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2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic analogue of the plant hormone auxin that is commonly used in many in vitro plant regeneration systems, such as somatic embryogenesis (SE). Its effectiveness in inducing SE, compared to the natural auxin indole-3-acetic acid (IAA), has been attributed to the stress triggered by this compound rather than its auxin activity. However, this hypothesis has never been thoroughly tested. Here we used a library of 40 2,4-D analogues to test the structure-activity relationship with respect to the capacity to induce SE and auxin activity in Arabidopsis thaliana. Four analogues induced SE as effectively as 2,4-D and 13 analogues induced SE but were less effective. Based on root growth inhibition and auxin response reporter expression, the 2,4-D analogues were classified into different groups, ranging from very active auxins to not active. A halogen at the 4-position of the aromatic ring was important for auxin activity, whereas a halogen at the 3-position resulted in reduced activity. Moreover, a small substitution at the carboxylate chain was tolerated, as was extending the carboxylate chain with two but not with one carbon. In the process, we also identified two 2,4-D analogues as efficient inducers of adventitious root formation and several possible anti-auxins. The auxin activity of the 2,4-D analogues was consistent with their simulated TIR1-Aux/IAA coreceptor binding characteristics. A strong correlation was observed between SE induction efficiency and auxin activity, indicating that the stress-related effects triggered by 2,4-D that are considered important for SE induction are down-stream of auxin signaling.

Understanding the mechanisms by which natural anti-freeze proteins protect cells and tissues from cold could help to improve the availability of donor organs for transplantation.

The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non‐saccharolytic, but recently it emerged that l‐fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l‐fucose utilisation. In the intestinal tract, l‐fucose is abundantly available in mucin O‐linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l‐fucose. We set out to determine how C. jejuni can gain access to these intestinal l‐fucosides. Growth of the fuc + C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l‐fucose while fucose permease knockout strains did not benefit from additional l‐fucose. With fucosidase assays and an activity‐based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco‐2 intestinal cells that express complex O‐linked glycan structures that contain l‐fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion.

The use of microreactor technology for the ring-opening polymerization of l-lactide catalyzed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene allows for rapid optimization of reaction parameters (reaction temperature and residence time). At moderate catalyst loading, good control over the polymerization is demonstrated by high conversion of monomer (>95%) and low polydispersity (

Alcohol protection and deprotection reactions, catalyzed by solid-supported sulfonic acid, have been investigated under continuous-flow conditions. Primary, secondary, benzylic and phenolic alcohols can be protected under these conditions by tetrahydropyranyl and several silyl ether moieties, generating synthetically useful amounts of material in short time. Furthermore, the described setup can be used to deprotect protected alcohols and be used in selective protection reactions. Because the solid-supported acid catalyst is continually reused in a packed-bed approach, workup is greatly simplified and in most cases only solvent removal is necessary, while reaching high turn-over numbers.

CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification, and degradation of complex carbohydrates. CAZypedia is closely connected with the actively-curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic, and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven, and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism - glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2) - is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator. Taking the best of both: Two established glucosylceramide synthase (GCS) inhibitors were merged via convergent synthesis to obtain hybrid compounds. Members of this 39-compound library have characteristics of both parent GCS inhibitors. No new GCS inhibitors were established, but a potent (200nm) acid glucosylceramidase (GBA1) inhibitor was identified. This adamantanemethyloxypenanoic acid pyrrolidene-substituted derivative of eliglustat can serve as a lead for further biomedical development of selective GBA1 modulators.

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.

A better characterization of nanometer-thick organic layers (monolayers) as used for engineering surface properties, biosensing, nanomedicine, and smart materials will widen their application. The aim of this study was to develop direct analysis in real time high-resolution mass spectrometry (DART-HRMS) into a new and complementary analytical tool for characterizing organic monolayers. To assess the scope and formulate general interpretation rules, DART-HRMS was used to analyze a diverse set of monolayers having different chemistries (amides, esters, amines, acids, alcohols, alkanes, ethers, thioethers, polymers, sugars) on five different substrates (Si, Si3N4, glass, Al2O3, Au). The substrate did not play a major role except in the case of gold, for which breaking of the weak Au-S bond that tethers the monolayer to the surface, was observed. For monolayers with stronger covalent interfacial bonds, fragmentation around terminal groups was found. For ester and amide-terminated monolayers, in situ hydrolysis during DART resulted in the detection of ions characteristic of the terminal groups (alcohol, amine, carboxylic acid). For ether and thioether-terminated layers, scission of C-O or C-S bonds also led to the release of the terminal part of the monolayer in a predictable manner. Only the spectra of alkane monolayers could not be interpreted. DART-HRMS allowed for the analysis of and distinction between monolayers containing biologically relevant mono or disaccharides. Overall, DART-HRMS is a promising surface analysis technique that combines detailed structural information on nanomaterials and ultrathin films with fast analyses under ambient conditions.

