No abstract

The first bacterial α2−6-sialyltransferase cloned from Photobacterium damselae (Pd2,6ST) has been widely applied for the synthesis of various α2−6-linked sialosides. However, the extreme substrate flexibility of Pd2,6ST makes it unsuitable for site-specific α2−6-sialylation of complex substrates containing multiple galactose and/or N-acetylgalactosamine units. To tackle this problem, a general redox-controlled site-specific sialylation strategy using Pd2,6ST is described. This approach features site-specific enzymatic oxidation of galactose units to mask the unwanted sialylation sites and precisely controlling the site-specific α2−6-sialylation at intact galactose or N-acetylgalactosamine units.

Enzymatic synthesis of all 15 naturally occurring human ABH antigens was achieved using a diversityoriented enzymatic modular assembly (EMA) strategy. Three enzyme modules were developed, each one-pot multienzyme module comprises a glycosyltransferase and one or two corresponding sugar nucleotide generating enzyme(s). These multienzyme cascade processes provide an efficient and convenient platform for collective synthesis of all 15 ABH antigens in three operationally simple steps from five readily available disaccharide acceptors and three simple free sugars as donor precursors.

Ganglioside GD2 is an attractive tumor-associated carbohydrate antigen for anti-cancer vaccine development. However,i ts lowi mmunogenicity and the significant side effects observed with anti-GD2 antibodies present significant obstacles for vaccines.T oo vercome these,anew GD2 derivative bearing an N-acetamide (NHAc) at its non-reducing end neuraminic acid (9NHAc-GD2) has been designed to mimic the 9-O-acetylated-GD2 (9OAc-GD2), aG D2 based antigen with ar estricted expression on tumor cells.9 NHAc-GD2 was synthesized efficiently via achemoenzymatic method and subsequently conjugated with ap owerfulc arrier bacteriophage Qb.M ouse immunization with the Qb-9NHAc-GD2 conjugate elicited strong and long-lasting IgG antibodies, which were highly selective toward9 NHAc-GD2 with little cross-recognition of GD2. Immunization of canines with Qb-9NHAc-GD2 showed the construct was immunogenic in canines with little adverse effects,p aving the wayf or future clinical translation to humans.

A β-galactoside α2,6-sialyltransferase from Photobacterium damselae (Pd2,6ST) that is capable of sialylating both terminal and internal galactose and N-acetylgalactosamine was herein redesigned for regioselectively producing terminal α2,6-sialosides. Guided by a recently developed bump-hole strategy, a series of mutations at Ala200 and Ser232 sites were created for reshaping the acceptor binding pocket. Finally, a Pd2,6ST double mutant A200Y/S232Y with an altered L-shaped acceptor binding pocket was identified to be a superior α2,6-sialyltransferase which can efficiently catalyze the regioselective α2,6-sialylation of galactose or N-acetylgalactosamine at the nonreducing end of a series of glycans. Meanwhile, A200Y/S232Y remains flexible donor substrate specificity and is able to transfer Neu5Ac, Neu5Gc, and KDN.