Sialylation Reaction Research Articles

Observations on chemical and enzymatic approaches to α-2,3-sialylated octyl β-lactoside

A comparison of chemical and chemo-enzymatic syntheses of α-2,3-sialylated octyl lactoside is reported. The chemical approach, starting from lactose and sialic acid, required 14 steps and proceeded in 5% overall yield; poor α-selectivity in the sialylation step necessitated a difficult and low yielding separation of anomers. A chemoenzymatic approach, employing recombinant Trypanosoma cruzi trans-sialidase to effect the key sialylation reaction, required 10 steps and gave a similar overall yield. Whereas the chemo-enzymatic synthesis required only three chromatographic purification steps overall, the chemical synthesis required at least nine.

Human milk oligosaccharides: an enzymatic protection step simplifies the synthesis of 3′- and 6′- O-sialyllactose and their analogues

We describe a chemo-enzymatic synthesis of 3′- and 6′- O-sialyllactose, two trisaccharides occurring in the ‘acidic fraction’ of the human milk oligosaccharides and endowed with potential antiadhesive activity. The key step is the highly regioselective 6′- O-acylation of benzyllactoside, which gave access to suitably protected lactose building blocks to be used as acceptors in the sialylation reaction. Moreover, the synthesis of the carboxymethyl and sulfo analogues of the title compounds is reported.

The effect of ammonia on the O‐linked glycosylation of granulocyte colony‐stimulating factor produced by chinese hamster ovary cells

Ammonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granuiocyte colony-stimulating factor (G-CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O-linked glycosylation pathway, the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine, is reduced by NH(4) (+) concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G-CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine occurs in the trans-Golgi compartment prior to the trans-Golgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion concentrations of 10 mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH-activity profile for the CHO O-linked alpha(2,6) sialytransferase using monosialylated G-CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0.Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammoniainduced increase in trans-Golgi pH could produce the observed decrease in G-CSF sialylation. (c) 1995 John Wiley & Sons, Inc.

13] Glycosyl phosphites as glycosylation reagents

This chapter describes the application of glycosyl phosphites to the synthesis of different types of glycosides. The development of new methods for the efficient construction of glycosidic bonds is an objective of current research in carbohydrate synthesis. The practical and stereo controlled synthesis of oligosaccharides containing sialic acid, for example, remains of particular interest because of their important roles in biological recognition and cellular communication. However, the steric hindrance of the anomeric center and the ease of elimination during activation and glycosylation make the sialylation reaction particularly difficult to execute in a high-yield and stereoselective manner. A novel and high-yield sialylation using sialyl phosphite as donor and trimethylsilyltrifluoromethane sulfonate (TMSOTf) as catalyst has been illustrated in the chapter.

Assay of Rat Brain Sialyltransferase Activities on Thin-Layer Plate

Abstract A modified method for measuring the activities of glycosyltransferases using high-performance thin-layer chromatographic (HPTLC) plates is described. In this method a mixture of glycosphingolipid acceptors were chromatographed on a silica gel coated plate and were incubated with the enzyme solution and an appropriate radio-active sugar nucleotide donor. Following incubation, the plate was washed with a buffer and the radiolabeled reaction products on the plate were scrapped off and the radioactivities determined using a liquid scintillation counter. Alternatively, the plate could be exposed to an X-ray film to reveal the radioactive products. This method was used to assay the activities of rat brain cytidine 5′-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. We found that this method was sensitive, fast and reliable and was capable of assaying However, we found that this kind of pretreatment could influence the sialylation reaction, especially when the subst...