Porous aluminum oxide (PAO), a nanostructured support for, among others, culturing microorganisms, was chemically modified in order to attach biomolecules that can selectively interact with target bacteria. We present the first comprehensive study of monolayer-modified PAO using conditions that are relevant to microbial growth with a range of functional groups (carboxylic acid, a-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane). Their stability was initially assessed in phosphate-buffered saline (pH 7.0) at room temperature. The most stable combination (PAO with phosphonic acids) was further studied over a range of physiological pHs (4–8) and temperatures (up to 80 °C). Varying the pH had no significant effect on the stability, but it gradually decreased with increasing temperature. The stability of phosphonic acid-modified PAO surfaces was shown to depend strongly on the other terminal group of the monolayer structure: in general, hydrophilic monolayers were less stable than hydrophobic monolayers. Finally, an alkyne-terminated PAO surface was reacted with an azide-linked mannose derivative. The resulting mannose-presenting PAO surface showed the clearly increased adherence of a mannose-binding bacterium, Lactobacillus plantarum, and also allowed for bacterial outgrowth.

Multivalency plays a major role in biological processes and particularly in the relationship between pathogenic microorganisms and their host that involves protein–glycan recognition. These interactions occur during the first steps of infection, for specific recognition between host and bacteria, but also at different stages of the immune response. The search for high-affinity ligands for studying such interactions involves the combination of carbohydrate head groups with different scaffolds and linkers generating multivalent glycocompounds with controlled spatial and topology parameters. By interfering with pathogen adhesion, such glycocompounds including glycopolymers, glycoclusters, glycodendrimers and glyconanoparticles have the potential to improve or replace antibiotic treatments that are now subverted by resistance. Multivalent glycoconjugates have also been used for stimulating the innate and adaptive immune systems, for example with carbohydrate-based vaccines. Bacteria present on their surfaces natural multivalent glycoconjugates such as lipopolysaccharides and S-layers that can also be exploited or targeted in anti-infectious strategies., The authors acknowledge the support from EU COST Actions CM1102 and COST BM1003. In addition, the work reviewed has been supported by the sources listed in the original publications cited and by institutions mentioned in the affiliation list., The authors also thank the Unit of Information Resources for Research of the Spanish National Research Council (URICI-CSIC) for the co-financing of this publication in Open Access.

Second-order reaction kinetics of known strain-promoted azide–alkyne cycloaddition (SPAAC) reactions were compared with theoretical data from a range of ab initio methods. This produced both detailed insights into the factors determining the reaction rates and two straightforward theoretical tools that can be used to predict a priori the reaction kinetics of novel cyclooctynes for strain-promoted cycloaddition reactions. Multiple structural and electronic effects contribute to the reactivity of various cyclooctynes. It is therefore hard to relate a physical or electronic property directly and independently to the reactivity of the cyclooctyne. However, we show that Hartree–Fock LUMO energies, which were acquired while calculating activation energies at the MP2 level of theory, correlate with second-order kinetic rate data and are therefore usable for reactivity predictions of cyclooctynes towards azides. Using this correlation, we developed a simple theoretical tool that can be used to predict the reaction kinetics of (novel) cyclooctynes for SPAAC reactions.

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection. Since antisense detection is also possible in the absence of the TO label, our sensing platform based on azido-d-ornithine containing PNA even allows for additional and more advanced functionalization and sensing strategies.

A new masked aldehyde-containing nitrone 1 that is easily available through a facile one-step procedure has been developed. It undergoes a [3 + 2]-thermal cycloaddition with a wide range of dipolarophiles, affording isoxazolidine cycloadducts that are suitable for versatile postcycloaddition modifications. The acetal cycloadducts are acid-stable, but allow for acetal hydrolysis under mildly basic conditions. The isoxazolidine ring can be opened via an efficient one-pot procedure to give amine-protected γ-alcohols that can be further converted to furanose derivatives.

