Synthesis of high specific activity ³⁵S-labelled N-methanesulfonyl farnesylcysteine and a photoactive analog

Prenylated cysteine analogs, which mimic the prenylated cysteine residue of prenylated GTP-binding proteins (G-proteins), have been used in a variety of contexts for the study of prenylated G-protein behavior. In earlier work in this area, we prepared the photoactive analog [³⁵S]4 and showed that it labelled RhoGDI upon photolysis; those results were consistent with the idea that GDI contains an isoprenoid binding site. Here, we describe the preparation of [³⁵S]N-methanesulfonyl labelled analogs (1a and 2a) of N-acetyl farnesylcysteine and its methyl ester together with an improved synthetic procedure for photoactive analogs 3 and 4; specific activities of ∼1100 Ci/mmol were achieved. Compounds 1a and 2a in unlabelled form were used as competitors in photolysis reactions to show that the methanesulfonamido group is a reasonable acetamide substitution. Additional experiments show that the photoactive ester [³⁵S]3 can cross-link GDI in both purified form and crude bacterial extract. However, the extent of cross-linking obtained with the ester ([³⁵S]3) is significantly less than that observed with the free acid ([³⁵S]4) despite the fact that the esterified form probably more closely reflects the structure of the C-terminus of a prenylated protein; using the GDI·Cdc42 co-crystal structure, the structural basis for these results is discussed.