Glycan‐Specific Metabolic Oligosaccharide Engineering of C7‐Substituted Sialic Acids

Abstract

Intact and integral glycosylation of membrane-associated as well as secreted glycoproteins has been shown to be essential for many aspects of the proper function of biological systems. Recombinantly expressed glycoproteins, such as antibodies, growth factors, hormones, vaccines, and contrast agents are key elements in medical applications. The quality of these therapeutically administered glycoproteins can be efficiently improved by the incorporation of chemically functionalized monosaccharides into their glycan moieties, a process denoted as metabolic oligosaccharide engineering (MOE). In addition to these pharmaceutical applications, MOE has greatly advanced diagnostics by localizing and visualizing glycans even in living animals. To date, a multitude of chemically modified monosaccharides have been designed for MOE applications. Owing to their terminal position at glycan structures of glycoproteins and relevance for cellular recognition, sialic acids and their metabolic precursor N-acetylmannosamine (ManNAc), are the most prominent targets for MOE. Several ManNAc derivatives with N-acetyl side-chain modifications have been synthesized and metabolically incorporated by the sialic acid biosynthetic pathway into a corresponding sialic acid C5 analogue (Figure 1). This approach was beneficial to extending the understanding of the biological role of the N-acyl side chain of sialic acids, for example, in virus infection or neuronal differentiation. Alternatively, C9 modifications of sialosides have also been achieved by directly administering synthetic sialic acid analogues. Additionally, selective cleavage of the glycol moiety led to a truncated sialic acid equipped glycans with an aldehyde for labeling reactions (Figure 1). All of these modifications address sialylation of both, Nand O-glycosylation of glycoproteins, to almost the same extent. Herein we investigate whether the biosynthetic machinery for sialic acids also tolerates other ManNAc derivatives as substrates, which are modified directly at the six-membered carbohydrate ring. The modification of the C4 position appeared most attractive, because it is not enzymatically modified during cellular glycoprotein production and would deliver previously unknown C7-modified sialic acid containing glycoproteins (Figure 1). To probe the biosynthetic promiscuity, we targeted a C4-modified ManNAc derivative, N-acetyl-4-azido-4-deoxymannosamine (4-azido-ManNAc, 1), in our study to enable postglycosylational conjugation and visualization by bioorthogonal reactions. N-acetyl-(1,3,6-O-acetyl)-4-azido-4-deoxy-mannosamine (Ac3-4-azido-ManNAc) was generated by an optimized literature method (Figure S1 in the Supporting InformaFigure 1. Methods for the structural modification of glycan-bound sialic acids by application of chemically modified ManNAc or direct periodate oxidation of glycan-bound sialic acids (left). Specific modification of the C7 position of sialic acids was achieved by C4-modified ManNAc in this study (right; note that to date these methods were carried individually, resulting in only one modification of a single sialic acid molecule).