Aloysius Siriwardena

Mechanistic insights into a Ca2+-dependent family of α-mannosidases in a human gut symbiont

Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are alpha-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca(2+)-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca(2+) plays a key catalytic role in helping distort the mannoside away from its ground-state (4)C(1) chair conformation toward the transition state.

The Reaction Coordinate of a Bacterial GH47 α‐Mannosidase: A Combined Quantum Mechanical and Structural Approach

Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic resolution structures along the reaction coordinate of an inverting α-mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α-D-mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.

Sugar‐Modified Foldamers as Conformationally Defined and Biologically Distinct Glycopeptide Mimics

It is predicted that over half of all eukaryotic proteins are glycosylated, and it is now well-established that co- and post-translational modification of proteins with glycans can have dramatic consequences on their folding, stability, and ultimately their function.[1] Considerable effort has thus been invested in delineating the impact of appended carbohydrates on the conformational preferences of proteins and peptides in solution and vice versa,[2] and also in understanding their interactions with their cognate receptors.[3] These endeavours are not straightforward, and success in rationalizing such processes has been possible only in a handful of well-studied cases.[4] Important insights into such questions have been gleaned from the study of glycoconjugate mimetics, whose interactions with cellular targets can impact a wide range of physiological phenomena, including fertilization, immune response, host–pathogen interactions, cell growth, and tumor metastasis.[1] However, attempts to successfully correlate biological functions of structurally well-defined glycopeptides with their secondary structures have been relatively sparse,[2–4] despite the importance of such targets in the quest for carbohydrate-based therapeutics.[5]

Unprecedented inhibition of glycosidase-catalyzed substrate hydrolysis by nanodiamond-grafted O-glycosides

We report herein the unprecedented finding that α-O-glucosides and also α-O-mannosides, when conjugated on nanodiamond particles (ND), are not only stable towards the hydrolytic action of the corresponding matching glycosidases, but are also endowed with the ability to inhibit them. Moreover, conjugation of the O-glycosides to ND (glyco-ND) sees them transformed into inhibitors of mismatching enzymes (for which they do not serve as substrates even when in their monovalent, free form). The effects of the glyco-NDs have been demonstrated on a panel of commercial glycosidases and the inhibition found to be competitive and reversible and not to be related to any denaturation of enzymes by the ND-conjugates. Values for Ki in the low micromolar range have been measured for certain glyco-ND (for example, a Ki value of 5.5 ± 0.2 μM was measured for the glucopyranosyl-coated NDs against the α-glucosidase from bakers yeast) and found to depend on both the identity of the enzyme and the glyco-ND. The latter Ki value compares well with that obtained for the natural glucosidase inhibitor, 1-deoxynojirimycin (Ki of 25 μM against the α-glucosidase from bakers yeast under identical assay conditions). The monovalent control O-glycosides was hydrolysed efficiently by the appropriate glycosidase. Glyco-ND bearing 50% loading of O-glycoside as well ND conjugated with both O-glucosides and O-mannosides (mixed) have also been assayed and shown also to inhibit the panel of glycosidases with potencies and selectivities different from those recorded for the 100% loaded ND and also from one another. The impact on factors such as glycotope density and heteromultivalency on inhibition is reminiscent of that typically encountered in carbohydrate–lectin recognition events. The abilities of the glyco-ND to bind, cross-link and aggregate concanavalin A, a lectin known to recognize both α-O-D-mannosides and α-O-D-glucosides, was assessed by a range of methods including an enzyme-linked lectin assay (ELLA), a two-site sandwich ELLA and a turbidimetry assay, respectively and indeed seen to reflect their expected per glycotope affinity enhancements as compared to monovalent controls: the high avidity of the lectin for each respective glycosylated ND particle was consistent with the manifestation of potent multivalent effects driving lectin recognition and binding.

Concise synthesis of C-1-cyano-iminosugars via a new Staudinger/aza Wittig/Strecker multicomponent reaction strategy.

A new Staudinger/aza Wittig/Strecker multicomponent reaction sequence to C-1-cyano iminoalditols has been developed. When applied to 5-azidodeoxy-d-xylose and -d-glucose as substrates the method leads smoothly in good yield and with excellent stereoselectivity to respectively, 1,5-dideoxy-1,5-imino-d-idurono nitrile and 2,6-didesoxy-2,6-imino-d-glycero-d-ido-heptononitrile.

Differentiation of Crohn’s Disease-Associated Isolates from Other Pathogenic Escherichia coli by Fimbrial Adhesion under Shear Force

Shear force exerted on uropathogenic Escherichia coli adhering to surfaces makes type-1 fimbriae stretch out like springs to catch on to mannosidic receptors. This mechanism is initiated by a disruption of the quaternary interactions between the lectin and the pilin of the two-domain FimH adhesin and transduces allosterically to the mannose-binding pocket of FimH to increase its affinity. Mannose-specific adhesion of 14 E. coli pathovars was measured under flow, using surface plasmon resonance detection on functionalized graphene-coated gold interfaces. Increasing the shear had important differential consequences on bacterial adhesion. Adherent-invasive E. coli, isolated from the feces and biopsies of Crohn’s disease patients, consistently changed their adhesion behavior less under shear and displayed lower SPR signals, compared to E. coli opportunistically infecting the urinary tract, intestines or loci of knee and hip prostheses. We exemplified this further with the extreme behaviors of the reference strains UTI89 and LF82. Whereas their FimA major pilins have identical sequences, FimH of LF82 E. coli is marked by the Thr158Pro mutation. Positioned in the inter-domain region known to carry hot spots of mutations in E. coli pathotypes, residue 158 is indicated to play a structural role in the allosteric regulation of type-1 fimbriae-mediated bacterial adhesion.

The Staudinger/aza-Wittig/Grignard reaction as key step for the concise synthesis of 1-C-Alkyl-iminoalditol glycomimetics

The scope of a one-pot tandem approach for the synthesis of C-1 alkyl iminoalditol derivatives with a Staudinger/aza-Wittig/Grignard cascade has been evaluated. The reaction conditions have been optimized for two azidodeoxy aldose substrates and a range of Grignard reagents. The nature of both, substrate as well as nucleophile, was found to control the stereoselectivity of the alkyl addition to the cyclic iminium intermediate at position C-1. This approach enabled the synthesis of a collection of C-alkyl iminoalditols, which were biologically evaluated as inhibitors against a set of standard glycoside hydrolases. All compounds were found to exhibit highly selective inhibition of β-glucosidase activity.

(1R,2S,4S,7aR)-1,2-Dihydroxy-7-thia-3a-thioniaperhydropentalene chloride: a new, biologically active pyrrolizidine alkaloid analogue

The cis-fused bicyclic sulfonium salt 8 has been synthesized in two steps from D-erythrose and shown selectively to inhibit human liver α-mannosidases.