25(C-1) 溝呂木-ヘック反応を用いたタンパク質への新規炭素 : 炭素結合導入(口頭発表の部)

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The development of carbon-carbon (C-C) bond formation allows chemists to build various molecules with excellent yield and high selectivity under mild conditions. However no one has accomplished a novel C-C bond formation to the desired site of proteins without losses of their tertiary structures and innate properties. In this contribution, we have succeeded in the first site-specific introduction of a new functional group into the Ras protein using Mizoroki-Heck reaction. We firstly incorporated, 4-iodophenylalanine, a chemoselective tag to Mizoroki-Heck reaction into the nonsense codon of c-Ha-ras by using in vitro protein synthesis. Site-specific incorporation was determined by both LC/MS and LC/MS/MS analyses. Secondly, we optimized reaction conditions for the post-translational modification of proteins under physiological conditions. Glu-4-iodoPhe-Thr tripeptide as a model of Ras carrying 4-iodophenylalanine (Iphe-Ras) was used for this optimization experiments. The coupling reaction has been completely done at 5℃ within 93 hour. In addition, Ras was not decomposed under the same conditions, and still retained its binding activity against the Ras-binding domain of Raf-1 (Raf-1-RBD). Then, Iphe-Ras was subjected to Mizoroki-Heck reaction with vinylated biotin at 5℃ for 50 hour. The covalent attachment of biotin to Ras was detected with Western blotting and the undisturbed activity of Ras after biotinylation was demonstrated with in vitro binding assays. This is the first report that a new C-C bond has been introduced into the target sites of proteins. It is reported that small peptides are chemoselectively modified in aqueous media by using organometallic catalysts. However, full preparation is not committed for preventing potential degradation and retention of fragile tertiary structures when proteins are used as a substrate in organometallic reactions. We have shown that Mizoroki-Heck reaction is benign enough to maintain activities of proteins. Our developed methodology enables us to take more advantage of intelligent materials or devices mimicking subtle biological processes near future.
天然有機化合物討論会の論文
2004-10-01

25(C-1) 溝呂木-ヘック反応を用いたタンパク質への新規炭素 : 炭素結合導入(口頭発表の部)

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