Synthesis and Characterization of EAK-YIGSR Peptide

The synthesis, purification and characterization of EAK-YIGSR were performed as previously reported [29 (link)]. Briefly, the peptide EAK-YIGSR (sequence: H-Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys-Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys-Tyr-Ile-Gly-Ser-Arg-NH₂) was synthesized on Rink Amide MBHA resin (0.52 mmol/g) using Fmoc chemistry by a Syro I synthesizer (Multisyntech, Witten, Germany). The side-chain protecting groups were: OtBu, Glu; Boc, Lys; Pbf, Arg; tBu, Ser and Tyr. All the couplings were double. After Fmoc-deprotection, the peptide was deblocked from the resin and deprotected from side-chain protecting groups using the mixture 1,9 mL TFA, 0.05 mL TES, 0.05 mL H₂O, for 1.5 h. The resin was filtered off and the solution was concentrated. The product was precipitated with diethyl ether and filtered. The identity of the crude peptide was determined by MALDI mass spectrometry (exp. mass = 2192.06 Da; theor. mass = 2191.45 Da; AB-SCIEX TOF-TOF 4800 instrument). The peptide EAK-YIGSR was purified by RP-HPLC and characterized by analytical RP-HPLC (conditions: Nova-Pak HR C₁₈ column (4 μm, 60 Å, 3.9 × 300 mm, Waters), eluent A: 0.05% TFA in H₂O; eluent B: 0.05% TFA in CH₃CN; gradient: from 18 to 26% di B in 24 min, flow rate: 1 mL/min; detector: 214 nm. t_R = 10.68 min). The integration of the chromatogram gave a 99% purity grade.