieved by treating the resin-bound peptides with 4 ml of 1:3 1 M NaOH:dioxane and then by 1 M HCl. The peptides were lyophilised and purified by preparative reversed-phase HPLC. The molecular mass of the peptides was determined by either MALDI-TOF or ESI mass spectrometry. The peptides that were synthesised on the HMBA-AM resin are listed below.

Pep1 – LR(Pbf)FF: ESI (m/z) [MH]⁺ 834.4; [MNa]⁺ 856.4.

Pep2 – D(OBtu)PLR(Pbf)FF: ESI (m/z) [MH]⁺ 1102.5; [MNa]⁺ 1124.5; [MK]⁺ 1140.5.

Pep3 – DPLRFF: ESI (m/z) [MH]⁺ 794.4; [MNa]⁺ 816.5; [MK]⁺ 832.5.

Pep4 – PLR(Pbf)FFD(OBtu): ESI (m/z) [MH]⁺ 1102.4; [MNa]⁺ 1124.4; [MK]⁺ 1140.0.

Pep5 – FFRLPD: ESI (m/z) [MH]⁺ 794.4.

Coupling of Fmoc-protected amino acids was achieved by using HBTU in DMF in combination with DIPEA, for a 1 h cycle. Fmoc deprotection was conducted with piperidine. N-terminus acetylation was carried out by using 10 ml of the acetylation solution, which contains 19 ml of acetic anhydride, 9 ml DIPEA, 6 mmol of HOBt, and 72 ml of NMP (N-methyl-2-pyrrolidone), for 35 min. This procedure was carried out twice. A 6-(Fmoc-amino)hexanoic acid (4 eq), which in some peptides were used as a linker, was coupled under HOBt (3 eq), Bop (3 eq), and DIPEA (6 eq) treatment in DMF for 1.5 h. Side-chain deprotection and peptide cleavage from the resin were carried out by treating the resin-bound peptides with 5 ml of 95% TFA, 2.5% TIS, and 2.5% H₂O. The peptides were washed three times with cold diethyl ether, vortexed, and then centrifuged for 5 min at 3500 rpm. The peptides were then purified by preparative reversed phase HPLC. The mass of the peptide was confirmed either by MALDI-TOF MS or by ESI mass spectrometry. The peptides that were synthesised using Rink amide resin are listed below.

Pep6 – Ac-LRFF-NH₂: ESI (m/z) [MH]⁺ 623; [MNa]⁺ 645^; [MK]⁺ 661.

Pep7 – Ac-LRFFDK-NH₂: ESI (m/z) [MH]⁺ 866.4.

Pep8 – Ac-DFFRLP-NH₂: ESI (m/z) [MH]⁺ 835.6.

Pep9 – Ac-FFRL-NH₂: ESI (m/z) [MH]⁺ 623.4.

Pep10 – Ac-KDFFRL-NH₂: [MH₂]²⁺ 433.8; [MH]⁺ 866.4.

Pep11 – Ac-FFRLPD-NH₂: ESI (m/z) [MH]⁺ 835.6.

Pep12 – Ac-PLR-NaI-D-NH₂: ESI (m/z) [MH]⁺ 738.4.

Pep13 – Ac-PLRFAD-NH₂: ESI (m/z) [MH]⁺ 759.4.

Pep14 – Ac-PLRAFD-NH₂: ESI (m/z) [MH]⁺ 759.4; [MNa]⁺ 781.4.

Pep15 – Ac-HKKIHKK-NH₂: ESI (m/z) [MH₃]³⁺ 320.7; [MH₂]²⁺ 480.5; [MH]⁺ 959.7.

Pep16 – Ac-NTLDPK-NH₂: ESI (m/z) [MH]⁺ 728.5.

Pep17 – Ac-PLAPYD-NH₂: ESI (m/z) [MH]⁺ 851.4.

Pep18 – Tetrzaol-FFRLP-NH₂: ESI (m/z) [MH]⁺ 788.3; [MNa]⁺ 810.4.

Pep19 – Ac-LRAA-NH₂: ESI (m/z) [MH]⁺ 471.