Reactions in bioorthogonal chemistry for detection of azide-modified sialic acid. (a) Two reactions involving bioorthogonal chemistry of azide-modified sialic acid (Sia) have been developed for in vivo imaging, the strain-promoted [3 + 2] cycloaddition, and the Staudinger ligation. In both cases a peracetylated analogue of N-acetylmannosamine (Ac4ManNAz) enters cells by enhanced passive diffusion and is deacetylated by intracellular carboxylesterases. The resulting ManNAz molecule is then converted to N-azidoacetyl sialic acid (SiaNAz) in the cytosol and transported to the nucleus, where it is activated with cytidine monophosphate (CMP) to form CMP-SiaNAz. CMP-SiaNAz is then incorporated into glycans as SiaNAz by the action of STs in the Golgi. The Staudinger ligation allows azide group detection of SiaNAz with a phosphine-substituted ester. The harmful byproduct nucleophilic aza-ylide is captured by intramolecular cyclization by an electrophilic trap (methyl ester) within the phosphine structure allowing it to be used in living animals without physiological harm because phosphine oxide is not produced. Regarding the strain-promoted [3 + 2] cycloaddition of azides the cyclooctyne molecule possesses a ring strain and electron-withdrawing fluorine substituents that together promote the [3 + 2] dipolar cycloaddition with azides to form regioisomeric mixtures of triazoles. This reaction occurs more rapidly than the Staudinger ligation, can be used in vivo, and does not require auxiliary reagents. First- and second-generation cyclooctyne (DIFO) compounds have been used in [3 + 2] cycloaddition. (b) Modification of phosphines or cyclooctynes with biotin and subsequent detection with fluorescent or radioisotope labelled avidins have been used for SiaNAz detection in vivo in animal models of lung cancer, a similar strategy could be employed using cyclooctynes.
