title: Chemical cell-surface receptor engineering using affinity-guided, multivalent organocatalysts.
abstract: Catalysts hold promise as tools for chemical protein modification. However, the application of catalysts or catalyst-mediated reactions to proteins has only recently begun to be addressed, mainly in in vitro systems. By radically improving the affinity-guided DMAP (4-dimethylaminopyridine) (AGD) catalysts that we previously reported (Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 245.), here we have developed a new organocatalyst-based approach that allows specific chemical acylation of a receptor protein on the surface of live cells. The catalysts consist of a set of 'multivalent' DMAP groups (the acyl transfer catalyst) fused to a ligand specific to the target protein. It was clearly demonstrated by in vitro experiments that the catalyst multivalency enables remarkable enhancement of protein acylation efficiency in the labeling of three different proteins: congerin II, a Src homology 2 (SH2) domain, and FKBP12. Using a multivalent AGD catalyst and optimized acyl donors containing a chosen probe, we successfully achieved selective chemical labeling of bradykinin B(2) receptor (B(2)R), a G-protein coupled receptor, on the live cell-surface. Furthermore, the present tool allowed us to construct a membrane protein (B(2)R)-based fluorescent biosensor, the fluorescence of which is enhanced (tuned on) in response to the antagonist ligand binding. The biosensor should be applicable to rapid and quantitative screening and assay of potent drug candidates in the cellular context. The design concept of the affinity-guided, multivalent catalysts should facilitate further development of diverse catalyst-based protein modification tools, providing new opportunities for organic chemistry in biological research.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors.forename: Hangxiang
authors.initials: H
authors.lastname: Wang
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Koshi
authors.forename: Daishiro
authors.initials: D
authors.lastname: Minato
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nonaka
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kiyonaka
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
authors.forename: Shinya
authors.initials: S
authors.lastname: Tsukiji
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
mesh_terms: D015761:4-Aminopyridine / Q000031:analogs & derivatives* / Q000737:chemistry; D015374:Biosensing Techniques; D002384:Catalysis; D037161:Galectins / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D015202:Protein Engineering; D043782:Receptor, Bradykinin B2 / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry*
chemical_list: D037161:Galectins; D008024:Ligands; D043782:Receptor, Bradykinin B2; C122080:congerin II protein, Conger eel; D015761:4-Aminopyridine; D022061:Tacrolimus Binding Protein 1A; C003885:4-dimethylaminopyridine