One of the confounding features of glycoproteins is the incredible diversity of specific molecular species that can exist even in a single cell. For each site on a protein that can be glycosylated, and often there are multiple sites, the number of glycans that can be attached can be large. Indeed, research suggests that cells create different glycoforms for a given protein as an important means of modulating the properties of that protein and its interactions with other biomolecules (Varki 1993). Glycan diversity as expressed in glycoforms may, in fact, help explain human complexity (Varki 2006; Bishop and Gagneux 2007).

The factors that govern glycan diversity pose significant challenges to the isolation, structural characterization, and synthesis of single glycoforms. The structural diversity of glycans arises from the various linkage combinations between the monosaccharides that make up glycans, and those linkages are determined by an array of more than 250 enzymes in the human secretory pathway that support glycan synthesis and processing, including a suite of glycosyltransferases that add sugars using activated sugar donors and glycosidases that cleave them (Ohtsubo and Marth 2006; Varki 2006). In this network, competing or overlapping substrate and donor specificities, substrate availability, and varying levels of enzyme expression, activation, and localization along the secretory pathway all contribute to functionally significant glycan heterogeneity (Lowe and Marth 2003). While such heterogeneous mixtures serve a purpose in biological systems, they are inadequate for the important task of establishing how the molecular architectures of glycans convey specific biological properties.

Access to homogeneous glycoproteins is necessary to first determine the molecular details of a glycan’s function and then to produce those glycoforms with the desired properties. Today, it is possible to isolate relatively simple homogeneous glycoforms using enzymatic trimming of glycoproteins (Schmaltz et al. 2011), but synthesizing more complex homogeneous glycoforms requires the development of more sophisticated methods for the chemoenzymatic manipulation of glycoproteins as well as new techniques for synthesizing them de novo. Establishment of single glycoform synthesis has been critical to the identification of glycoforms with distinct properties, such as enhanced glycoprotein stability (Hanson et al. 2009; Price et al. 2010; Culyba et al. 2011), altered binding or immunogenic properties (Dwek 1996), and increased therapeutic efficacy (Arnold et al. 2007; Jefferis 2009)—to name a few. The identification of desired glycoforms will also spur large-scale production needs. For example, glycoproteins with increased α-2-3 sialylation of N-glycans

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