☆ 4.4 Article

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

CHEMBIOCHEM (2021)

Journal

CHEMBIOCHEM

Volume 22, Issue 7, Pages 1243-1251

Publisher

WILEY-V C H VERLAG GMBH

DOI: 10.1002/cbic.202000715

Keywords

bioorthogonal chemistry; carbohydrates; DMB labeling; metabolic engineering; sialic acids

Funding

Deutsche Forschungsgemeinschaft (SPP 1623) [SFB 969]
Ministerium fur Wissenschaft, Forschung und Kunst Baden-Wurttemberg
University of Konstanz
Konstanz Research School Chemical Biology
Projekt DEAL

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Abstract

Metabolic glycoengineering (MGE) is a method utilized to incorporate chemical reporter groups into cellular glycans, allowing for visualization and isolation of glycans and potentially targeting drugs to cancer cells. The study introduced a new labeling reaction using 1,2-diamino-4,5-methylenedioxybenzene for quantification of sialic acid derivates after MGE. It also explored the effects of various ManNAc derivatives on sialic acid production in different cell types.

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.

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Chemical methods for measuring RNA expression with metabolic labeling

Monika Singha, Leslie Spitalny, Kim Nguyen, Abigail Vandewalle, Robert C. Spitale

Summary: This article outlines the current state of employing chemical approaches for cell-specific RNA expression studies, including the utility of metabolic labels, approaches for characterizing metabolic incorporation and enrichment of labeled RNAs, and how these approaches have been used to study biological systems by providing mechanistic insights into transcriptional dynamics. Further efforts in this area will focus on developing novel chemical handles for RNA enrichment and profiling, as well as innovative approaches to control cell-specific incorporation of chemically modified metabolic probes. These advancements in RNA metabolic labeling techniques enable sensitive detection of RNA expression dynamics within relatively small subsets of cells in living tissues and organisms, crucial for understanding complex developmental and pathological processes.

WILEY INTERDISCIPLINARY REVIEWS-RNA (2021)