The peptide chain was synthesized by SPPS on Rink Amide MBHA resin. Each amino acid coupling step was carried out with Fmoc-protected amino acid (2 eq), DIC (4 eq), ethyl cyanohydroxyiminoacetate (2 eq) in DMF at 75 °C (155 W) for 0.25 min and at 90 °C (30 W) for further 2 min. For the penultimate lysine, the coupling was carried out using Fmoc-Lys(Boc)-OH (2 eq), DIC (4 eq), ethyl cyanohydroxyiminoacetate (2 eq) at 75 °C (155 W) for 0.5 min and at 90 °C (30 W) for further 4 min. For the last lysine, the coupling was carried out using Fmoc-Lys(Mtt)-OH (3 eq), DIC (6 eq), ethyl cyanohydroxyiminoacetate (3 eq) at 75 °C (155 W) for 1 min and at 90 °C (30 W) for further 7 min. After each coupling step, the respective Fmoc protecting group was removed by 10% Piperazine (w/v) in ethanol:NMP (1:9). After the final Fmoc deprotection, the peptidyl-resin was removed from the automated synthesizer and the N-terminal amine capped using acetic anhydride (50 eq) and pyridine (50 eq) in 8 mL of DMF, for 30 min for 2 times. For the Mtt group cleavage, the resin was treated with 1% TFA (v/v) in DCM for 2 min and then the solution filtrated off; this procedure was repeated 12 times. Then, glutamic acid was coupled using 4 eq of Fmoc-Glu(OtBu)-OH, 4 eq of (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 4 eq of hydroxybenzotriazole (HOBt) and 8 eq of N,N-diisopropylethylamine (DIPEA). Deprotection of the Fmoc protecting group was achieved by treatment with 5 mL of 20% (v/v) piperidine in DMF, for 10 min for 2 times. For the pteroic acid coupling, 2 eq of pteroic acid were suspended in 10 mL of DMSO and heated at 50 °C for 1 h. 8 eq of DIPEA and 2 eq of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) were added to the suspension which was then reacted with the peptidyl-resin for 16 h, at 40 °C. The pteroic acid coupling was then repeated for a further 16 h, at 40 °C. Cleavage from the resin was performed in TFA/H2O/TIS 95:2.5:2.5 solution for 2 h. The resin was removed by filtration and the filtrate concentrated by N2 flow. The crude product was precipitated with cold diethyl ether, centrifuged (4,000 g for 10 min) and the supernatant discarded. The precipitate was dissolved in 2 mL of CH3CN + 0.1% TFA, diluted with 13 mL of H2O + 0.1% TFA, filtrated through a 0.22 µm syringe filter and purified by RP-HPLC.