Schistosomiasis is an acute and chronic disease caused by blood dwelling parasitic trematodes of the genus Schistosoma, and it is classified as the second most socioeconomically devastating... Show moreSchistosomiasis is an acute and chronic disease caused by blood dwelling parasitic trematodes of the genus Schistosoma, and it is classified as the second most socioeconomically devastating parasitic disease, second only to malaria. Currently the wormload is determined by the Kato-Katz method, which is not always reliable. In order to prepare diagnostic tools able to capture specific anti-carbohydrate antibodies or develop conjugate vaccines targeting these carbohydrate structures, sufficient amounts of well-defined fragments are needed. This thesis describes the synthesis of several glycans of these glycans present on the S. mansoni parasite, focusing mainly on the Circulating Anodic Antigen (CAA) and glycans bearing the unique α-(1-2)-L-Fucose-α-(1-2)-L-Fucose motifs. These glycans have been attached to gold nanoparticles and these particles were screened against several monoclonal antibodies and sera of individuals suffering from schistosomiasis. Show less
FiveC-glycosyl functionalized lysine building blocks, featuringC-glycosidic derivatives of alpha-rhamnose, alpha-mannose, alpha-galactose, beta-galactose, and beta-N-acetyl glucosamine have been... Show moreFiveC-glycosyl functionalized lysine building blocks, featuringC-glycosidic derivatives of alpha-rhamnose, alpha-mannose, alpha-galactose, beta-galactose, and beta-N-acetyl glucosamine have been designed and synthesized. These derivatives, equipped with acid-labile protecting groups, are eminently suitable for solid-phase synthesis of multivalent glycopeptides. The lysine building blocks were prepared fromC-allyl glycosides that underwent a Grubbs cross-metathesis with an acrylate, followed by a reduction of the C=C double bond in the resulting alpha,beta-unsaturated esters, and liberation of the carboxylate to allow condensation with a lysine side chain. The thus obtainedC-glycosides, five in total, were applied in the solid-phase peptide synthesis (SPPS) of three glycopeptides, showing the potential of the described building blocks in the assembly of well-defined mimics of homo- and heteromultivalent glycopeptides and glycoclusters. Show less
Purpose Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-conceptin vivostudy aims to validate the use of aberrant Lewis glycans as target for... Show morePurpose Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-conceptin vivostudy aims to validate the use of aberrant Lewis glycans as target for antibody-based, real-time imaging of gastrointestinal cancers. Procedures Immunohistochemical (IHC) staining with monoclonal antibody FG88.2, targeting Lewis(a/c/x), was performed on gastrointestinal tumors and their healthy counterparts. Then, FG88.2 and its chimeric human/mouse variant CH88.2 were conjugated with near-infrared fluorescent (NIRF) IRDye 800CW for real-time imaging. Specific binding was evaluatedin vitroon human gastrointestinal cancer cell lines with cell-based plate assays, flow cytometry, and immune-fluorescence microscopy. Subsequently, mice bearing human colon and pancreatic subcutaneous tumors were imagedin vivoafter intravenous administration of 1 nmol (150 mu g) CH88.2-800CW with the clinical Artemis NIRF imaging system using the Pearl Trilogy small animal imager as reference. One week post-injection of the tracer, tumors and organs were resected and tracer uptake was analyzedex vivo. Results IHC analysis showed strong FG88.2 staining on colonic, gastric, and pancreatic tumors, while staining on their normal tissue counterparts was limited. Next, human cancer cell lines HT-29 (colon) and BxPC-3 and PANC-1 (both pancreatic) were identified as respectively high, moderate, and low Lewis(a/c/x)-expressing. Using the clinical NIRF camera system for tumor-bearing mice, a mean tumor-to-background ratio (TBR) of 2.2 +/- 0.3 (Pearl: 3.1 +/- 0.8) was observed in the HT-29 tumors and a TBR of 1.8 +/- 0.3 (Pearl: 1.9 +/- 0.5) was achieved in the moderate expression BxPC-3 model. In both models, tumors could be adequately localized and delineated by NIRF for up to 1 week.Ex vivoanalysis confirmed full tumor penetration of the tracer and low fluorescence signals in other organs. Conclusions Using a novel chimeric Lewis(a/c/x)-targeting tracer in combination with a clinical NIRF imager, we demonstrate the potential of targeting Lewis glycans for fluorescence-guided surgery of gastrointestinal tumors. Show less
