Photocrosslinking sialic acid (SiaDAz)

While we have previously prepared SiaDAz synthetically and added it to cultured cells, we find that it is more practical to use an analog of ManNAc, which is a committed precursor of Neu5Ac synthesis. The ManNAc analog (Ac4ManNDAz) contains the diazirine substituent and also has acetyl-protecting groups on all hydroxyl groups. These protecting groups reduce the polarity of the compound, allowing Ac4ManNDAz to readily enter cells where it is deprotected to ManNDAz. Enzymes in the sialic acid biosynthetic pathway convert ManNDAz to CMP-SiaDAz, and add SiaDAz to glycoproteins and glycolipids in place of other sialic acids, like Neu5Ac. Cells can then be subjected to UV (~350 nm) irradiation, resulting in covalent crosslinking between SiaDAz-containing glycoconjugates and nearby molecules. These covalent complexes can be analyzed by immunoblot and/or mass spectrometry. For example, we have used SiaDAz crosslinking to demonstrate that cholera toxin subunit B (CTB) can crosslink to both the glycolipid GM1 and fucosylated glycoproteins such as CEACAM5. One limitation of SiaDAz crosslinking technology is that the level of SiaDAz incorporation varies among different cell types, ranging from 0 – 75 % replacement of cell surface Neu5Ac. In general, we find that crosslinking can be achieved with incorporation levels of ~10 % or greater. We can help you measure SiaDAz incorporation levels in your cell line of interest.