Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids

Natural and synthetically modified cytidine monophosphate activated sialic acids (CMP-Sias) are essential research assets in the field of glycobiology: among other applications, they can be used to probe glycans, detect sialylation defects at the cell surface or carry out detailed studies of sialyltransferase activities. However, these chemical tools are notoriously unstable because of hydrolytic decomposition, and are very time-consuming and costly to obtain. They are nigh impossible to store with satisfactory purity, and their preparation requires multiple laborious purification steps that usually lead to heavy product loss. Using in situ time-resolved ³¹P phosphorus nuclear magnetic resonance (³¹P NMR), we precisely established the kinetics of formation and degradation of a number of CMP-Sias including CMP-Neu5Ac, CMP-Neu5Gc, CMP-SiaNAl and CMP-SiaNAz in several experimental conditions. ³¹P NMR can be carried out in undeuterated solvents and is a sensitive and nondestructive technique that allows for direct in situ monitoring and optimization of chemo-enzymatic syntheses that involve phosphorus-containing species. Thus, we showed that CMP-sialic acid derivatives can be robustly obtained in high yields using the readily available Neisseria meningitidis CMP-sialic acid synthase. This integrated workflow takes less than an hour, and the freshly prepared CMP-Sias can be directly transferred to sialylation biological assays without any purification step.