The glycosylation reaction is a pivotal reaction in creating new and complex oligosaccharides. These biologically relevant compounds must be obtained in enough quantity and in pure, well... Show moreThe glycosylation reaction is a pivotal reaction in creating new and complex oligosaccharides. These biologically relevant compounds must be obtained in enough quantity and in pure, well-defined form. Organic synthesis can provide these compounds, but efficient methods regarding their selective synthesis are often absent. Every saccharide building block is coupled to another in a glycosylation reaction, which must be both regioselective and stereoselective. For the stereoselectivity of this reaction common protocols are not generally applicable. This thesis investigates the stereoselectivity of the glycosylation reaction from the standpoint of the sugar acceptor, rather than from the sugar donor which has been the focal point for many decades. The results obtained showed that a scale of selectivities can be obtained by changing the reactivity of the acceptor. The trends within the acceptor reactivity are valid for all donors studied, but some donors are more susceptible than others to changes in acceptor reactivity. In the second part of this thesis the spatial identity of intermediates in the glycosylation reaction are investigated. Experimental and computational techniques combined resulted in an effective model to understand the glycosylation reaction on the SN1-side of the reaction spectrum when different functional groups are located on the carbohydrate donor. Show less
Beenakker, T.J.M.; Wander, D.P.A.; Codée, J.D.C.; Aerts, J.M.F.G.; Marel, G.A. van der; Overkleeft, H.S. 2017
Cyclophellitol and cyclophellitol aziridine are potent and irreversible inhibitors of retaining β‐glucosidases. They preferentially adopt a 4H3 half‐chair conformation, thereby mimicking the... Show moreCyclophellitol and cyclophellitol aziridine are potent and irreversible inhibitors of retaining β‐glucosidases. They preferentially adopt a 4H3 half‐chair conformation, thereby mimicking the substrate‐transition‐state conformation characteristic of retaining β‐glucosidases. As a consequence, both compounds bind tightly to the enzyme active site, and attack of the catalytic nucleophile onto the epoxide/aziridine results in enzyme deactivation. Replacement of the epoxide oxygen in cyclophellitol by a (substituted) carbon yielded carba‐cyclophellitols, a conceptually new class of inhibitors of retaining β‐glucosidases, as we demonstrated in a recent communication. In this paper, in‐depth synthetic studies of this class of compounds are described, and the preparation of a comprehensive set of structurally and configurationally new carba‐cyclophellitols is presented. Show less
The glycosylation, the reaction which forms a bond between sugar molecules (the donor and the acceptor), is the central reaction in carbohydrate chemistry. Despite tremendous advances in... Show more The glycosylation, the reaction which forms a bond between sugar molecules (the donor and the acceptor), is the central reaction in carbohydrate chemistry. Despite tremendous advances in the past decades, however, the glycosylation reaction remains relatively poorly understood. Especially the formation of 1,2-cis glycosidic linkages remains a significant challenge. This thesis describes an investigation of the influence of reactivity and selectivity of several classes of carbohydrate donors and –acceptors on the selectivity in glycosylation reactions. Special emphasis was placed on the influence of protecting groups on the donor, and nucleophilicity of the acceptor, two major factors that play a tremendous role in the outcome of a glycosylation reaction. The obtained knowledge has been applied in the synthesis of complex carbohydrate molecules, native to pathogens such as Staphylococcus aureus, of which antibiotic-resistant forms such as MRSA present significant danger in hospitals, and the parasite Schistosoma mansoni, causative agent of the neglected tropical disease schistosomiasis. The synthesis of these molecules can play a part in the development of vaccines targeted against these pathogens. Show less
Volbeda, A.G.; Reintjens, N.R.M.; Overkleeft, H.S.; Marel, G.A. van der; Codée, J.D.C. 2016
Carbohydrates or sugars, the most diverse class of biopolymers, are involved in many different biological processes. To be able to study these processes, well defined sugar structures are required.... Show moreCarbohydrates or sugars, the most diverse class of biopolymers, are involved in many different biological processes. To be able to study these processes, well defined sugar structures are required. The synthesis of these sugar structures is at this moment far from ideal and therefore requires fundamental research, in particular towards the glycosylation reaction. In this reaction a positively charged oxocarbenium ion can be considered as the product forming intermediate, the cation is however commonly reasoned to lead to non-selective reactions and product mixtures. Chemical calculations on the oxocarbenium ion combined with model glycosylations proved the contrary, namely that these oxocarbenium ions are in fact selective. With these results, insight into the glycosylation mechanism is improved. The orientation of specific substituents on the sugar ring proved to have a profound influence on the stability of the oxocarbenium ion and thereby on the stereochemical outcome of glycosylations. Show less
