We have shown that 4-dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthesis approach, reacts exceptionally fast in the absence of a CuI catalyst with azido-containing compounds to give stable triazoles. Chemical modifications of DIBO, such as oxidation of the alcohol to a ketone, increased the rate of strain promoted azide–alkyne cycloadditions (SPAAC). Installment of a ketone or oxime in the cyclooctyne ring resulted in fluorescent active compounds whereas this property was absent in the corresponding cycloaddition adducts; this provides the first example of a metal-free alkyne–azide fluoro-switch click reaction. The alcohol or ketone functions of the cyclooctynes offer a chemical handle to install a variety of different tags, and thereby facilitate biological studies. It was found that DIBO modified with biotin combined with metabolic labeling with an azido-containing monosaccharide can determine relative quantities of sialic acid of living cells that have defects in glycosylation (Lec CHO cells). A combined use of metabolic labeling/SPAAC and lectin staining of cells that have defects in the conserved oligomeric Golgi (COG) complex revealed that such defects have a greater impact on O-glycan sialylation than galactosylation, whereas sialylation and galactosylation of N-glycans was similarly impacted. These results highlight the fact that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells might have a means to selectively alter specific glycan structures of glycoproteins.