Peptide synthesis and purification

The hBD-3 analog peptides of 45 amino acids were synthesized using Fmoc chemistry. Two isotope-labeling schemes were used to provide site-specific resolution and ensure coverage of the whole span of the peptide sequence. The VALIG sample contains ¹³C, ¹⁵N-labeled residues V13, A19, L21, I30, and G37, while IVLG sample contains ¹³C, ¹⁵N-labeled residues I3, V20, L24, and G31. ¹³C,¹⁵N-labeled amino acids were protected by Fmoc in lab, with purity of > 90% as monitored by ¹H solution-NMR spectroscopy. The peptides were synthesized using Fmoc solid-phase methods using 0.1 mM scale with five-fold excess input for the labeled residues to ensure efficiency in the double couplings. The Fmoc group was then removed with 20% piperidine twice (3 min and 10 min) at room temperature, and the resin was washed with DMF and DCM. The peptide was cleaved from the resin and sidechain deprotected using TFA/EDT/water (4 mL, 94:3:3) for 5 hr. The cleavage reaction was repeated for 10 min. The crude peptide was then purified by HPLC. Eluents for A is 0.1% TFA in water and that for B is 0.1% TFA in acetonitrile. MALDI–TOF analysis was performed to verify the molecular weight of peptides. In total, 5184.8 Da for VALIG and 5181.0 Da for IVLG are the match to the calculated molecular weight. The purity is higher than 98% for both peptides.