Affinity-Guided Oxime Chemistry for Selective Protein Acylation in Live Tissue Systems

  • Tomonori Tamura
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
  • Zhining Song
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
  • Kazuma Amaike
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
  • Shin Lee
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
  • Sifei Yin
    Magdalene College, University of Cambridge, Cambridge CB3 0AG, United Kingdom
  • Shigeki Kiyonaka
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
  • Itaru Hamachi
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

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説明

Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.

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