SYNTHESIS OF AN O-PALMITOYLATED 44-RESIDUE PEPTIDE AMIDE (PLTX-II) BLOCKING PRESYNAPTIC CALCIUM CHANNELS IN DROSOPHILA

PLTX II, a presynaptic calcium channel blocker in Drosophila isolated from the plectreurys spider venom, is a 44-residue peptide containing ten Cys residues and an O-palmitoylated threonine amide at the carboxy -terminus. In this study, the palmitoylated peptide was synthesized in solution by applying our maximum protection strategy using the HF met hod at the final deprotecting step. Before designing the synthesis, we examined the stability of the palmitoyl moiety under the conditions f or the synthesis of the peptide using several model peptides. The O-pa lmitoyl group was confirmed to be stable during elongation of the the peptide bonds, but was partially removable during the deprotection rea ction in HE The depalmitoylation reaction in HF was temperature- and t ime-dependent. Therefore, the decision was made to protect the Asp res idues with benzyl ester, since it is more susceptible to HF than cyclo hexyl ester, which is now commonly used in the Boc-based, solid-phase synthesis. Thus, the HF reaction was carried out at -10 degrees or -15 degrees C for 1 h in order to reduce the extent of the depalmitoylati on reaction. The resulting palmitoylated and depalmitoylated products were separated the remaining Acm groups were removed using Hg(OAc)(2), and then the completely deprotected peptides were folded to their nat ive forms. The final palmitoylated peptide was proven to be identical with the natural one using various HPLC systems and by bioassay. Both synthetic PLTX II and depalmitoylated PLTX II exhibited almost identic al CD spectra, indicating that removal of the palmitoyl moiety from th e PLTX II molecule did not cause significant conformational change, al though the depalmitoylated peptide was inactive even when administered to the assay system for PLTX II at four orders of magnitude higher th an the effective dose of the native PLTX II.