Synthesis of β-(1→6)-linked N-acetyl-d-glucosamine oligosaccharide substrates and their hydrolysis by Dispersin B

Abstract

Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB. The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also examined using mutant enzymes.

PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms

“…Furthermore, Bb -GH and Ec -GH lack hydrolytic activity on p NP-glycoside substrates and short PNAG oligomers and contain a pronounced and well-conserved substrate-binding groove suited to binding long stretches of polymer [ 24 ]. This is in contrast to the only other characterized PNAG hydrolase, DspB, which displays both exo and endo -acting hydrolysis of PNAG and has a shallow substrate-binding groove [ 53 , 54 ]. However, it does correlate with the bacterial cellulose and Psl biosynthesis systems, as BcsZ and PslG are both endo -acting glycoside hydrolases with long and deep substrate-binding clefts [ 25 , 26 ].…”

Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds

“…Indeed, microorganisms themselves utilize specific glycoside hydrolases to initiate dispersal events. For example, dispersin B is a ␤-hexosaminidase produced by the Gram-negative bacterium Aggregatibacter actinomycetemcomitans in order to disperse adherent cells from a mature biofilm (12). It has been shown that exogenous addition of dispersin B is capable of preventing and disrupting biofilms in vitro and in vivo (13)(14)(15)(16)(17).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Fluorescence-labeled hexasaccharide as a substrate for the processing enzymeglucosidase I. Facile synthesis of 1,2-cis glucosidic linkage (Iino et al, 2012) Glycosaminoglycan disaccharides TOF Use of a glucuronate glycosyl donor with a 2-O-acyl-6,3-lactone structure (Furukawa et al, 2011b) -Glycosyl amides TOF Synthesis from N-glycosyl dinitrobenzenesulfonamides (Gaitonde & Sucheck, 2012) Glycosyl disulfides MALDI (DHB) -glycosyl disulfides prepared in a few hours from per-O-Ac precursors (Adinolfi et al, 2011) -Glycosyl imidinium triflate TOF Elaboration to disarmed-armed iterative glycosylation (Lin et al, 2012c) Heparosan oligosaccharides TOF (DHB) Synthesis by Pasteurella multocida PmHS2 single-action transferases (Chavaroche et al, 2012) Chitosan oligosaccharides Effects on activation of murine spleen CD11c+ dendritic cells via toll-like receptor 4 TOF (Fekete et al, 2011) Low-MW chitosan oligosaccharides TOF By chitosanase hydrolysis of chitosan. Anti-inflammatory effects (Chung et al, 2012a) Mannosides with hydrophobic substituents TOF Synthesis as DC-SIGN antagonists (Obermajer et al, 2011) 15 N, 13 C 2 8-sialic acid oligomers TOF Evidence for helical structure in a tetramer of 2 8 sialic acid (Battistel et al, 2012) Oligo( -D-glycosyl phosphate) derivatives TOF Use of a phosphoramidite method via boranophosphate intermediates (Fujita et al, 2011b) Orthogonally protected disaccharide MALDI For synthesis of 6-sulfated derivatives with terminal ethylamine (Liu et al, 2012g) Pustulooligosaccharides ( (Yang et al, 2011f) Tri-, penta-, and heptasaccharides, with orthogonally N-protected amino residues TOF Synthesis of four bifunctionalized orthogonally N-protected oligosaccharides derived from lactose and mannose, intended as cross-linking derivatives (Fyrner et al, 2012a) Poly-N-acetyllactosamineextended TOF (DHB) For recognition studies by human and avian influenza A virus hemagglutinins …”