title: 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.
authors.affiliation: Department of Biophysics and Biochemistry, School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kodama
authors.forename: Seketsu
authors.initials: S
authors.lastname: Fukuzawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakayama
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Natsuko
authors.initials: N
authors.lastname: Matsuda
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Koji
authors.initials: K
authors.lastname: Takio
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tachibana
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D002244:Carbon / Q000737:chemistry*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry / Q000379:methods; D009942:Organometallic Compounds / Q000737:chemistry*; D010165:Palladium / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D009942:Organometallic Compounds; D011506:Proteins; D010165:Palladium; D002244:Carbon