β-Glycosyl Azides as Substrates for α-Glycosynthases: Preparation of Efficient α-L-Fucosynthases

Abstract

Fucose-containing oligosaccharides play a central role in physio-pathological events, and fucosylated oligosaccharides have interesting potential applications in biomedicine. No methods for the large-scale production of oligosaccharides are currently available, but the chemo-enzymatic approach is very promising. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, have been demonstrated to be an interesting alternative. However, examples of glycosynthases available so far are restricted to a limited number of glycosidases families and to only one retaining alpha-glycosynthase. We show here that new mutants of two alpha-L-fucosidases are efficient alpha-L-fucosynthases. The approach shown utilized beta-L-fucopyranosyl azide as donor substrate leading to transglycosylation yields up to 91%. This is the first method exploiting a beta-glycosyl azide donor for alpha-glycosynthases; its applicability to the glycosynthetic methodology in a wider perspective is presented.

Efficient synthesis of α‐galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α‐galactosidase (BtGH97b)

“…8D). The β-glucosyl moiety at subsite +1 was tightly surrounded by 5 these amino-acid residues, and the carboxy group of Glu174 established a hydrogen bond with 6 the O2 atom (Fig. 8C, D).…”

“…4 A single trisaccharide species (ESI-MS: m/z 527.16 for C18H32O16 + Na + ) was produced 5 when lactose was used as the acceptor molecule. The NMR spectra indicated that the trisaccharide 6 was β-lactosyl α-D-galactopyranoside [α-D-Galp-(1↔1)-β-D-Glcp-(4←1)β-D-Galp; Gal-Lac], 7 which respective occupancies of β-glucosyl and β-galactosyl moieties at subsites +1 and +2 8 formed. The α-(1↔1)-β linkage was determined by the HMBC-correlation between H-1 (J = 8.2 9 Hz) of the glucose residue and C-1 of the galactose residue, and that between H-1 (J = 3.8 Hz) of 10 the galactose residue and C-1 of the glucose residue (Fig.…”

“…In addition, the poor reactivity of the 3 ternary complex (enzyme • β-galactosyl azide • lactose) might result from scarce ability of lactose 4 to act as the acceptor as well as the stabile β-GalN3, because the yield of the product from lactose 5 in the presence of the azide ion was markedly low. 6 Additional lactose acceptor causes a significant decrease in kcat for the liberation of fluoride 7 from α-GalF in the azide-rescued reaction (Fig. 6B) …”

“…6D). The α-GalF concentration (10 19 mM) used was deemed to be greater than Km (Km = 0.136 mM with formate, Km = 0.101 mM with 20 azide; suggested that the chemical-rescued transglycosylation of BtGH97b-D415G followed the scheme 6 in Fig. 7.…”

“…The enzyme certainly hydrolyzes β-Glc-(1→4)-β-Glc linkages, and 3 the results suggest that the mixture contains non-reducing α-D-Galp(1↔1)β-D-Glcp-(4←1)β-D- 4 Glcp resulting from binding of the reducing and non-reducing ends of glucose moieties of 5 acceptor cellobiose at subsites +1 and +2, respectively. The unhydrolyzed oligosaccharide was 6 subjected to methylation analysis. 2,3,4,6-tetra-O-methyl-galactose, 2,3,4-tri-O-methyl-glucose, 7 and 2,3,6-tri-O-methyl-glucose were detected by TLC (Fig.…”