Synthesis of O-Linked glycopeptides using enzymatic catalysis.
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Enzymes have many advantages over conventional synthetic methods. They can be used without the need of a protecting group strategy whilst maintaining high stereo- and regiochemistry. Over the past few decades, the use of enzymes has become commonplace because of their catalytic efficiency and their ability to be used under ‘green conditions’. The use of glycosyl hydrolases (glycosidases), enzymes that catalyse the energetically favourable hydrolysis of glycosidic linkages, is also becoming increasingly widespread. In particular, the construction of glycosidic linkages using glycosidases has slowly gained prominence, however the tendency for the product of the reverse hydrolysis to be a substrate for the natural enzymatic hydrolysis reaction has plagued this process with low yields. To remedy this, glycosynthases, a class of mutant glycosidases, have been engineered to eliminate hydrolytic activity. In the presence of an activated donor substrate and an appropriate acceptor, glycosynthases are able to catalyze the formation of the glycosidic linkage and not the hydrolysis leading to higher product yields. The work in this thesis focuses on the synthesis of activated O-glycan donors, and the expression, purification, and mutagenesis of glycosidase enzymes of the super-family GH101. Subsequent kinetic assays and glycosylation investigations with various acceptors using the wild type and mutant enzymes are described. Also, work on the synthesis of glycosides in water is presented.