Bioorthogonal Chemistry for Protein Labeling in Live Cells
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Chapter one provides an overview of bioorthogonal reactions commonly used in the chemical biology field. Chapter two describes the synthesis of a BODIPY-tetrazole and its use as an “off-on” reporter of hydrogen peroxide in live cells. The BODIPY–pyrazoline sensor is generated in situ in cellular systems using photoclick chemistry, and subsequent oxidation of the pyrazoline to a pyrazole serves as the basis for detection of hydrogen peroxide. Chapter three focuses on the optimization of photoclick chemistry in the context of the tetrazole structure. A sterically shielded nitrile imine was designed that favors the 1,3-dipolar cycloaddition over the competing nucleophilic addition. The utility of this sterically shielded nitrile imine in rapid (∼1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was demonstrated. Chapter four focuses on bioorthogonal modification of class B G-Protein Coupled Receptors (GPCRs), specifically the glucagon-like peptide 1 receptor (GLP-1R). A FRET-based approach was undertaken with a goal to study the biophysical properties on the native receptor in living cells. A strained alkene amino acid was site-specifically incorporated into the extracellular loops of GLP-1R using amber codon suppression. Dual labeling of GLP-1R was achieved using SNAP-tag technology along with bioorthogonal tetrazine ligation.