Ethan D. Goddard-Borger

Sensitivities of some imidazole-1-sulfonyl azide salts.

Imidazole-1-sulfonyl azide hydrochloride, an inexpensive and effective diazotransfer reagent, was recently found to be impact sensitive. To identify safer-to-handle forms of this reagent, several different salts of imidazole-1-sulfonyl azide were prepared, and their sensitivity to heat, impact, friction, and electrostatic discharge was quantitatively determined. A number of these salts exhibited improved properties and can be considered safer than the aforementioned hydrochloride. The solid-state structures of the chloride and less sensitive hydrogen sulfate were determined by single-crystal X-ray diffraction in an effort to provide some insight into the different properties of the materials.

Rapid Assembly of a Library of Lipophilic Iminosugars via the Thiol–Ene Reaction Yields Promising Pharmacological Chaperones for the Treatment of Gaucher Disease

A highly divergent route to lipophilic iminosugars that utilizes the thiol-ene reaction was developed to enable the rapid synthesis of a collection of 16 dideoxyiminoxylitols bearing various different lipophilic substituents. Enzyme kinetic analyses revealed that a number of these products are potent, low-nanomolar inhibitors of human glucocerebrosidase that stabilize the enzyme to thermal denaturation by up to 20 K. Cell based assays conducted on Gaucher disease patient derived fibroblasts demonstrated that administration of the compounds can increase lysosomal glucocerebrosidase activity levels by therapeutically relevant amounts, as much as 3.2-fold in cells homozygous for the p.N370S mutation and 1.4-fold in cells homozygous for the p.L444P mutation. Several compounds elicited this increase in enzyme activity over a relatively wide dosage range. The data assembled here illustrate how the lipophilic moiety common to many glucocerebrosidase inhibitors might be used to optimize a lead compounds ability to chaperone the protein in cellulo. The flexibility of this synthetic strategy makes it an attractive approach to the rapid optimization of glycosidase inhibitor potency and pharmacokinetic behavior.

Insights into mucopolysaccharidosis I from the structure and action of α-L-iduronidase

Mucopolysaccharidosis type I (MPS I), caused by mutations in the gene encoding α-L-iduronidase (IDUA), is one of approximately 70 genetic disorders collectively known as the lysosomal storage diseases. To gain insight into the basis for MPS I, we have crystallized human IDUA produced in an Arabidopsis thaliana cgl mutant. IDUA consists of a TIM barrel domain containing the catalytic site, a β-sandwich domain and a fibronectin-like domain. Structures of IDUA bound to induronate analogues illustrate the Michaelis complex and reveal a 2,5B conformation in the glycosyl-enzyme intermediate, that suggest a retaining double displacement reaction employing the nucleophilic Glu299 and the general acid/base Glu182. Surprisingly, the N-glycan attached to Asn372 interacts with iduronate analogues in the active site and is required for enzymatic activity. Finally, these IDUA structures and biochemical analysis of the disease-relevant Pro533Arg mutation have enabled us to correlate the effects of mutations in IDUA to clinical phenotypes.

Structure-activity relationship of karrikin germination stimulants

Karrikins (2H-furo[2,3-c]pyran-2-ones) are potent smoke-derived germination promoters for a diverse range of plant species but, to date, their mode of action remains unknown. This paper reports the structure-activity relationship of numerous karrikin analogues to increase understanding of the key structural features of the molecule that are required for biological activity. The results demonstrate that modification at the C5 position is preferred over modification at the C3, C4, or C7 positions for retaining the highest bioactivity.

An Expeditious Synthesis of Isofagomine

Isofagomine 1 (Scheme 1), a prominent member of the aza sugar family, was proposed and first synthesized by Lundt, Bols, and co-workers in 1994.[1] Subsequently, the molecule attracted considerable synthetic attention[2] owing to its strong inhibition of a range of enzymes that hydrolyze, in particular, the β-glycosidic linkage. One of the most expeditious syntheses of 1 starts from benzyl α-l-xylopyranoside and introduces the required nitrogen and extra carbon atom in the form of the nitrile 2.[2] This synthesis very closely follows our route to isofagomine, starting with benzyl β-l-xylopyranoside;[3] we have recently reported some

A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism

The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.

Potent human α-amylase inhibition by the β-defensin-like protein helianthamide

Selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent (Ki = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Stichodactyla helianthus. Active helianthamide was produced in Escherichia coli via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitory motif. Helianthamide represents the first of a novel class of glycosidase inhibitors and provides an unusual example of functional malleability of the β-defensin fold, which is rarely seen outside of its traditional role in antimicrobial peptides.

Glycosynthase-mediated assembly of xylanase substrates and inhibitors.

An exo‐β‐xylosidase mutant with glycosynthase activity was created to aid in the synthesis of xylanase substrates and inhibitors. Simple monosaccharides were easily elaborated into di‐, tri‐ and tetrasaccharides by using this enzyme. Some products proved to be surprisingly potent inhibitors of xylanases from glycoside hydrolase families 10 and 11.

Protein O-fucosylation in Plasmodium falciparum ensures efficient infection of mosquito and vertebrate hosts

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.

Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut.

The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.