Synthesis and Characterization of Glycosylated Antimicrobial Peptide

Lasioglossin III (VNWKKILGKIIKVVK-NH₂) peptide and its glycosylated form g-LL-III were chemically synthesized by solid phase peptide synthesis (SPPS) and purified by reverse phase HPLC (RP-HPLC). All the Fmoc-protected amino acids were purchased from Novabiochem (Merck). The asparagine residue functionalized with N-γ-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-beta-D-glucopyranosyl) was purchased from IRIS Biotech. Piperidine, N,N-diisorpyplethylamine (DIEA) and triisopropylsilane (TIS) were purchased from Sigma-Aldrich, as well as the resin and the carboxyl group activators. Trifluoroacetic acid (TFA) and all the solvents used in the synthesis and purification were supplied by Romil. The coupling reagents HATU and HOBt were purchased from Anaspec. Dimethyl formamide (DMF) and N-methyl pyrrolidone (NMP) were used as chromatographic grade solvents and were purchased from Romil.
The lipids 1-palmytoil-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1-palmytoil-2-oleyl-sn-glycero-3-rac-phosphoglycerol (POPG) and 1,2-dipalmytoil-sn-glycero-3-phospocholine (DPPC) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA) and were used without further purification. Chloroform, methanol, ethanol and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich Chemical. The fluorescent probe Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphtylamine) and the fluorophore 1, 4-bis(5-phenyloxazole-2-yl)benzene (DM-POPOP) for lifetime experiments were purchased from Sigma Aldrich. The biophysical experiments were performed in a 10 mM phosphate buffer solution at pH 7.4.