Peptide synthesis and lipid conjugation.

Peptides were synthesized on rink amide 4-methylbenzhydrylamine (MBHA) resin using a standard solid-phase 9-fluorenylmethoxycarbonyl (FMOC) method, as described previously (36). For lipopeptides, each template peptide contains a lysine residue at its C terminus with a 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) side chain-protecting group, enabling the conjugation of a fatty acid group requiring a deprotection step in a solution of 2% hydrazinehydrate-N,N-dimethylformamide (DMF) (36); C34-Chol was produced by chemoselective thioether conjugation between the peptide precursor that has a C-terminal cysteine residue and the cholesterol derivative, as described previously (31). For fluorescein (FITC)-labeled peptides, FITC was conjugated to the N terminus of the peptides through a 6-aminohexanoic acid (AHX) linker. All peptides were N-terminally acetylated and C-terminally amidated, and they were purified by reverse-phase high-performance liquid chromatography (HPLC) to more than 95% homogeneity, followed by characterization with mass spectrometry.