Strain-promoted bioorthogonal cycloadditions to investigate sialyltransferases promiscuity towards unnatural sialic...

Bioorthogonal chemical reporters have revolutionized the study of glycosylation by enabling the selective labeling, visualization, and enrichment of glycans in living systems. Monosaccharide analogs bearing small, inert bioorthogonal tags such as azides, alkynes, cyclopropenes or sydnones, are metabolically incorporated into glycoconjugates by the cell's endogenous glycosyltransferases (GTs). Subsequent chemoselective ligation (e.g., strain-promoted alkyne-azide cycloaddition or inverse electron-demand Diels-Alder reactions) permits downstream detection or pull-down with minimal perturbation to cellular physiology. This chapter demonstrates the impact of a sydnone bioorthogonal chemical reporter on sialyltransferases (STs) substrate tolerance and catalytic efficiency. While many STs display surprising promiscuity toward modified CMP-sialic acid donors, incorporation efficiencies vary widely depending on the position and steric demand of the bioorthogonal handle, and can alter kinetic parameters, and influence donor/acceptor specificity. Herein, we present detailed protocols for assessing the impact of bioorthogonal chemical reporters, with a focus on 1,3-dipoles, on the activity of recombinant human sialyltransferases. The methods include qualitative evaluation by in-gel fluorescence imaging and quantitative analysis through determination of enzyme-specific kinetic parameters. These protocols can be extended to other glycosyltransferases collectively expanding the enzymatic toolkit for glycobiology, enabling additional mechanistic studies in the interrogation of glycosylation dynamics and GT function.