Search

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 (

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.

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.

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.

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.

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.

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.

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.

Phosphatidyl ethanolamine-binding proteins (PEBPs) are implicated in various critical physiological processes in all eukaryotes. Among them is Flowering Locus T (FT), the protein recently discovered as the vital flowering hormone in plants. Small molecule inhibitors and activators of FT could provide control over plant flowering and are therefore an interesting target for industrial agriculture. No small molecule inhibitors or activators are known for FT, but for a structurally similar PEBP, RKIP, an inhibitor called locostatin has been reported to covalently bind in the RKIP ligand binding pocket. Herein, we report the synthesis of novel locostatin-based chemical PEBP probes and evaluate their ability and selectivity towards the binding of FT and RKIP.

A growing body of evidence implicates ceramide and/or its glycosphingolipid metabolites in the pathogenesis of insulin resistance. We have developed a highly specific small molecule inhibitor of glucosylceramide synthase, an enzyme that catalyzes a necessary step in the conversion of ceramide to glycosphingolipids. In cultured 3T3-L1 adipocytes, the iminosugar derivative N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM) counteracted tumor necrosis factor-α–induced abnormalities in glycosphingolipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. When administered to mice and rats, AMP-DNM significantly reduced glycosphingolipid but not ceramide concentrations in various tissues. Treatment of ob/ob mice with AMP-DNM normalized their elevated tissue glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced A1C, and improved insulin sensitivity in muscle and liver. Similarly beneficial metabolic effects were seen in high fat–fed mice and ZDF rats. These findings provide further evidence that glycosphingolipid metabolites of ceramide may be involved in mediating the link between obesity and insulin resistance and that interference with glycosphingolipid biosynthesis might present a novel approach to the therapy of states of impaired insulin action such as type 2 diabetes.

Chapter 1 provides a perspective of synthetic organic chemistry as a discipline involved in the design, synthesis and evaluation of complex molecules. The reader is introduced with a brief history of synthetic organic chemistry, all the while dealing with different aspects of synthetic organic chemistry. These aspects include design, synthesis and evaluation of complex molecules, which are described with representative examples. For instance, chapter 1 described different strategies to design antibiotics like penicillin. Furthermore, efforts at the interface of chemistry and biology has led to the emerging of a new subdisciplines such as chemical biology. Although this discipline is relatively new, the scientific community has witnessed many breakthrough discoveries and some of these discoveries are highlighted in this chapter. After the general introduction presented in chapter 1, the following chapters of this thesis focus on the design, synthesis and evaluation of small molecular probes towards the study of proteins and glycans in plant and mammalian cells. Most of these probes are complex glycomimetic small molecules that in many instances are prepared with a nitrone-olefin [3+2] cycloaddition as a key step. In chapter 2 , a novel nitrone is presented for the nitrone-olefin [3+2] cycloaddition reaction. This reaction is a powerful tool in synthetic organic chemistry for the synthesis of a wide range of complex molecules. The versatile nature of the reaction is illustrated by the synthesis of several classes of natural product such as vitamins and alkaloids – all complex molecules containing multiple neighboring chiral centers – through the nitrone-olefin [3+2] cycloaddition. However, most nitrones that show good regio- and stereoselectivity are limited in their synthetic versatility, as subsequent synthetic modification of the cycloaddition products are limited. The nitrone described in chapter 2 is a novel masked aldehyde-containing nitrone. This nitrone is prepared by a simple and scalable procedure and can be combined with a diverse set of olefins and other dipolarophiles to afford a broad range of cycloadducts. These cycloadducts can be considered as a masked form of amino-aldehydes, which makes them interesting from a synthetic point of view as illustrated by several postcycloaddition modifications. The nitrone-olefin [3+2] cycloaddition is also utilized in chapter 3 for the synthesis of glycomimetic building blocks. Glycomimetics such as iminosugars and pipecolic acids are found in nature and possess a variety of biological activities. The potential of glycomimetics have led to the development of drugs for the treatment of diseases such as type 2 diabetes, Gaucher disease and HIV. Chapter 3 describes how glycomimetic building blocks can be obtained through a nitrone-olefin [3+2] cycloaddition, providing different bicyclic isoxazolidines. These cycloadducts are synthetically versatile, as we report a set of reactions that allow selective modification at each functional position. Accordingly, these versatile bicyclic isoxazolidines enable the synthesis of different glycomimetic building blocks. For example, we were able to make a library of pipecolic acid derivatives – a popular drug-motif – via a one-pot Staudinger/aza-Wittig/Ugi three-component reaction. The bicyclic isoxazolidines, discussed in chapter 3, are also reported in chapter 4 . This chapter describes the development of a synthetic route towards an activity-based probe (ABP) to study the enzymatic activity of neuraminidases. Neuraminidases are a class of enzymes found in a range of organisms including mammals. The importance of neuraminidases is illustrated by the existence of a neuraminidase-related genetic disease, sialidosis. With no cure for this fatal disease being very limited, there is keen interest in the discovery of novel mechanisms to restore neuraminidase activity. The poor enzyme-activity of a different enzyme-related disease, Gaucher disease, could be improved through the identification of a small molecule that stabilizes and/or promotes the folding of the active enzyme. The validation of this small molecule was aided greatly by the development of a sensitive ABP targeting this disease specific enzyme. Accordingly, the development of a neuramidase-ABP would provide a diagnostic tool to study the activity of neuramidases and ultimately help identify small molecules that could increase the activity of mutant-neuraminidase molecules by stabilizing and/or promoting the folding of this enzyme. The synthesis of a carbocyclic neuraminidase ABP was the goal of chapter 4 and was approached by starting with the nitrone-olefin [3+2] cycloaddition as the key-step. This reaction provided a bicyclic cycloadduct that was used for the development of a synthetic route, which led to a high yielding and practical synthesis of an advance intermediate possessing the majority of the stereochemistry of the required carbocyclic neuraminidase ABP. Chapter 5 describes the synthesis and evaluation of chemical tools to study phoshatidyl ethanolamine-binding proteins (PEBPs). This family of proteins is found in a variety of organisms including mammals and plants. This family includes FLOWERING LOCUS T (FT), a signaling protein that acts as a vital flowering hormone in plants. No small molecule inhibitors for FT are known, but an inhibitor called locostatin has been reported to bind in the highly conserved ligand binding site of a structurally related protein. Based on this conservation and overall structurally similarity with FT it was hypothesized that locostatin or derivatives thereof could covalently bind in the ligand binding pocket of FT and hence affect flowering. Chapter 5 reports on the synthesis of novel locostatin-based chemical PEBP probes, followed by evaluation for their ability and selectivity towards FT and a mammalian PEBP. Chemical tools were also used to study plants in chapter 6 , although different aspects of plants were investigated. This chapter describes the direct molecular imaging of carbohydrates (glycans) in Arabidopsis thaliana. Glycans play a crucial but not fully understood role in plant health and development. The formation of glycans is not genetically encoded, which makes it impossible to image them in vivo using genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycans that are metabolically incorporated in plants via the roots, which may then be visualized with copper-catalyzed click labeling. However, this labelling-technique is toxic to plants and future applications would benefit from bio-orthoganol copper-free labeling techniques. Chapter 6 shows, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods. These methods are bio-orthogonal and lead to more uniform labeling. Furthermore, this chapter also describes the metabolic incorporation of five novel monosaccharide probes in Arabidopsis thaliana roots and their imaging after (copper-free) fluorescent labeling. Finally, chapter 7 contains a general discussion, critically summarizing the body of this thesis along with additional ideas and recommendations for further research.

