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
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
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
