Sialic Acid Glycoengineering Using an Unnatural Sialic Acid for the Detection of Sialoglycan Biosynthesis Defects and On-Cell Synthesis of Siglec Ligands

Abstract

Sialoglycans play a vital role in physiology, and aberrant sialoglycan expression is associated with a broad spectrum of diseases. Since biosynthesis of sialoglycans is only partially regulated at the genetic level, chemical tools are crucial to study their function. Here, we report the development of propargyloxycarbonyl sialic acid (Ac5NeuNPoc) as a powerful tool for sialic acid glycoengineering. Ac5NeuNPoc showed strongly increased labeling efficiency and exhibited less toxicity compared to those of widely used mannosamine analogues in vitro and was also more efficiently incorporated into sialoglycans in vivo. Unlike mannosamine analogues, Ac5NeuNPoc was exclusively utilized in the sialoglycan biosynthesis pathway, allowing a genetic defect in sialic acid biosynthesis to be specifically detected. Furthermore, Ac5NeuNPoc-based sialic acid glycoengineering enabled the on-cell synthesis of high-affinity Siglec-7 ligands and the identification of a novel Siglec-2 ligand. Thus, Ac5NeuNPoc glycoengineering is a highly efficient, nontoxic, and selective approach to study and modulate sialoglycan interactions on living cells.

Metabolic sialic acid blockade lowers the activation threshold of moDCs for TLR stimulation

“…Together, the direct inhibition of sialyltransferases and the indirect inhibition of sialic acid synthesis result in metabolic sialic acid blockade in the cell (Figure 1a). Previously, we have reported that Ac 5 3F ax Neu5Ac blocks sialic acid incorporation into surface glycans of the monocytic THP‐1 cell line 19 . Here we assessed if the sialic acid mimetic blocks sialoglycan expression in freshly generated moDCs.…”

“…We have previously shown that a fluorinated sialic acid mimetic, Ac 5 3F ax Neu5Ac, blocks sialic acid expression in human and mouse tumor cell lines with high efficiency and specificity 15 , 19 , 20 , 21 . Most notably, this inhibitor caused a prolonged inhibition of the sialic acid biosynthesis (about 48 h to recover 50% sialylation) and is chemically pure and free from endotoxins.…”

Metabolic Oligosaccharide Engineering with Alkyne Sialic Acids Confers Neuraminidase Resistance and Inhibits Influenza Reproduction

“…9,10 Both sialic acid analogues are well-tolerated by the cellular sialylation pathway, and these small modifications to the C-5 position are generally thought to be nonintrusive. There is evidence, however, that the modification of sialic acids at the C-5 position affects their recognition and cleavage by bacterial neuraminidases.…”

“…In contrast to our previously published comparison of Ac 5 SiaNAz and Ac 5 SiaNPoc (see ref (9)), the click reaction was now done at 37 °C instead of at room temperature, which greatly increased the fluorescence signal and gave usual click efficiencies of 80% or higher (see ref (10)).…”

Metabolisches Glykoengineering mit N‐Acyl‐Seiten‐ ketten‐modifizierten Mannosaminen

“…Es wurde gezeigt, dass durch den Gebrauch von N ‐Propargylcarbonylmannosamin (ManNProc) oder N ‐Propargylcarbonylsialinsäure (Neu5Proc) Zellen kreiert werden können, die hoch‐affine Liganden für Siglecs exprimieren (High‐affinity siglec ligand‐expressing cells, HASLECs) . In dieser Studie wurden die vorbehandelten, Neu5Proc tragenden Zellen über CuAAC mit bestimmten Azid‐Gruppen verbunden, welche die Bindungsaffinität zu spezifischen Siglec‐Subtypen stark erhöhen .…”