The processes of glycosidic bond formation and destruction are a central theme in glycochemistry and glycobiology, and form the basis of the research described in this Thesis. In the first part,... Show moreThe processes of glycosidic bond formation and destruction are a central theme in glycochemistry and glycobiology, and form the basis of the research described in this Thesis. In the first part, studies towards the stereoselective construction of two complex bacterial oligosaccharide fragments are described. These fragments contain mannuronic acid residues connected through a beta-linkage, which is amongst the most challenging linkages to construct synthetically. In the second part, the use of mannuronic acid building blocks in the automated synthesis of alginates using solid-phase chemistry is presented. Using a second-generation carbohydrate synthesizer, alginate fragments up to 12 residues were assembled with high stereoselectivity. Using the same automated set-up, fragments of hyaluronic acid of up to 15 residues were synthesized. In the third part, 2-deoxy-2-fluoroglucosides are investigated as activity-based probes for glucocerebrosidase, a retaining beta-glucosidase enzyme. Show less
Protecting groups play a key role in the synthesis of complex natural products.This holds especially true for the synthesis of oligosaccharides, of which the monomeric carbohydrate building blocks... Show moreProtecting groups play a key role in the synthesis of complex natural products.This holds especially true for the synthesis of oligosaccharides, of which the monomeric carbohydrate building blocks usually contain up to five different hydroxyl functions. The discrimination of these hydroxyl functions requires a careful protecting group strategy and typically involves multistep protocols.This thesis describes the prepartion, installation, their use in the synthesis of stereoselective glycosidic bonds. Although protecting groups primarily function to mask a given functionality on the carbohydrate core, they also have a profound effect on the overall reactivity of a carbohydrate building block and can control the stereochemical outcome of a glycosylation reaction. Furthermore protecting groups can be used to introduce extra functionality on the carbohydrate core, such as visualization and/or purification handles. Fluorous solid phase extraction (FSPE) is an emerging tecnique, in which compounds are seperated on the basis of flourous content. The compound bearing fluorous tags which are difficult to purify by routine methods can easily be purified. Show less
The first half of this thesis describes the synthesis of several conformationally restricted alkylated and bicyclic sugar amino acids (SAAs). The second half of the thesis describes the application... Show moreThe first half of this thesis describes the synthesis of several conformationally restricted alkylated and bicyclic sugar amino acids (SAAs). The second half of the thesis describes the application of the SAAs and their intermediates presented in the first half, as components of tools applied for the probing of biological systems. Show less
In this thesis new strategies towards biologically active oligosaccharides are described. In addition a detailed mechanistic study is performed to investigate the stereodirecting capacity of... Show moreIn this thesis new strategies towards biologically active oligosaccharides are described. In addition a detailed mechanistic study is performed to investigate the stereodirecting capacity of glycosyl C-5 substituents in systems that were devoid of any other stereodirecting factors. The postulated mechanism described here can aid in the design of glycosylation strategies. Show less
This Thesis reports on research aimed at the assembly of acidic and zwitterionic polysaccharides of bacterial origin, using suitably protected 1-thioglycoside residues. Thioglycosides are... Show moreThis Thesis reports on research aimed at the assembly of acidic and zwitterionic polysaccharides of bacterial origin, using suitably protected 1-thioglycoside residues. Thioglycosides are attractive monosaccharide building blocks because of their high stability towards the diverse reaction conditions used in functional group manipulations. Recently, the use of 1-thioglycosides has been stimulated by the advent of the 1-benzenesulfinylpiperidine (BSP)/triflic anhydride (Tf2O) and the diphenylsulfoxide (Ph2SO)/Tf2O activator system. These sulfonium ion based activators proved to be efficient promoters for glycosylations in which inreactive (disarmed) 1-thioglycosides are employed. Furthermore, one-pot orthogonal and chemoselective oligosaccharide synthesis strategies that involve the use of 1-thioglycosides in combination with the above mentioned sulfonium based activator systems have proven to be feasible. Show less
