Chemo-Enzymatic Synthesis of Glycans: Overview

Abstract

The synthesis of glycans has a long history, and its advancement continues to evolve along with the methods available for the analysis of glycans (see reviews Koeller and Wong 2001; Hsu et al. 2011; Paulsen 1982; Schmidt et al. 1999). This chapter covers various aspects of methodology development, including the development of glycosylation reagents and protecting groups, and programmable one-pot synthesis (Zhang et al. 1999) as well as automated solid-phase synthesis of oligosaccharides (Plante et al. 2001). The use of enzymes in the synthesis of complex glycans has not been fully appreciated until in the 1980s when glycosyltransferases were shown to be useful for the synthesis of glycans if coupled with regeneration of sugar nucleotides (Wong et al. 1982). Since then, various strategies and methods have been further developed to improve the methodology (Hsu et al. 2011; Wang et al. 2013), including enzyme expression and its improvement with directed evolution and reactor configuration. With regard to reactor configuration, the enzymes could be used as free forms in a homogeneous system or immobilized to beads or solid supports or polymers in order to recover the enzymes for reuse. In general, the use of free enzymes in a homogeneous system has been most common and practical and could be carried out in a one-pot manner for the gram-scale or kilogram-scale synthesis of oligosaccharides such as sialyl Lewis x tetrasaccharide (Ichikawa et al. 1992) and Globo H or SSEA4 hexasaccharide (Tsai et al. 2013). In addition to glycosyltransferases, glycosidases have been used in a kinetic mode to conduct glycan synthesis, though often leading to a mixture of products. The design of glycosynthase with mutation of one of the two aspartyl groups involved in the glycosidic cleavage to another residue such as alanine and together with the use of glycosyl fluoride as donor was shown to eliminate the problem of by-product formation (Hancock et al. 2006). Glycans could be further modified by sulfation or other reactions, and the enzymes involved in the modification have been used in the synthesis of glycan derivatives. A typical example is the synthesis of heparan sulfates and analogs (Xu et al. 2011). In this case, glycosyltransferases, epimerases, and sulfotransferases have been utilized in the synthesis of complex glycan sulfates and related substances. In this regard, the use of sulfotransferase coupled with regeneration of its cofactor phosphoadenosine phosphosulfate (PAPS) from its by-product phosphoadenosine phosphate (PAP) would be necessary to make the process practical for large-scale synthesis (see review Hsu et al. 2011). These enzymatic methods could also be used in conjunction with the synthesis of glycopeptides and glycoproteins and could be carried out in a cellular process through pathway engineering. With continual improvement in the field, it is expected that in the future, efficient