Exploring Regioselective Fucosylation Catalyzed by Bacterial Glycosyltransferases through Substrate Promiscuity and Acceptor-Mediated Glycosylation

Abstract

Fucose in fucosylated glycans is primarily assembled at multiple sets of N-acetyllactosamine (LN) and lacto-N-biose (LNB) units, which are known as Lewis antigens. The synthesis of these compounds is difficult due to the diversity of Lewis antigens on complex glycans. A promising strategy is to perform enzymatic fucosylation with oligosaccharide backbones because the method is stereoselective and synthetically efficient. However, purification is difficult with this method because heterogeneous glycans are produced. Herein, we developed a general strategy to perform sitespecific fucosylation on poly LN or LN/LNB hybrid backbones. We found that trifluoroacetyl glucosamine (GlcNTFA) can function as a GlcNAc substituent and be incorporated into the LN/LNB chain. The TFA group was readily converted to a butyloxycarbonyl (Boc) group, and the resulting GlcNBoc can be accepted by bacterial galactosyltransferases and provides steric hindrance to prevent fucosyltransferase recognition. Thus, enzymatic fucosylation can only occur specifically at GlcNAc sites within GlcNHBoc-containing poly LN/LNB. We demonstrated the feasibility of this strategy by efficiently synthesizing myeloglycans (dodecasaccharides) and internal/terminal fucosylated human milk oligosaccharides. This approach offers a solution to bypass complex chemical glycosylations and control the selectivity of enzymatic fucosylations. Thus, the method shows significant potential for the future synthesis of branched glycans, such as N-glycans and O-glycans.

A Biomimetic Synthetic Strategy Can Provide Keratan Sulfate I and II Oligosaccharides with Diverse Fucosylation and Sulfation Patterns

“…These enzymes can install glycosidic linkages in a regio- and stereospecific manner and reactions can be driven to completion by using alkaline phosphatases to hydrolyze nucleotide products that can act as product inhibitor. − The efficiency of glycosyltransferase mediated glycan assembly has made it possible to prepare complex oligosaccharides in an automated fashion. − Complex glycans can have several acceptor sites for a given glycosyltransferase, making it difficult to prepare discrete compounds. Site selective glycosylations can, however, be accomplished by careful synthetic planning, the use of unnatural sugar nucleotide donors, , chemical modifications, − or temporary monosaccharide blocking groups …”