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Abstract

Characteristic peptides, which have the same lipid modifications as their parent proteins and labels, by which they can be traced, for example, the biotin group, are efficient reagents for the study of signal transduction processes using lipid-modified Ras proteins Such peptide conjugates often contain both acid- and base-labile groups, and their synthesis calls for the application of protecting groups that can be removed selectively and under extremely mild conditions. These criteria are met by the enzyme-labile choline (Cho) ester group. Choline esters can be cleaved at pH 6.5 and room temperature by employing the enzyme butyrylcholine esterase from horse serum. By using this enzymatic protecting group technique as the key step, access to the characteristic S-palmitoylated and S-farnesylated C terminus of the human N-Ras protein was successfully achieved. The conditions under which these enzymatic deprotections proceed are so mild that no undesired side reaction is observed (that is no hydrolysis or beta elimination of the thioester and no acid-mediated attack on the double beads of the farnesyl group). In addition to this technique, the allyl ester group was removed by means of Pd-0-mediated allyl transfer to accepting nucleophiles. This reaction was exploited to construct biotin-labeled lipidated peptides, which correspond to the C terminus of N-Ras, and labeled analogues thereof. The lipidated and biotinylated peptides served as anchors for a protein moiety in an artifical membrane in a BIAcore surface plasmon resonance system. The stability of the membrane insertion was monitored by surface plasmon resonance after the binding of streptavidin to the biotin heads of farnesylated or farnesylated and palmitoylated peptides. Thus, it was shown that a double hydrophobic modification of the peptides is necessary to obtain stable insertion of the conjugates.