Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine–alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we labeled sialic acids on the surface of living cells. Screening of several alkenes showed that a previously reported carbamate‐linked methylcyclopropene reporter that is well suited for the inverse‐electron‐demand Diels–Alder (DAinv) reaction has a surprisingly low reactivity in the photoclick reaction. Thus, for the first time, we were able to triply label glycans by a combination of DAinv, photoclick, and copper‐free click chemistry.

Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse‐electron‐demand Diels–Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene‐modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2‐diamino‐4,5‐methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.

Sialic acids play an important role in numerous cell adhesion processes, and sialylation levels are known to be altered under certain pathogenic conditions, such as cancer. Metabolic glycoengineering with mannosamine derivatives is a convenient way to introduce non‐natural chemical reporter groups into sialylated glycoconjugates, offering the opportunity to label sialic acids by using bioorthogonal ligation chemistry. The labeling intensity depends not only on the rate of the ligation reaction but also on the extent to which the natural sialic acids are replaced by the modified ones; that is, the incorporation efficiency. Here, we present a comparative study of eight mannosamine derivatives featuring terminal alkenes as chemical reporter groups that can be labeled by an inverse‐electron‐demand Diels–Alder (DAinv) reaction. The derivatives differed in chain length as well as the type of linkage (carbamates, amides, and a urea) that connects the terminal alkene to the sugar. As a general trend, increasing chain lengths resulted in higher DAinv reactivity and, at the same time, reduced incorporation efficiency. Carbamates were better accepted than amides with the same chain length; nevertheless, the latter resulted in more intense cell‐surface staining, visible by live‐cell fluorescence microscopy. A urea derivative was also shown to be accepted.

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.

While there are many methods to quantify the synthesis, localization, and pool sizes of proteins and DNA during physiological responses and toxicological stress, only few approaches allow following the fate of carbohydrates. One of them is metabolic glycoengineering (MGE), which makes use of chemically modified sugars (CMS) that enter the cellular biosynthesis pathways leading to glycoproteins and glycolipids. The CMS can subsequently be coupled (via bio-orthogonal chemical reactions) to tags that are quantifiable by microscopic imaging. We asked here, whether MGE can be used in a quantitative and time-resolved way to study neuronal glycoprotein synthesis and its impairment. We focused on the detection of sialic acid (Sia), by feeding human neurons the biosynthetic precursor N-acetyl-mannosamine, modified by an azide tag. Using this system, we identified non-toxic conditions that allowed live cell labeling with high spatial and temporal resolution, as well as the quantification of cell surface Sia. Using combinations of immunostaining, chromatography, and western blotting, we quantified the percentage of cellular label incorporation and effects on glycoproteins such as polysialylated neural cell adhesion molecule. A specific imaging algorithm was used to quantify Sia incorporation into neuronal projections, as potential measure of complex cell function in toxicological studies. When various toxicants were studied, we identified a subgroup (mitochondrial respiration inhibitors) that affected neurite glycan levels several hours before any other viability parameter was affected. The MGE-based neurotoxicity assay, thus allowed the identification of subtle impairments of neurochemical function with very high sensitivity.

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The inside back cover picture shows the principle of metabolic glycoengineering that has it roots in Werner Reutter's research of 1992. Modified carbohydrate derivatives cross the cell membrane (symbolized by the Old Rhine Bridge in Konstanz) and enter the glycan biosynthesis pathway, which eventually leads to the presentation of the modified glycans as part of cell‐surface proteins and lipids. There, they can undergo a bioorthogonal ligation reaction, for example a tetrazine ligation, allowing them to be visualized by fluorescence microscopy. More information can be found in the full paper by V. Wittmann et al. on page 1242 in Issue 13, 2017 (DOI: 10.1002/cbic.201700002).

The inside cover picture shows how metabolic glycoengineering can be explained in a creative way (idea: Sven Epple). Modified monosaccharides are used to engineer the cell surfaces’ glycans with functional groups. Surprisingly, bulky norbornenes can also be used as modifiers. In a second step, these modifications are “colored” through a bioorthogonal ligation reaction with a tetrazine or cyclooctyne that is linked to a label. More information can be found in the full paper by V. Wittmann et al. on page 1374 in Issue 14, 2016 (DOI: 10.1002/cbic.201600197).