Carles Malet

From β‐glucanase to β‐glucansynthase: glycosyl transfer to α‐glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile

Removal of the catalytic nucleophile Glu134 of the retaining 1,3‐1,4‐β‐glucanase from Bacillus licheniformis by mutation to alanine yields an enzyme with no glycosidase activity. The mutant is able to catalyze the regio‐ and stereospecific glycosylation of α‐laminaribiosyl fluoride with different glucoside acceptors through a single‐step inverting mechanism. The main advantage of the mutant as glycosylation catalyst with respect to the kinetically controlled transglycosylation using the wild‐type enzyme is that the reaction products cannot be hydrolyzed by the mutant enzyme, and glycosylation yields rise to 90%.

Probing the acceptor substrate binding site of Trypanosoma cruzi trans-sialidase with systematically modified substrates and glycoside libraries

Trypanosoma cruzi trans-sialidase is a versatile catalyst for enzymatic α-2,3-sialylation reactions.

Generation of molecular diversity on N-acetyllactosamine via O-cyanomethyl ethers

Abstract The ability to generate molecular diversity around a natural carbohydrate ligand taking advantage of the rich chemistry of the nitrile functional group was demonstrated by synthesizing 24 derivatives of N-acetyllactosamine (LacNAc). The disaccharides prepared carried carboxymethyl, amidinomethyl, aminoethyl, and carbamoylmethyl substituents projecting from each of the six OH groups. The resulting LacNAc derivatives present new structural features with either negatively charged, positively charged, or polar-neutral small substituents sampling the entire periphery of the molecule. These new derivatives should be useful probes for studying carbohydrate-protein interactions in general, and for designing inhibitors of N-acetyllactosamine-binding proteins in particular.

Acceptor hydroxyl group mapping for calf thymus α-(1 → 3) - galactosyltransferase and enzymatic synthesis of α-d-Galp-(1 → 3)-β-d-Gal p-(1 → 4)-βd-GlcpNAc analogs

Abstract The epitope of the acceptor substrate for α -(1 → 3)-galactosyltransferase from calf thymus has been mapped by using a series of mono-deoxygenated and mono- O -alkylated Type II (β- d - Gal p-(1 → 4)-β- d - Glc p NAc ) disaccharides. The 4-OH group of the β- d -galactopyranosyl residue is a key polar group essential for glycosyl transfer, tolerating neither deoxygenation nor O -alkylation. Substitution at positions 6 and 6′ by a variety of polar alkyl substituents was readily tolerated, allowing the preparative enzymatic synthesis of a series of trisaccharide derivatives carrying polar substituents on each of these hydroxyl groups. These new analogs are potential inhibitors of Clostridium difficile toxin A and of a human anti-α-Gal antibody.

A specific chromophoric substrate for activity assays of 1,3-1,4-β-d-glucan 4-glucanohydrolases

The synthesis of 4-methylumbelliferyl 3-beta-O-cellobiosyl-beta-D-glucopyranoside (3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-beta-D-glucan 4-glucanohydrolases are described. The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose beta-D-glucan polysaccharide is degraded down to a tri- and tetrasaccharide by an extracellular extract of recombinant E. coli expressing and secreting Bacillus licheniformis 1,3-1,4-beta-glucanase. The trisaccharide 1 is further chemically transformed into the title compound. Its use as substrate for an enzyme activity assay, the specificity of cleavage, and kinetic parameters are reported. As it undergoes a single glycosidic bond hydrolysis with release of 4-methylumbelliferone, direct UV monitoring of the reaction provides a sensitive kinetic assay of the enzyme action.