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

✍ Scribed by Tamara A. Kale; Conrad Raab; Nathan Yu; Evelyn Aquino; Dennis C. Dean; Mark D. Distefano

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: French
• Weight: 321 KB
• Volume: 46
• Category: Article
• ISSN: 0022-2135
• DOI: 10.1002/jlcr.638

✦ Synopsis

Abstract

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 [^35^S]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 [^35^S]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 [^35^S]3 can cross‐link GDI in both purified form and crude bacterial extract. However, the extent of cross‐linking obtained with the ester ([^35^S]3) is significantly less than that observed with the free acid ([^35^S]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. Copyright © 2002 John Wiley & Sons, Ltd.