Glucosylceramide synthase (GCS) is an approved drug target for the treatment of Gaucher disease and is considered as a valid target for combating other human pathologies, including type 2 diabetes. The clinical drug N-butyldeoxynojirimycin (Zavesca) is thought to inhibit through mimicry of its substrate, ceramide. In this work we demonstrate that, in contrast to what is proposed in this model, the C2-hydroxyl of the deoxynojirimycin core is important for GCS inhibition. Here we show that C6-OH appears of less important, which may set guidelines for the development of GCS inhibitors that have less affinity (in comparison with Zavesca) for other glycoprocessing enzymes, in particular those hydrolases that act on glucosylceramide.

The structure–activity relationship of lipophilic aza-C-glycosides as inhibitors of the three enzymes of glucosylceramide metabolism is investigated. A library of β-aza-C-glycosides was synthesized with variations in N-alkylation and the linker length/type to the lipophilic moiety. A cross-metathesis reaction was used to prepare a second library of α-aza-C-glycosides with D-gluco, L-ido and D-xylo iminosugar cores possessing analogous linker variations. Evaluation of both libraries did not reveal a potent or selective inhibitor of glucosylceramide synthase. However, β-aza-C-glycoside 43 was found to be a selective inhibitor of β-glucosidase 2. The α-aza-C-glycosides – especially with a D-xylo core (e.g. 80) – proved to be very potent and selective inhibitors of glucocerebrosidase.

The lipophilic iminosugar N-[5-(adamantan-1-ylmethoxy)pentyl]-1-deoxynojirimycin (2, AMP-DNM) potently controls hyperglycemia in obese rodent models of insulin resistance. The reduction of visceral glycosphingolipids by 2 is thought to underlie its beneficial action. It cannot, however, be excluded that concomitant inhibition of intestinal glycosidases and associated buffering of carbohydrate assimilation add to this. To firmly establish the mode of action of 2, we developed a panel of lipophilic iminosugars varying in configuration at C-4/C-5 and N-substitution of the iminosugar. From these we identified the l-ido derivative of 2, l-ido-AMP-DNM (4), as a selective inhibitor of glycosphingolipid synthesis. Compound 4 lowered visceral glycosphingolipids in ob/ob mice and ZDF rats on a par with 2. In contrast to 2, 4 did not inhibit sucrase activity or sucrose assimilation. Treatment with 4 was significantly less effective in reducing blood glucose and HbA1c. We conclude that the combination of reduction of glycosphingolipids in tissue and buffering of carbohydrate assimilation by 2 produces a superior glucose homeostasis.

Die Entdeckung der Glycosphingolipide wird im Allgemeinen Johan L. W. Thudichum zugeschrieben, der 1884 uber die chemische Zusammensetzung des Gehirns publizierte. In seinen Studien isolierte er aus ethanolischen Hirnextrakten mehrere Verbindungen, die er als Cerebroside bezeichnete. Eine dieser Verbindungen, Phrenosin (heute als Galactosylceramid bekannt), unterwarf er einer sauren Hydrolyse, aus der drei Komponenten hervorgingen. Eine Komponente identifizierte er als Fettsaure, eine andere als Isomer zur D-Glucose, heute als D-Galactose bekannt. Die dritte Komponente mit “alkaloidaler Natur” gab Thudichum “viele Ratsel” auf, weshalb er sie Sphingosin nannte, nach dem mythologischen Ratsel der Sphinx. Derzeit sind Sphingolipide und ihre glycosidierten Derivate Gegenstand intensiver Forschung mit dem Ziel, ihre Funktion fur die strukturelle Integritat der Zellmembran, ihre Teilnahme in Erkennungs- und Signalubertragungsvorgangen und besonders ihre Beteiligung an pathologischen Prozessen, die als Ursache menschlicher Krankheiten (z. B. von Sphingolipidosen und Typ-2-Diabetes) gelten, aufzuklaren. Dieser Aufsatz wird auf neuere Befunde zur Biosynthese, Funktion und zum Abbau von Glycosphingolipiden im Menschen eingehen, mit Fokus auf das Glycosphingolipidglucosylceramid. Besondere Aufmerksamkeit richtet sich auf die klinische Bedeutung von Verbindungen, die mit den Faktoren des Glycosphingolipid-Metabolismus wechselwirken.

