Abstract
N-acetylneuraminic acid (Neu5Ac) represents the most common terminal carbohydrate residue in many mammalian glycoconjugates and is directly involved in a number of different physiological as well as pathological cellular processes. Endogenous sialic acids derive from the biosynthetic precursor molecule N-acetyl-D-mannosamine (ManNAc). Interestingly, N-acyl-analogues of D-mannosamine (ManN) can also be incorporated and converted into corresponding artificial sialic acids by eukaryotic cells. Within this study, we optimized a protocol for the chemical synthesis of various peracetylated ManN derivatives resulting in yields of approximately 100%. Correct molecular structures of the obtained products ManNAc, N-propanoyl-ManN (ManNProp) and N-butyl-ManN (ManNBut) were verified by GC-, ESI-MS- and NMR-analyses. By applying these substances to human umbilical vein endothelial cells (HUVECs), we could show that each derivative was metabolized to the corresponding N-acylneuraminic acid variant and subsequently incorporated into nascent glycoproteins. To investigate whether natural and/or artificial sialic acid precursors are able to modulate the angiogenic capacity of HUVECs, a spheroid assay was performed. By this means, an increase in total capillary length has been observed when cells incorporated N-butylneuraminic acid (Neu5But) into their glycoconjugates. In contrast, the natural precursor ManNAc inhibited the growth of capillaries. Thus, sialic acid precursors may represent useful agents to modulate blood vessel formation.
Highlights
Sialic acids are commonly found as terminal residues of carbohydrate moieties of both glycoproteins as well as glycolipids [1,2,3]
We divided the synthesis into three independent reaction steps to increase the overall yields (Figure 2A), since in our hands only 50% of ManN was converted into the respective artificial sialic acid precursors, when the published protocol was employed
Analogues by registration of their sodium adducts ([M+Na]+) in Extracted ion chromatograms (EIC) of m/z 462 and 476 (Figure 5B) as well as by displaying the characteristic fragmentation pattern of respective precursor molecules (Figure 5C). These findings demonstrated that incorporation of artificial sialic acid precursors into glycoproteins takes place resulting in an alteration of cell sialylation in vitro
Summary
Sialic acids are commonly found as terminal residues of carbohydrate moieties of both glycoproteins as well as glycolipids [1,2,3]. Many studies have elucidated these acidic monosaccharides to be crucially involved in different biological processes [4] In this context, for example, sialylLewisX-dependent homing of leukocytes via selectins represents one of the best characterized mechanisms [5]. Since addition of N-acyl-ManN evades this checkpoint, levels of artificial sialic acids increase proportional to the amount of ManN derivatives applied [7]. In terms of their biological functions, sialic acids on endothelial cells were reported to influence the initiation of lumen formation in developing blood vessels [9]. To investigate the impact of artificial sialic acids on capillary sprouting, human umbilical vein endothelial cells (HUVECs) were treated with ManNAc or artificial sialic acid precursors and respective capillary sprouting was analyzed after stimulation with basic fibroblast growth factor (bFGF)
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