Synthesis of Substituted Alkanethiols Intended for Protein Immobilization -Chelate Associated Photochemistry (CAP)

The first and main part of this thesis is focused on the design and synthesis of photo-activable and metal chelating alkanethiols. Chelate associated photochemistry (CAP) is a novel concept of combining two well-known protein (ligand) immobilization strategies to obtain a sensor surface of covalently bound ligand with defined orientation. This includes nitrilotriacetic acid (NTA) which is used to capture and pre-orientate histidine-tagged proteins to the sensor surface, followed by UV activation of a neighboring photo-crosslinking agent, benzophenone (BP), to covalently bind the ligand in this favorable orientation. Our results (paper 1) indicate that up to 55% more activity of the ligand is achieved with the CAP concept compared to the activity of the randomly oriented ligand (immobilized only by BP). This also yields a surface that is more robust compared to if only NTA is used. The photo cross-linking with benzophenone (BP) adduct is limited to a distance range of 3Å, it is therefore favorable to capture the ligand before reacting with surface bound BP-adduct. The surface consists of an excess of ethylene glycols (known for its protein-repellent properties) to prevent non-specific protein binding, thereby increase the specificity of the sensor surface. With this obtained surface chemistry we hope to contribute to the development of large-scale screening systems and microarrays based on His-tagged labeled biomolecules. This will be used in a number of applications such as proteomics-related applications, including drug discovery, the discovery of lead compounds and characterization of protein-protein interactions. The second part of this thesis describes the effect of the synthetic N-(3-oxododecanoyl)-Lhomoserine lactone (30-C12-HSL) on eukaryotic cells. 30-C12-HSL is a natural occurring signal substance in the bacterium Pseudomonas aeruginosa, and this signal molecule is involved in the regulation of bacterial growth. Pseudomonas aeruginosa has been considered as a common cause of infections in hospitals, especially in patients with compromised immune systems. Since the 30-C12-HSL can diffuse freely cross the cell membranes, it is expected to have influence on the host cell behaviour. Herein, we study how the eukaryotic cells respond to the bacterial signal molecule, 30-C12-HSL. Our results (paper 2) indicate that 30-C12-HSL disrupt the adherens junctions in human epithelial cells. The disruption is caused by a hyperphosphorylation of the adherens junction proteins (protein complex between epithelial tissues). This suggests the bacterial signals are sensed by that the host cells.