The discovery of the glycosphingolipids is generally attributed to Johan L. W. Thudichum, who in 1884 published on the chemical composition of the brain. In his studies he isolated several compounds from ethanolic brain extracts which he coined cerebrosides. He subjected one of these, phrenosin (now known as galactosylceramide), to acid hydrolysis, and this produced three distinct components. One he identified as a fatty acid and another proved to be an isomer of D-glucose, which is now known as D-galactose. The third component, with an "alkaloidal nature", presented "many enigmas" to Thudichum, and therefore he named it sphingosine, after the mythological riddle of the Sphinx. Today, sphingolipids and their glycosidated derivatives are the subjects of intense study aimed at elucidating their role in the structural integrity of the cell membrane, their participation in recognition and signaling events, and in particular their involvement in pathological processes that are at the basis of human disease (for example, sphingolipidoses and diabetes type 2). This Review details some of the recent findings on the biosynthesis, function, and degradation of glycosphingolipids in man, with a focus on the glycosphingolipid glucosylceramide. Special attention is paid to the clinical relevance of compounds directed at interfering with the factors responsible for glycosphingolipid metabolism.

A library of polyhydroxylated pyrrolidines was obtained by performing a tandem Staudinger/aza-Wittig/Ugi three-component reaction on a L-ribose-derived 4-azido aldehyde. In this paper we describe the effect of Lewis acids on the diastereoselectivity of the final Ugi three-component reaction step that the intermediate (D-lyxo-pyrroline) cyclic imine undergoes. When the Ugi reaction was performed in methanol almost exclusively pyrrolidines with a 2,3-cis relationship were formed. However, a significant amount of 2,3-trans product was formed upon addition of Lewis acids to the Ugi reaction mixture. The scope of this effect is explored by evaluating a diverse set of Lewis acids in combination with variation of other reaction parameters and the carboxylic acid/isocyanide component. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

A synthetic route for the preparation of glucosylceramide synthase inhibitor N-[5-(adamantan-1-yl-methoxy)-pentyl]-1-deoxynojirimycin methanesulfonic acid salt (AMP-DNM) has been developed. Herein we report the development and optimization of this synthetic route from its initial version in an academic research laboratory at milligram-scale to the final optimized route that was implemented in a cGMP miniplant on kilogram-scale. The definitive route starts with the separate synthesis of building blocks 2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin and 5-(adamantan-1-yl-methoxy)-pentanal. The aldehyde was synthesized from 1,5-pentanediol in five steps and 45% overall yield. Protected 1-deoxynojirimycin was prepared by a successive hemiacetal reduction/Swern oxidation/double reductive aniination sequence of 2,3,4,5-tetra-O-benzyl-D-glucopyranose in 52% overall yield. Reductive amination of the two building blocks produced the benzyl-protected penultimate that was isolated as its crystalline (+)DTTA salt in 68% yield. Hydrogenolysis of the penultimate and crystallization of the end product as its methanesulfonic acid salt produced AMP-DNM in 76% yield with a purity of > 99.5%. The described route enables the production of multikilogram amounts of inhibitor AMP-DNM as a stable crystalline solid with high purity under cGMP control

Starting from a chiral furanone, the nitrone-olefin [3 + 2] cycloaddition can be used to obtain bicyclic isoxazolidines for which we report a set of reactions to selectively modify each functional position. These synthetically versatile bicyclic isoxazolidines allowed us to obtain complex glycomimetic building blocks, like iminosugars, via multicomponent chemistry. For example, a library of 20 pipecolic acid derivatives, a recurring motif in various prescription drugs, could be obtained via a one-pot Staudinger/aza-Wittig/Ugi three-component reaction of a bicyclic isoxazolidine-derived azido-hemiacetal. Notably, specific pipecolic acids in this library were obtained via hydrolysis of an unique tricyclic imidate side product of the Ugi reaction. The azido-hemiacetal was also converted into an aza-C-glycoside iminosugar via an unprecendented one-pot Staudinger/aza-Wittig/Mannich reaction.

In this article, we present a straightforward synthesis of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives. The used synthetic routes are flexible and can be used to create a wide variety of lipophilic mono- and difunctionalized 1-deoxynojirimycin derivatives. The compounds reported here are lipophilic iminosugar based on lead compound 4, a potent inhibitor of the three enzymes involved in the metabolism of the glycosphingolipid glucosylceramide. Iminosugar-based inhibitors of glucosylceramide synthase, one of these three enzymes, have attracted increasing interest over the past decade due to the crucial role of this enzyme in glycosphingolipid biosynthesis. Combined with the fact that an increasing number of pathological processes are being linked to excessive glycosphingolipid levels, glucosylceramide synthase becomes a very attractive therapeutic and research target. Our results presented here demonstrate that relocating the lipophilic moiety from the nitrogen atom to other positions on the 1-deoxynojirimycin ring system does not lead to a more potent or selective inhibitor of glucosylceramide synthase. The beta-aza-C-glycoside analogue (17) retained the best inhibitory potency for glucosylceramide synthase and is a more potent inhibitor than the therapeutic agent N-butyl-1-deoxynojirimycin (3), marketed as treatment for Gaucher disease under the commercial name Zavesca.

