Photo-triggering of the metal-free azide to acetylene cycloaddition reaction was achieved by masking the triple bond of dibenzocyclooctynes as cyclopropenone. Such masked cyclooctynes do not react with azides in the dark. Irradiation of cyclopropenones results in the efficient (Φ355 = 0.33) and clean regeneration of the corresponding dibenzocyclooctynes, which then undergo facile catalyst-free cycloadditions with azides to give corresponding triazoles under ambient conditions. In-situ light activation of a cyclopropenone linked to biotin made it possible to label living cells expressing glycoproteins containing N-azidoacetyl-sialic acid. The cyclopropenone-based photo-triggered click chemistry offers exciting opportunities to label living organisms in a temporally and spatially controlled manner and may facilitate the preparation of microarrays.

Although metal free cycloadditions of cyclooctynes and azides to give stable 1,2,3-triazoles have found wide utility in chemical biology and material sciences, there is an urgent need for faster and more versatile bioorthogonal reactions. We have found that nitrile oxides and diazocarbonyl derivatives undergo facile 1,3-dipolar cycloadditions with cyclooctynes. Cycloadditions with diazocarbonyl derivatives exhibited similar kinetics compared to azides whereas the reaction rates of cycloadditions with nitrile oxides were much faster. Nitrile oxides could conveniently be prepared by direct oxidation of the corresponding oximes with BAIB and these conditions made it possible to perform oxime formation, oxidation and cycloaddition as a one-pot procedure. The methodology was employed to functionalize the anomeric center of carbohydrates with various tags. Furthermore, oximes and azides provide an orthogonal pair of functional groups for sequential metal free click reactions and this feature makes it possible to multi-functionalize biomolecules and materials by a simple synthetic procedure that does not require toxic metal catalysts.

We have shown that 4-Dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthetic 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, thereby providing 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, thereby facilitating 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 that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides that are being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells may have a means to selectively alter specific glycan structures of glycoproteins.

SEEL cell surface glycans N-Glycans of living cells could be selectively tagged by exogenously administered recombinant ST6Gal I sialyltransferase and azido-modified CMP-Neu5Ac followed by a strain promoted cycloaddition using biotin modified dibenzylcyclooctynol (DIBO). The methodology will make it possible to dissect the mechanisms that underlie altered glycoconjugate recycling and storage in disease and identify the glycoconjugates whose cell surface localization or secretion are affected.