Chemoenzymatic synthesis of GDP- l -fucose and the Lewis X glycan derivatives

Abstract

Lewis X (Le x )-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Le x and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5-diphosphate-␤-L-fucose (GDPfucose), the universal fucosyl donor, the Le x trisaccharide, and their C-5 substituted derivatives can be synthesized on preparative scales, using a chemoenzymatic approach. This method exploits L-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts L-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) intermediate. Combining the activities of FKP and a Helicobacter pylori ␣1,3 fucosyltransferase, we prepared a library of Le x trisaccharide glycans bearing a wide variety of functional groups at the fucose C-5 position. These neoglycoconjugates will be invaluable tools for studying Le x -mediated biological processes.glycobiology ͉ catalysis ͉ enzyme

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

“…The fermentation could be performed in a large scale and used for industrial application. In addition to a better productivity, the established schemes in this work have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue [15,16]. The modulation strategy for guanosine nucleotide biosynthesis in this work may also be a reference for the fermentation processes of FOSs such as 2 -fucosyllactose to further improve the productivities.…”

“…has been demonstrated [15]. Furthermore, Fkp has been recombinantly expressed and purified, and then used in the in vitro synthesis reactions for GDP-fucose or FOSs [16,17]. Although Fkp-catalyzed synthesis of GDP-fucose in test tube is straightforward and efficient, precious start materials including adenosine 5 -triphosphate (ATP) and guanosine 5 -triphosphate (GTP) have to be used with purified enzymes.…”

1,3-1,4-α-l-Fucosynthase That Specifically Introduces Lewis a/x Antigens into Type-1/2 Chains

“…Glycosyltransferases generally have strict regio-and acceptor specificity and are therefore good catalysts for defined oligosaccharide syntheses. Efficient preparations of Le x and H antigen oligosaccharides using ␣-1,3-and ␣-1,2-fucosyltransferases from H. pylori have been reported (11)(12)(13). However, as described in the literature (11), the use of glycosyltransferases requires an expensive sugar nucleotide or a complex system for nucleotide recycling.…”

Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection

“…The solution was vortexed and then incubated at room temperature for 10 min. Insoluble material was removed by centrifugation at 5000 rpm for 10 min [22]. Supernatant was transferred to a new tube for purification.…”