Peracetylated N-alpha-azidoacetylmannosamine (Ac(4)ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by approximately 25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by approximately 75%. In conclusion, exposure of cells to Ac(4)ManNAz allows in vivo chemical tagging of gangliosides.

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.

This chapter commences with an overview of the different methods most commonly used to prepare glycosurfaces. The discussion on these methods is divided over three sections, each reflecting one of the three distinct approaches used to create glycosurfaces: direct formation of carbohydrate-containing self-assembled monolayers (SAMs), use of secondary (or tertiary) reactions to install a carbohydrate on a preformed SAM, and noncovalent immobilization of carbohydrates on a surface. Next, the chapter focuses on several key surface analysis techniques that can be used to characterize a prepared glycosurface and the type of information that can be obtained from each technique. Finally, it discusses several other applications of glycosurfaces in microbiology, focusing on binding, capture, and sensing of bacteria and bacterial toxins and on the multivalency effects that exert a large influence on the interaction between carbohydrates and proteins in biological systems and on fabricated glycosurfaces.

Porous aluminum oxide (PAO) is a nanoporous material used for various (bio)technological applications, and tailoring its surface properties via covalent modification is a way to expand and refine its application. Specific and complex chemical modification of the PAO surface requires a stepwise approach in which a secondary reaction on a stable initial modification is necessary to achieve the desired terminal molecular architecture and reactivity. We here show that the straightforward initial modification of the bare PAO surface with bromo-terminated phosphonic acid allows for the subsequent preparation of PAO with a wide scope of terminal reactive groups, making it suitable for (bio)functionalization. Starting from the initial bromo-terminated PAO, we prepared PAO surfaces presenting various terminal functional groups, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS). We also show that this wide scope of easily accessible tailored reactive PAO surfaces can be used for subsequent modification with (bio)molecules, including carbohydrate derivatives and fluorescently labeled proteins.

The preparation of highly functionalized pyrrolidines by a tandem Staudinger/aza-Wittig/ Ugi Three-Component Reaction (SAWU-3CR) is presented. The SAWU-3CR was applied on two different carbohydrate derived 4-azidopentanals and two libraries of chemically diverse products were prepared.

In this paper, a glycosylation-based approach towards novel aminoglycoside analogues is presented. Three different cyclitol building blocks were condensed with different thioglycosides to give, after removal of the protective groups, several neamine-type analogs varying in the positioning and the stereochemistry of the amino functions. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Enantiomerically pure 4,6-diaminocyclohexenols are obtained from carbohydrate derived 1,7-dienes by ring-closing metathesis and palladium catalyzed allylic amination using o-nitrobenzenesulfonylamides as nucleophiles. In the latter reaction the use of a cyclic carbonate as a leaving group proved to be essential to facilitate a smooth substitution. The obtained compounds were converted into orthogonally protected diaminocyclitols, which are stereoisomers of the naturally occurring 2-deoxystreptamine, a constituent of aminoglycoside antibiotics.

A new masked aldehyde-containing nitrone 1 that is easily available through a facile one-step procedure has been developed. It undergoes a [3 + 2]-thermal cycloaddition with a wide range of dipolarophiles, affording isoxazolidine cycloadducts that are suitable for versatile postcycloaddition modifications. The acetal cycloadducts are acid-stable, but allow for acetal hydrolysis under mildly basic conditions. The isoxazolidine ring can be opened via an efficient one-pot procedure to give amine-protected γ-alcohols that can be further converted to furanose derivatives.

