Synthesis and Analysis of Specific Covalent Inhibitors of endo‐Xyloglucanases

Abstract

A series of N-bromoacetylglycosylamines and bromoketone C-glycosides were synthesised from complex xyloglucan oligosaccharide (XyGO) scaffolds as specific active-site affinity labels for endo-xyloglucanases. Compounds based on XXXG (Xyl3 Glc4 ) and XLLG (Xyl3 Glc4 Gal2 ) oligosaccharides exhibited strikingly higher affinities and higher rates of irreversible inhibition than known cellobiosyl and new lactosyl disaccharide congeners when tested with endo-xyloglucanases from two distinct glycoside hydrolase (GH) families. Intact-protein mass spectrometry indicated that inactivation with XyGO derivatives generally resulted in a 1:1 labelling stoichiometry. Together, these results indicate that XyGO-based affinity reagents have significant potential as inhibitors and proteomic reagents for the identification and analysis of diverse xyloglucan-active enzymes in nature, to facilitate industrial enzyme applications.

Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases

“…The surface‐cysteine variant VvEG16(−V152/C22S/C188S) was originally produced in an unsuccessful attempt to eliminate over‐labeling by a XXXG‐ N ‐bromoacetylglycosylamine inhibitor (Fenger and Brumer, ). Fortuitous crystallization of this variant in complex with the heptasaccharide XXXG in the negative subsites revealed identical interactions to those observed in the VvEG16(ΔV152/E89A):XyGO complex (Figure S14; Table S3).…”

Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor ofendo-xyloglucanases

“…The chemo-enzymatic synthesis of the heptasaccharide XXXG (2F)-β-DNP has enabled the production of one of the most structurally complex 2-deoxy-2-fluorosugar mechanism-based inactivators to date. Possessing active-site-residue specificity advantages over analogous xyloglucan oligosaccharide bromoketone C-glycoside and N-bromoacetylglycosylamine inhibitors, 63 we anticipate that XXXG(2F)-β-DNP may find continued use in structure-function analyses of endo-(xylo)glucanases from diverse GH families. In particular, N-bromoacetylglycosyl-amines generally appear to react with catalytic acid-base residues in configuration-retaining GHs, 46,48,64 contrasting the exclusive catalytic nucleophile specificity of the Withers-type fluorosugar inhibitors.…”

Chemoselective Reactions for the Synthesis of Glycoconjugates from Unprotected Carbohydrates

“…Recently, Fenger et al. have reported the synthesis of β‐C‐glycosyl bromopropanones as inhibitors of endo ‐xyloglucanases . The synthesis consisted of two steps.…”

“…[207] Recently,F enger et al have reported the synthesiso fb-Cglycosyl bromopropanones as inhibitors of endo-xyloglucanases. [208] The synthesis consisted of two steps. Lubineau'sreaction afforded the corresponding b-C-glycosyl propanones, which were subsequently a-brominated by treatment with bromine in methanol at 40 8C.…”

“…They have also shown that the barbiturate moiety can be hydrolyzed, [206] Scheme53. b-C-Glycosyl bromopropanonesp repared by Fenger et al [208] Scheme54. One-pot azide nucleophilic substitution and subsequent Cu I -catalyzed azide-alkyne cycloaddition from 1-bromo-3-(b-d-glucosyl)propan-2one.…”