Dienophile‐Modified Mannosamine Derivatives for Metabolic Labeling of Sialic Acids: A Comparative Study

Abstract

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.

Potent Metabolic Sialylation Inhibitors Based on C-5-Modified Fluorinated Sialic Acids

“…31 This finding is also supported by a later study on the metabolic precursor of sialic acid, where an increased metabolic incorporation was observed for mannosamine carbamates over amides with the same side chain length. 32 In addition to linkage type, linear 2–3 carbon substituents ( 7 , 8 , 10 , and 18 ) on the carbamate seem to afford more potent inhibitors than 1 ( 9 ) or 4 ( 15 ) carbons, or the introduction of a more bulky substituent ( 11–14 ). In an attempt to modulate carbamate hydrogen bonding properties, 16–18 were synthesized, but these did not further improve the inhibitory effect observed with 7 .…”

Metabolic precision labeling enables selective probing of O-linked N -acetylgalactosamine glycosylation

“…Protein-based reporter reagents have enabled the study of glycobiology but rarely probe nonaccessible glycan core structures. Thus far, the forays made into developing chemical tools have yielded an arsenal of monosaccharide analogs: for instance, of ManNAc/Sia ( 8 , 56 – 58 ), GlcNAc ( 18 – 20 , 35 ), Fuc ( 59 ), Gal ( 60 , 61 ), and GalNAc/GlcNAc ( 10 , 15 , 17 , 62 , 63 ). Probes are typically selected based on their labeling intensity, which in turn, is often a function of poor glycan specificity.…”

Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity

“…After corresponding treatment times were over, cells were washed twice with PBS, scraped-off the well surface, and pelleted. Afterwards, release of Sia and DMB labeling was performed as described earlier (Dold et al 2017). Briefly, to release the Sia, each cell pellet was resuspended in 300 µl AcOH (3 M) and incubated for 90 min at 80 °C inside a thermomixer at 300 rpm.…”

“…As final step of the basic characterization of MGE for human neurons, we determined what proportion of Sia was modified through MGE in LUHMES. The incorporation efficiency of the tagged Sia precursor was quantified by a 1,2-diamino-4,5-methylenedioxybenzene (DMB) labeling strategy (Dold et al 2017). After 24 h treatment with the azide-tagged mannosamine (Ac 4 ManNAz), 38% of neuronal Sia was modified with an azide group (Fig.…”

“…modified Sia) are positioned at the same site and with the same chemical connections as their natural counterpart (Sia) in a non-modified cell. MGE substrates have been developed to be used in a highly specific way by mammalian cells to substitute N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose, Sia or O-linked N-acetyl-glucosamine (O-GlcNAc) (Campbell et al 2007;Dold et al 2017;Doll et al 2016;Du et al 2009;Niederwieser et al 2013;Nischan and Kohler 2016). For instance, N-acetylmannosamine (ManNAc) with an azide (Az) modification (N-azidoacetylmannosamine (ManNAz)) is converted within cells to azide-modified Sia (SiaAz).…”