This article covers recent developments in the design and application of activity-based probes (ABPs) for glycosidases, with emphasis on the different enzymes involved in metabolism of glucosylceramide in humans. Described are the various catalytic reaction mechanisms employed by inverting and retaining glycosidases. An understanding of catalysis at the molecular level has stimulated the design of different types of ABPs for glycosidases. Such compounds range from (1) transition-state mimics tagged with reactive moieties, which associate with the target active site—forming covalent bonds in a relatively nonspecific manner in or near the catalytic pocket—to (2) enzyme substrates that exploit the catalytic mechanism of retaining glycosidase targets to release a highly reactive species within the active site of the enzyme, to (3) probes based on mechanism-based, covalent, and irreversible glycosidase inhibitors. Some applications in biochemical and biological research of the activity-based glycosidase probes are discussed, including specific quantitative visualization of active enzyme molecules in vitro and in vivo, and as strategies for unambiguously identifying catalytic residues in glycosidases in vitro.

Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC50) of 104 pM for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.

Conjugation of a known, mechanism-based glycosidase inhibitor to sensitive fluorophores yielded unexpectedly potent and selective probes for quantifying active lysosomal glucocerebrosidase. These conjugates could prove to be invaluable tools for diagnosing and studying Gaucher disease.

Tuning the surface properties of mesoporous silica is desirable for many applications, such as catalysis. We present a new modification method of calcined mesoporous silica, based on the thermal grafting of 1,7-octadiene. This easy method generates a high loading of terminal alkenes. We show that the modified surface is directly available for thiol-ene click chemistry, which enables a wide variety of molecules to be attached to this alkene-functionalized mesoporous silica.

The rate of formation of covalently linked organic monolayers on HF-etched silicon carbide (SiC) is greatly increased by microwave irradiation. Upon microwave treatment for 60 min at 100 °C (60 W), 1-alkenes yield densely packed, covalently attached monolayers on flat SiC surfaces, a process that typically takes 16 h at 130 °C under thermal conditions. This approach was extended to SiC microparticles. The monolayers were characterized by X-ray photoelectron spectroscopy and static water contact angle measurements. The microwave-assisted reaction is compatible with terminal functionalities such as alkenes that enable subsequent versatile “click” chemistry reactions, further broadening the range and applicability of chemically modified SiC surfaces.

This article covers recent developments in the design and application of activity-based probes (ABPs) for glycosidases, with emphasis on the different enzymes involved in metabolism of glucosylceramide in humans. Described are the various catalytic reaction mechanisms employed by inverting and retaining glycosidases. An understanding of catalysis at the molecular level has stimulated the design of different types of ABPs for glycosidases. Such compounds range from (1) transition-state mimics tagged with reactive moieties, which associate with the target active site—forming covalent bonds in a relatively nonspecific manner in or near the catalytic pocket—to (2) enzyme substrates that exploit the catalytic mechanism of retaining glycosidase targets to release a highly reactive species within the active site of the enzyme, to (3) probes based on mechanism-based, covalent, and irreversible glycosidase inhibitors. Some applications in biochemical and biological research of the activity-based glycosidase probes are discussed, including specific quantitative visualization of active enzyme molecules in vitro and in vivo, and as strategies for unambiguously identifying catalytic residues in glycosidases in vitro.

Adding to the Antiviral Arsenal The envelope of influenza virus contains two immunodominant glycoproteins: hemagglutinin and neuraminidase (NA). Existing antivirals like zanamivir (Relenza) and oseltamivir (Tamiflu) target NA; however, the development of drug resistance is a problem. Kim et al. (p. 71 , published online 21 February) now report a different class of NA inhibitors. NA catalyzes the removal of sialic acids from the surface of host cells to initiate entry. Discovery of a NA–sialic acid intermediate led to the production of sialic acid analogs that bound covalently to NA and inhibited its enzymatic activity. These compounds showed activity against a wide variety of influenza strains, inhibited viral replication in cell culture, and were able to protect mice against influenza infection. Protection of mice was equivalent to protection seen from zanamivir. Moreover, the compounds showed activity against drug-resistant strains in vitro. These compounds represent a potentially useful addition to the arsenal of antivirals used to treat influenza infection.

Eight symmetric and pentavalent corannulene derivatives were functionalized with galactose and the ganglioside GM1-oligosaccharide (GM1os) via copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions. The compounds were evaluated for their ability to inhibit the binding of the pentavalent cholera toxin to its natural ligand, ganglioside GM1. In this assay, all ganglioside GM1os-sym-corannulenes proved to be highly potent nanomolar inhibitors of cholera toxin.

Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100000 compared to a monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB inhibitors. This journal is © The Royal Society of Chemistry 2013.

When this Letter was first published the author, Tom Wennekes, was omitted from the authorship list. The correct authorship is printed above.