Synthesis with Glycosynthases: Cello-Oligomers of Isofagomine and a Tetrahydrooxazine as Cellulase Inhibitors

Abstract

Isofagomine and a carbohydrate-like tetrahydrooxazine, as their N-benzyloxycarbonyl derivatives, have been subjected to a glycosynthase in the presence of α-D-glucopyranosyl fluoride as a glucosyl donor. In each case, after protecting group removal, a mixture of 1,4-β-linked di-, tri-, and tetra-'saccharides' was obtained. These novel oligosaccharide derivatives were tested as inhibitors of the endo-glycanase Cex from Cellulomonasfimi. Affinities increased progressively as additional D-glucosyl residues were incorporated, which is consistent with the known substrate specificity of this enzyme.

Specificity Fingerprinting of Retaining β‐1,4‐Glycanases in theCellulomonas fimiSecretome Using Two Fluorescent Mechanism‐Based Probes

“…[18,36] The syntheses of the xylobiose-derived isofagomine (X 2 IF) and the cellobiose-derived isofagomine (CIF) have also been described previously. [34,35] Porcine modified trypsin was from Promega (Nepean, Ontario, Canada). All other chemicals were obtained from Sigma/Aldrich Chemical Co. (Oakville, Ontario, Canada) and were of reagent grade.…”

“…The iminosugar glycosidase inhibitor, xylobiose-derived isofagomine (X 2 IF), preferentially inhibits GH10 glycanases (nanomolar K i ) over GH11 glycanases (millimolar K i ) due to subtle differences in the transition states and active-site structures between enzymes from these two families. [34] We preincubated the secretomes with X 2 IF or a cellobiose-derived isofagomine [35] (CIF), and then competed these with both of our fluorescent ABPs over a limited incubation time. The intensities of all of the fluorescent bands harbouring XylC, Cex and/or Cfx or their CDs either decreased or were totally ablated upon competition with either X 2 IF or CIF as a result of active-site-directed competition (Figure 3), which is consistent with the mixed-specificities of the target enzymes.…”

The Structural Basis of Glycosidase Inhibition by Five‐Membered Iminocyclitols: The Clan A Glycoside Hydrolase Endoglycoceramidase as a Model System

“…500 compound) "in-house" library was screened for EGC inhibition. Three hits were discovered: cellobiose-like isofagomine 1 b, [12] cellobiose-like imidazole 2 b, [13] and the five-membered iminocyclitol 3 b. [9] Kinetic analyses of these derivatives at the pH optimum of the enzyme (pH 5) showed them to be competitive inhibitors with K i values of 5 mm, 0.5 mm, and 10 mm, respectively.…”

Robert Vyent Stick: A Colourful Character

“…Thus, the previously mentioned isofagomine and tetrahydrooxazine were elongated to oligosaccharide inhibitors for oligosaccharide-processing enzymes. [23] These compounds were suddenly in great demand and led to the establishment of a very productive collaboration with an X-ray crystallographer and biochemist, Professor Gideon Davies. These compounds were used to define and dissect various facets of inhibitor binding to various enzymes and led to a series of high profile publications and numerous crystallographic determinations of enzyme-inhibitor complexes and definition of some unusual aspects of conformational change that occur during catalysis.…”