title: Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids.
abstract: 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-resolved31P 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.
authors.affiliation: Université Lille 1, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Bât. C9, Cité Scientifique, F-59000 Lille, France.
authors.forename: Pierre-André
authors.initials: PA
authors.lastname: Gilormini
authors.affiliation: Université Lille 1, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Bât. C9, Cité Scientifique, F-59000 Lille, France cedric.lion@univ-lille1.fr.
authors.forename: Cédric
authors.initials: C
authors.lastname: Lion
authors.affiliation: Université Lille 1, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Bât. C9, Cité Scientifique, F-59000 Lille, France.
authors.forename: Maxence
authors.initials: M
authors.lastname: Noel
authors.affiliation: Université Lille 1, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Bât. C9, Cité Scientifique, F-59000 Lille, France.
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Krzewinski-Recchi
authors.affiliation: Université Lille 1, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Bât. C9, Cité Scientifique, F-59000 Lille, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Harduin-Lepers
authors.affiliation: Université Lille 1, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Bât. C9, Cité Scientifique, F-59000 Lille, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Guérardel
authors.affiliation: Université Lille 1, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Bât. C9, Cité Scientifique, F-59000 Lille, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Biot
mesh_terms: D003568:Cytidine Monophosphate / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry*; D015335:Molecular Probes / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry*; D000216:N-Acylneuraminate Cytidylyltransferase / Q000378:metabolism; D009345:Neisseria meningitidis / Q000201:enzymology; D011134:Polysaccharides / Q000032:analysis*; D012794:Sialic Acids / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D015335:Molecular Probes; D011134:Polysaccharides; D012794:Sialic Acids; D000216:N-Acylneuraminate Cytidylyltransferase; D003568:Cytidine Monophosphate
keywords: 
  31P NMR; CMP-sialic acid; CMP-sialic acid synthase; metabolic glycan engineering; sialylation