494
Bioconjugate Chem. 2003, 14, 494−499
Simultaneous Lipidation of a Characterized Peptide Mixture by Chemoselective Ligation Line Bourel-Bonnet,* Dominique Bonnet, Fre´de´ric Malingue, He´le`ne Gras-Masse, and Oleg Melnyk* UMR 8525 CNRS-Universite´ de Lille 2-Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue du Pr. Calmette, BP 245, 59021 Lille, France. Received September 24, 2002; Revised Manuscript Received November 12, 2002
The modification of a peptide antigen by a fatty acid such as palmitic acid is now recognized as a mean to induce cellular responses. Mixtures of lipopeptides, obtained by combining individually synthesized compounds, were shown to be promising synthetic vaccine candidates. Usually, in lipopeptide synthesis, the fatty acyl moiety is introduced on the crude peptide chain using solid-phase methods. The separation of the target compound from impurities by RP-HPLC is often complicated by the amphiphilic properties of lipopeptides and results in low overall yields. To overcome the difficulties associated with lipopeptide synthesis and mixture preparation, we have developed a method where the fatty acyl moiety is site-specifically and collectively introduced in solution onto a mixture of individually prepurified peptides. The lipidation is based on the quasistoichiometric and highyielding ligation of a glyoxylyl lipid with hydrazinoacetyl peptides. The hydrazone constructs were prepared in a salt-free medium and could be isolated by direct lyophilization of the reaction mixture. This process is compatible with cysteinyl peptides, and no aggregation nor degradation could be observed.
INTRODUCTION
Synthetic vaccines composed of multiepitopic lipopeptides were found to induce virus-specific T-cell responses in primates (1-3) or in humans (4). Recently, the tolerance and the immunogenicity of 6 HIV-1-derived synthetic lipopeptides used in a phase I clinical trial confirmed the potential of this approach (5). Peptides can be easily modified by a palmitoyl group using standard solid-phase protocols as shown in Scheme 1 (6-8). An orthogonally protected lysine residue is inserted during the peptide assembly. Selective removal of the protecting group P′ is followed by acylation with a palmitic acid activated ester. Peptide deprotection and cleavage afford the crude lipopeptide which is then purified by preparative RP-HPLC. The main limitation of this approach is the last RPHPLC purification step, which is often tricky due to the generally low solubility of lipopeptides and to the line broadening induced by the fatty acid chain. Thus, not surprisingly, the major difficulty encountered during lipopeptides production is associated with the preparative RP-HPLC steps. Since the separation of the target compound from the byproducts can be achieved only with a limited column loading, a considerable part of the labor cost is devoted to the repetition of the chromatographic runs. The overall yields are generally modest for lab-scale productions (∼10% at 10-100 mg scale) and much lower on a 1 g scale (
