Imaging Cell Surface Glycosylation in Vivo Using “Double Click” Chemistry

[Image: see text] Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell–cell communication and cell migration. We report here imaging of cell surface glycosylation in live mice using double click chemistry. Cell surface glycans were metabolically labeled using peracetylated azido-labeled N-acetylgalactosamine and then reacted, in the first click reaction, with either a cyclooctyne, in a Huisgen [3 + 2] cycloaddition, or with a Staudinger phosphine, via Staudinger ligation. The second click reaction was a [4 + 2] inverse electron demand Diels–Alder reaction between a trans-cyclooctene and a tetrazine, where the latter reagent had been fluorescently labeled with a far-red fluorophore. After administration of the fluorescent tetrazine, the bifunctional cyclooctyne-cyclooctene produced significant azido sugar-dependent fluorescence labeling of tumor, kidney, liver, spleen, and small intestine in vivo, where the kidney and tumor could be imaged noninvasively in the live mouse.