Continuous flow chemistry is an enabling technique in organic chemistry. Advantages include extremely fast mixing and heat transfer capabilities as well as rapid screening of reaction conditions. Combining continuous flow chemistry with solid-supported organocatalysis presents challenges that have been investigated in this thesis. We have developed a technique to modify the surface of silicon carbide with alkenes using microwaves. Silicon carbide is an extremely tough bio-compatible material and is of interest as a solid support. By using monolayers of bifunctional alkenes we were able to perform follow-up reactions to attach other molecules of interest, thereby further modifying the surface chemistry of silicon carbide. The use of microwaves speeds up the attachment of monolayers by as much as sixteen times. We have developed a new method for the surface functionalization of MCM-41, a type of mesoporous silica. Mesoporous silica is an interesting solid support for catalysts because of its large surface area. 1,7-octadiene can be covalently attached under mild conditions (24 h, 100 ºC). Follow-up thiol-ene click reactions can be performed using thiols. The use of 1,2-ethanedithiol leads to a material that can be reacted with the organocatalyst quinine, giving catalytically active mesoporous silica. This material was tested in flow and yielded full conversion of starting materials for the thio-Michael addition of 3-methoxythiophenol and 1-cyclohex-2-enone. Sulfonic acid supported on mesoporous silica (SBA-15) made via co-condensation of a thiol-containing silica precursor was used in a packed-bed continuous flow setup. This solid-supported sulfonic acid efficiently catalyzes the protection and deprotection of various alcohols as the tetrahydropyranyl-, trimethylsilyl and dimethylphenylsilyl derivatives under continuous flow conditions. By passing the starting material (primary, secondary, benzylic and phenolic alcohols) together with the appropriate protecting group over the packed bed more than 100 mg of product was produced in flow. Finally, the ring-opening polymerization of L-lactide using an organocatalyst was investigated. Using low catalyst loading and short residence times (0.25– 1.2% TBD catalyst, residence times as short as 2 s) well-defined poly(lactic acid) (PLA) is obtained with high (95–100%) monomer conversions and PDIs of typically 1.2. Use of the microreactor allows for a rapid screening of optimal reaction conditions, consistently yielding the optimal values for high conversion and low polydispersity. This quickly revealed that longer residence times will give rise to higher conversions and a concomitantly broader molecular weight distribution due to transesterification of the polymer backbone by TBD. The organocatalytic, metal-free continuous flow method described is rapid and mild enough to allow the use of a PEG-5,000 macroinitiator (yielding block copolymers), exo-BCN alcohol and even a base-labile tetrazine-derived alcohol, the latter of which cannot be used under traditional batch conditions. The resulting BCN-PLA and tetrazine-PLA materials were readily functionalized with small molecules and large polymers bearing azides and norbornenes via SPAAC and inverse electron demand Diels-Alder click chemistry. Despite the challenges that arise, the combination of continuous flow chemistry and (solid-supported) organocatalysis is a powerful concept that warrants further investigation.

Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis. After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2. Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated. In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface. With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system. In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide. In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment.