RESUMO
We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.
Assuntos
Acetilglucosamina/análogos & derivados , Corantes Fluorescentes/química , Proteínas/análise , Acetilglucosamina/análise , Acetilglucosamina/metabolismo , Animais , Biotina/análogos & derivados , Biotina/química , Células Cultivadas , Fluorescência , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Glicosilação , Células HeLa , Humanos , Neurônios/química , Neurônios/metabolismo , Proteínas/metabolismo , Proteômica/métodos , RatosRESUMO
Enantioenriched pyranones are important intermediates in the synthesis of natural products and the generation of compound libraries. A one-pot method for their synthesis is outlined. Catalytic asymmetric alkylation of 2-furfurals in the presence of catalytic (-)-MIB generates enantioenriched furyl zinc alkoxides. Addition of water/THF followed by NBS results in formation of pyranones with ee's >90% and yields between 46-77%.