Metabolic Glycoengineering with Azide‐ and Alkene‐Modified Hexosamines: Quantification of Sialic Acid Levels

Abstract

Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method has found broad application for the visualization and isolation of glycans allowing their biological roles to be probed. Furthermore, targeting of drugs to cancer cells that present high concentrations of sialic acids on their surface is an attractive approach. We report the application of a labeling reaction using 1,2‐diamino‐4,5‐methylenedioxybenzene for the quantification of sialic acid derivates after MGE with various azide‐ and alkene‐modified ManNAc, GlcNAc, and GalNAc derivatives. We followed the time course of sialic acid production and were able to detect sialic acids modified with the chemical reporter group – not only after addition of ManNAc derivatives to the cell culture. A cyclopropane‐modified ManNAc derivative, being a model for the corresponding cyclopropene analog, which undergoes fast inverse‐electron‐demand Diels‐Alder reactions with 1,2,4,5‐tetrazines, resulted in the highest incorporation efficiency. Furthermore, we investigated whether feeding the cells with natural and unnatural ManNAc derivative results in increased levels of sialic acids and found that this is strongly dependent on the investigated cell type and cell fraction. For HEK 293T cells, a strong increase in free sialic acids in the cell interior was found, whereas cell‐surface sialic acid levels are only moderately increased.