Qtof ms

Column chromatography was performed on 200–300 mesh silica gel (Qingdao Marine Chemical Co. Ltd., Qingdao, China). All purifications were monitored by TLC using commercially available glass plates pre-coated with silica gel 60 G (E. Merck Co., Billerica, Germany) and LC-6AD pHPLC (Shimadzu, Kyoto, Japan) with a UV SPD-20A detector using a reversed-phase C18 column (5 μm, 21.2 × 150 mm; Phenomenex Inc., Torrance, CA, USA). All the silica gel and GF₂₅₄ plates were treated with citrate buffer (pH 2.0). The analytical HPLC was performed using an Agilent LC1100 (Agilent Technologies, Santa Clara, CA, USA) equipped with a MWD detector using a reversed-phase C18 column (5 μm, 4.6 × 250 mm; Phenomenex Inc., Torrance, CA, USA). ¹H- and ¹³C-NMR data were all obtained on an Advance DRX-500 spectrometer (Bruker, Rheinstetten, Germany) in CDCl₃ with TMS as an internal standard. HRESIMS was performed on a QTOF-MS (AB SCIEX, Framingham, MA, USA). Anti-influenza virus H5N1 activity was assayed with a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, CAUSA).

FCLAPs were synthesized according to the standard Fmoc solid‐phase peptide synthesis protocols. The fidelity of the peptides was determined by a triple‐quadrupole time‐of‐flight mass spectrometry (Q‐TOF MS; AB SCIEX, Framingham, MA, USA). The purity was confirmed to be more than 95% by an Agilent 1200 high‐performance liquid chromatography system (HPLC; Agilent technologies, Santa Clara, CA, USA) with an analytical reverse‐phase Agilent ZORBAX SB‐Aq column (4.6 × 250 mm, 5 µm). The peptide detected at 220 nm was eluted with a gradient of acetonitrile (containing 0.1% trifluoroacetic acid) from 5% to 95% at a flow rate of 1 mL min⁻¹. Peptide theoretical MW, net charge, and hydrophobic ratio were analyzed using the online tool http://heliquest.ipmc.cnrs.fr/.