On April 16, 2025, a transformative study by the Macauley lab at the University of Alberta, published in RSC Chemical Biology, unveiled 4-azido sialic acid (4AzNeu5Ac) as a novel glycobiology reagent that overcomes longstanding synthetic barriers in metabolic oligosaccharide engineering. This research successfully functionalized the challenging C4 position of sialic acid using a strategic protective group strategy involving thioglycoside stabilization, preventing decomposition and enabling efficient synthesis. The team demonstrated robust metabolic incorporation of peracetylated 4AzNeu5Ac into cellular glycans of U937 cells, achieving near-equivalent efficiency to C5-modified analogs (only 21% signal reduction) through SPAAC click chemistry with DBCO-biotin probes.

Background

In the intricate world of glycobiology, the ability to track, visualize, and manipulate cell-surface glycans is paramount to understanding their critical roles in health and disease. Sialic acids, which often cap these glycans, are particularly important, serving as key recognition points in cell-cell communication and immune responses. For decades, metabolic oligosaccharide engineering (MOE) has been a cornerstone technique, allowing scientists to introduce chemical reporters, like azides or alkynes, into cellular glycans for subsequent visualization or functionalization. Traditionally, these "bioorthogonal handles" have been appended to the C5 or, to a lesser extent, the C7 and C9 positions of sialic acid. The researchers unveil the successful synthesis and application of a sialic acid reporter functionalized at the previously challenging C4 position: 4-azido sialic acid (4AzNeu5Ac).

A Synthetic Challenge Overcome

The journey to 4AzNeu5Ac was fraught with synthetic hurdles. While installing an azide at C5, C7, or C9 can be achieved by modifying the biosynthetic precursor ManNAc, the C4 position requires direct modification of the sialic acid skeleton itself. Previous attempts often led to low yields or decomposition via a base-catalyzed "peeling" reaction, where the sugar backbone degrades under alkaline conditions. The research team ingeniously circumvented this problem by employing a strategic protective group strategy. They installed a thioglycoside at the anomeric center, which acted as an anchor, stabilizing the molecule during the critical deprotection steps. This clever approach provided efficient access to both the protected version, ideal for cellular uptake, and the fully deprotected 4AzNeu5Ac, essential for enzymatic studies. This synthetic victory opened the door to a suite of previously inaccessible applications.

Robust Incorporation into Cellular Glycans

The first critical test was whether cells could metabolically incorporate this new reporter. The team fed peracetylated 4AzNeu5Ac to U937 cells and, using a strain-promoted azide-alkyne cycloaddition (SPAAC) "click" reaction with a DBCO-biotin probe, they successfully labeled the cell-surface glycans. Impressively, the incorporation efficiency of 4AzNeu5Ac was nearly as high as the gold-standard C5-modified analog (Neu5Az), with only a 21% reduction in signal at the highest concentration. Flow cytometry and microscopy confirmed that 4AzNeu5Ac robustly decorated the cell surface, demonstrating that the mammalian metabolic machinery—including the CMP-sialic acid synthetase and Golgi transporter—readily accepts this C4-modified variant. This finding significantly expands the MOE toolkit, offering a valuable alternative when the C5 position is reserved for other functional groups, such as photocrosslinkers or biotin.

Fig.1 Metabolic incorporation of peracetylated 4AzNeu5Ac (6) and Neu5Az (7) into cellular glycans. (Gray, et al., 2025)

Enzymatic Transfer and Ligand Binding Studies

Access to deprotected 4AzNeu5Ac enabled even more sophisticated applications. The researchers enzymatically activated it to form CMP-4AzNeu5Ac and demonstrated that a human sialyltransferase, ST3GAL1, could transfer this unnatural donor onto a disaccharide acceptor to create a trisaccharide sialoside. This proved that key glycosylation enzymes tolerate the C4-azide modification. The team then investigated the biological implications of this modification by testing the binding affinity of the newly synthesized 4AzNeu5Ac-containing trisaccharide to Siglec-7, an immunoregulatory receptor. Using a sophisticated concentration-independent native mass spectrometry (COIN-nMS) technique, they determined that the C4-azide modification did not significantly perturb binding to Siglec-7 compared to the unmodified sialoside. This suggests that Siglec-7's binding pocket can accommodate modifications at the solvent-facing C4 position, a finding with exciting implications for designing high-affinity ligands for immunomodulation.

Bridging Discovery and Application with GlycoCLICK™

The successful development and application of 4AzNeu5Ac exemplify the power of advanced glycochemical synthesis to drive biological discovery. This research opens new avenues for probing sialic acid biology, from tracking glycan dynamics in live cells to designing novel therapeutic agents that target Siglec and other sialic-acid-binding proteins.

At CD BioGlyco, we are at the forefront of translating such groundbreaking scientific advancements into practical tools and services. Our platform specializes in leveraging click chemistry methodologies for a wide range of glycobiology applications. The challenges overcome in the synthesis of 4AzNeu5Ac align directly with our expertise.

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Conclusion

The study by Gray et al. is more than just a synthetic achievement; it is a significant expansion of the glycochemist's toolbox. By demonstrating the metabolic incorporation, enzymatic transfer, and tolerated receptor binding of 4AzNeu5Ac, the researchers have provided a new versatile handle for interrogating sialic acid function. As research continues to explore the potential of C4-modified sialic acids, the integrated expertise and innovative services offered by CD BioGlyco are invaluable for scientists aiming to push the boundaries of glycobiology from fundamental research to therapeutic innovation. This work underscores a vibrant synergy between academic discovery and specialized technical platforms in advancing our understanding of the sugar code of life.