Regioselective Carbon–Carbon Bond Formation in Proteins with Palladium Catalysis; New Protein Chemistry by Organometallic Chemistry

Abstract

Palladium‐catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki–Heck reaction for site‐specific carbon–carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His₆‐fused Ras protein containing 4‐iodo‐L‐phenylalanine at position 32 (iF32‐Ras‐His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32‐Ras‐His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography–mass spectrometry (LC‐MS). The regioselectivity of the Mizoroki–Heck reaction was furthermore confirmed by LC‐MS/MS analysis. However, in addition to the biotinylated product (bF32‐Ras‐His), a dehalogenated product (F32‐Ras‐His) was detected by LC‐MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki–Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras‐binding domain as its downstream target, Raf‐1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.