An efficient and elegant synthesis of alpha(2,8)-oligosialosides is described. The 5-N,4-O-carbonyl-protected sialyl donor undergoes alpha-sialylation in CH2Cl2 to give alpha(2,8)- and alpha(2,9)-disialosides in excellent yields. The 5-N,4-O-carbonyl protecting group was effective in improving the reactivity of the C8 hydroxyl groups toward glycosylation. Using the sialyl building block, the synthesis of tetra-alpha(2,8)-sialic acid was accomplished by using a simple glycosidation and deprotection protocol.
In this report, we describe an efficient convergent synthesis of the GP1c glycolipid epitope, which is one of the most complex c-series gangliosides. The alpha(2,3) and alpha(2,8) sialylations were accomplished by use of 5N,4O-carbonyl and 7,8-O-isopropyliden as well as 5N,4O-carbonyl- and 7,8-di-O-chloroacetyl-protected sialyl donors in good yields with excellent alpha-selectivity, respectively. The two sialyl donors enable synthesis of the di- and trisialylgalactosides by simple glycosylation and deprotection. We synthesized the protected GP1c glycolipid epitope, which is a compact, rigid, branched structure, via direct coupling of tetarasaccharide and pentasaccharide units.
An efficient stereoselective synthesis of alpha(2,9) tetra- to disialic acids 1-3, using the 5,4-N,O-carbonyl protected thiosialoside 4, is described. The cyclic protecting group was effective for alpha-sialylation without the need for acetonitrile as the solvent. The donor 4 enabled the formation of a tetramer in excellent yield and selectivity. Deprotection of the cyclic protecting groups of the protected di- to tetrasialica acids proceeded smoothly to give the fully deprotected alpha(2,9) tetra- to disialic acids 1-3.
The monoclonal antibody mAb.A2B5 is a marker for the detection of oligodendrocyte progenitor cells that differentiate into type-2 astrocytes and oligodendrocytes. It is also a useful antibody for separating these cells from other lineage populations. The epitope of this antibody is considered to be the gangliosides GT3 and GQ1c. In this study, we sought to define more precisely the structure of the epitope. Accordingly, we chemically synthesized defined oligosialic acid structures linked to phosphatidylethanolamine and bovine serum albumin and used these to determine the antigenic specificity. mAb.A2B5 recognized the Neu5Acalpha2-->8Neu5Acalpha2-->8Neu5Acalpha--> structure on both glycolipids and glycoproteins. We then examined whether the mAb.A2B5 epitope exists on glycoproteins in developing mouse brains. Western blot analyses revealed the expression of four glycoproteins reactive with the mAb.A2B5, and their expression was dependent on the stage of neural development. All the immunoreactivity in these glycoproteins with mAb.A2B5 disappeared after sialidase treatment and were resistant to chloroform/methanol extraction. These epitopes were also detected in brain homogenates from both GD3 synthetase-null and GD3/GD2 synthetase double null mice. These findings show that the alpha2,8-trisialic acid (triSia) unit recognized by mAb.A2B5 resides not only on gangliosides but also on glycoproteins in developing mouse brain. We postulate that the triSia structure on glycoproteins may be involved in oligodendrocyte differentiation, similar to the case with the alpha2,8-triSia structure on gangliosides. Real time polymerase chain reaction analysis of the developmental expression of all known ST8Sia genes, which are responsible for the biosynthesis of alpha2,8-linked Sia residues, showed that ST8Sia III gene expression correlated with expression of the triSia epitope. We suggest that ST8Sia III is the principal sialyltransferase responsible for synthesis of the alpha2,8-triSia units on glycoproteins.
Siglec-7 is a human CD33-like siglec, and is localised predominantly on human natural killer (NK) cells and monocytes. Siglec-7 is considered to function as an immunoreceptor in a sialic acid-dependent manner. However, the underlying mechanisms linking sialic acid-binding and function remain unknown. Here, to gain new insights into the ligand-binding properties of Siglec-7, we carried out in silico analysis and site-directed mutagenesis, and found a new sialic acid-binding region (site 2 containing R67) in addition to the well-known primary ligand-binding region (site 1 containing R124). This was supported by equilibrium dialysis, STD-NMR experiments, and inhibition analysis of GD3-binding toward Siglec-7 using synthetic sialoglycoconjugates and a comprehensive set of ganglioside-based glycoconjugates. Our results suggest that the two ligand-binding sites are potentially controlled by each other due to the flexible conformation of the C-C′ loop of Siglec-7. Siglecs are sialic acid (Sia)-binding immunoglobulin-like lectins and the majority of them function as transmembrane receptors in the immune system via recognition of Sia residues 1. The expression pattern of siglecs is often cell type-specific. Through cytosolic regions or adaptor proteins, they are able to regulate inhibitory or activating signals. Siglecs contain a Sia-binding N-terminal V-set domain followed by 1 to 16 C2-set Ig-domains that are considered to act as spacers or regulators of oligomerisation. Siglecs differ from most other mammalian Sia-binding lectins, such as selectins, with respect to the absolute requirement for Sia, but the details of their molecular interactions, including specific natural ligands and ligand-binding mechanisms, remain largely unclear. These are thought to be important for immune cell functions via Sia-binding because disrupting the interactions between Sias and siglecs can alter responses to infection, inflammation, autoimmunity, and cancer 1. Therefore, understanding the role of ligand-binding in biological functions of siglecs is important. The binding affinity between siglecs and naturally occurring sialylated glycoconjugates is known to be relatively weak (0.1-3 mM) 2 , and therefore biological interactions between siglecs and their ligands depend on clustering and multivalent binding. For example, mucins can display multiple ligands, and gangliosides can be clustered within lipid microdomains. To conquer the low affinity of Sia ligands for siglecs, recent research in Sia chemistry has focussed on the development of biochemical probes toward siglecs with high affinity and specificity 2-6. Attempts to increase the affinity were successful, and in some cases materials were used for clinical therapy 7,8. However, the precise details of how siglecs engage their ligands remain unclear. Siglec-7 (CDw328) is the major siglec expressed by NK cells where it can function as an inhibitory receptor 4,9,10. Siglec-7 consists of an extracellular N-terminal V-set Ig domain that is responsible for a Sia-binding, two
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