A Photoactivatable Glycoenzyme Platform for Spatiotemporally Controlled, Protein-Selective, and In Vivo Glycan...

Precise control of cell-surface glycosylation remains challenging due to the dynamic and spatially complex nature of glycans. Here, we present GLOBE, a platform for optical regulation of glycoenzyme activity via site-specific incorporation of photocaged unnatural amino acids. Using galactose oxidase (GAO) as a model, o-nitrobenzyl-tyrosine (ONBY) installed at a proximal tyrosine suppresses activity until photoactivation, enabling rapid cell-surface glycan oxidation with micrometer-scale spatial and temporal resolution. Coupling GAO with MUC1-targeting aptamer produces binding-gated, protein-selective glycan oxidation, achieving precise control in mixed cellular populations. GLOBE is further extended to in situ suppression of a sialidase catalytic domain, and integrated into an orthogonal photoresponsive cascade, where sialic acid removal exposes substrates for subsequent GAO oxidation, enhancing labeling efficiency. Finally, upconversion nanoparticle-assisted photoactivation enables in vivo glycan editing in a murine tumor model, overcoming UV tissue penetration limits. Together, GLOBE provides a modular, spatiotemporally programmable framework for probing and manipulating dynamic glycosylation at single-cell and tissue levels, offering a versatile strategy to interrogate and engineer the complex sugar code underlying biological and pathological processes.