Endo-S2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins

A homogeneous composition comprising a modified core-fucosylated glycoprotein or nonfucosylated glycoprotein with an oligosaccharide component comprising at least 75% of the same number and types of sugar residues, wherein the core-fucosylated glycoproteins or nonfucosylated glycoproteins are prepared by the method of:

• providing a fucosylated or nonfucosylated N-acetylglucosamine (GlcNAc)-acceptor protein;
• and
• enzymatically reacting the fucosylated or nonfucosylated GlcNAc acceptor protein with a Streptococcus pyogenes (serotype M49) Endoglycosidase-S2 (Endo-S2) Asp184 mutant enzyme selected from the group consisting of D184M (SEQ ID NO:
• 7), D184E (SEQ ID NO:
• 8), D184C (SEQ ID NO:
• 6), D184G (SEQ ID NO:
• 9), D184A (SEQ ID NO:
• 2), D184N (SEQ ID NO:
• 3), D184Q (SEQ ID NO:
• 4), D184S (SEQ ID NO:
• 16), and D184T (SEQ ID NO. 17), wherein the mutant enzyme exhibits increased transglycosylation efficiency relative to a wild-type Endo-S2 enzyme with an activated oligosaccharide donor, wherein the activated oligosaccharide donor is a synthetic oligosaccharide oxazoline comprising an oxazoline and an oligosaccharide moiety comprising a predetermined number and type of sugar residues, wherein via an enzymatic reaction, the activated oligosaccharide moiety is covalently linked to the fucosylated or nonfucosylated GlcNAc acceptor protein, thereby preparing the modified fucosylated or nonfucosylated glycoprotein;
• and wherein the homogeneous composition comprises a higher percent of modified core-fucosylated glycoprotein or nonfucosylated glycoprotein with an oligosaccharide component than an homogeneous composition prepared by the same method in the presence of an Endo-S mutant.