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We present experimental proof that so-called "flowerlike micelles" exist and that they have some distinctly different properties compared to their "starlike" counterparts. Amphiphilic AB diblock and BAB triblock copolymers consisting of poly(ethylene glycol) (PEG) as hydrophilic A block and thermosensitive poly(N-isopropylacrylamide) (pNIPAm) B block(s) were synthesized via atom transfer radical polymerization (ATRP). In aqueous solutions, both block copolymer types form micelles above the cloud point of pNIPAm. Static and dynamic light scattering measurements in combination with NMR relaxation experiments proved the existence of flowerlike micelles based on pNIPAm(16kDa)-PEG(4kDa)-pNIPAm(16kDa) which had a smaller radius and lower mass and aggregation number than starlike micelles based on mPEG(2kDa)-pNIPAm(16kDa). Furthermore, the PEG surface density was much lower for the flowerlike micelles, which we attribute to the looped configuration of the hydrophilic PEG block. (1)H NMR relaxation measurements showed biphasic T(2) relaxation for PEG, indicating rigid PEG segments close to the micelle core and more flexible distal segments. Even the flexible distal segments were shown to have a lower mobility in the flowerlike micelles compared to the starlike micelles, indicating strain due to loop formation. Taken together, it is demonstrated that self-assemblies of BAB triblock copolymers have their hydrophilic block in a looped conformation and thus indeed adopt a flowerlike conformation.

Post-translational modifications are modifications of peptides or proteins after they have been synthesized in the ribosomes. These post-translational modifications (PTM’s) are intended to augment functional diversity of the 20 natural L-amino acids of which proteins are constructed. PTM’s have important functions and most are crucial for the tasks of the modified proteins. Sulfation was recognized as a post-translational modification of proteins in 1954 and it was slowly realized that tyrosine sulfation is a widespread modification of excreted and membrane bound peptides and proteins. Sulfation is catalyzed by two membrane bound tyrosylprotein sulfotransferases (TPST) located in the trans Golgi, which use PAPS as universal sulfate donor. Although sulfation is believed to be a widespread and biologically very important PTM, it has not been studied in great detail. This is mainly because of the instability of the sulfate group, especially under acidic conditions. In this thesis a new sequence independent synthesis method for the site selective introduction of multiple sulfated tyrosine residues into bioactive peptides is described. This method is applied on the synthesis of sulfated Leu-enkephalin and a collection of site selective sulfated N-terminal fragments of the Complement factor 5b Receptor (C5aR). This N-terminal part of the C5aR forms the binding site of the Chemotaxis Inhibitory Proteins of Staphylococcus aureus (CHIPS), an immunomodulatory protein excreted by S. aureus to prevent the C5aR from being activated by its natural ligand C5a. By preventing the activation of the C5aR, CHIPS can prevent the recruitment of specific white blood cells to the site of infection. The (over)activation of the C5a-receptor has been linked to several serious inflammatory diseases and therefore CHIPS and mimics of CHIPS could form a very interesting new class of anti-inflammatory compounds. The synthesized sulfated fragments of the C5aR are used to study the binding of CHIPS to the C5a-Receptor by ITC and NMR spectroscopy. The structure of CHIPS in complex with the N-terminus of the C5aR is elucidated, which reveals crucial structural elements within CHIPS for tight binding to the C5aR. These structural elements are selected and combined to design and synthesize new classes of C5aR inhibitors. One class based on the versatile TAC-scaffold were successfully synthesized but appeared to show no detectable affinity towards the sulfated C5aR fragments. A promising class of CHIPS mimics, based on a shortened CHIPS fragment including the fragments which interact with the C5aR, showed a low micromolar affinity towards the sulfated N-terminal part of the C5aR. The designed mimic CHOPS is a promising lead structure for development of new anti-inflammatory drug candidates.

Previously we have shown that the recombinantly produced SA2 amphiphilic oligopeptide (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu-COOH) self-assembles into nanovesicles (van Hell et al. 2007). In this study, the intermolecular interactions that contribute to the formation of such peptide vesicles are examined. First, analysis of a 3-hydroxyflavone fluorescent probe inserted into the peptide assemblies demonstrated that the peptide self-assembly is based on hydrophobic clustering. The polarity of this hydrophobic microenvironment was comparable to that of negatively charged lipid bilayers. A substantial level of hydration at the hydrophilic-hydrophobic interface was detected, as was further confirmed by tryptophan fluorescence analysis. However, organic solvents such as acetonitrile, tetrahydrofuran, or ethanol could not disrupt SA2 oligopeptide vesicles, whereas these solvents fully disintegrated lipid vesicles. Instead, the SA2 assembly immediately disintegrated in hydrogen breaking solvents such dimethylsulfoxide and dimethylformamide, suggesting the involvement of additional intermolecular interactions via hydrogen bonding. Circular dichroism and Fourier transform infrared spectroscopy excluded well-defined patterns of intramolecular hydrogen bonding and indicated the polyproline type II as the dominant SA2 peptide conformation, which enables intermolecular hydrogen bonding. All-atom computational simulations were used to confirm the presence of such intermolecular hydrogen bonds and degrees of hydration. On the basis of the experimental and computational data presented, we propose a model of an interdigitated peptide assembly that involves intermolecular hydrogen bonding in addition to hydrophobic interactions that stabilize SA2 oligopeptide vesicles.

The Spleen tyrosine kinase (Syk) protein functions as a switch in a number of receptor signaling cascades. One of these cascades is the high affinity IgE receptor (Fc?RI) signaling pathway. Fc?RI consists of an ?-, ?- and two ?-chains. The ?- and ?-chains have intracellular an Immunoreceptor Tyrosine based Activation Motif (ITAM). The ITAM sequence consists of Tyr-Xxx-Xxx-(Leu/Ile)-(Xxx6-8)-Tyr-Xxx-Xxx-(Leu/Ile), in which Xxx can be any amino acid and the Tyr-Xxx-Xxx-(Leu/Ile) sequences are, if phosphorylated, the binding epitopes of the SH2 domains of Syk. Upon IgE stimulation of the receptor, the ITAM motive is doubly phosphorylated. After this phosphorylation, Syk is recruited to the membrane where it becomes active. The first event in this activation process is the binding of the tandem SH2 domain (tSH2) of Syk to the phosphorylated ITAM motive on the ?-chain. Syk activation eventually leads to degranulation of the cell and thus release of mediators. Overstimulation of this pathway leads to allergic responses and therefore Syk is an interesting target for anti-allergic therapy. Recently, we have found that binding of Syk tSH2 to an ITAM peptide causes a significant change in the dynamics of tSH2. This change could imply that a change in the inter SH2 distance is necessary for Syk activation. To gain more insight into the functioning of Syk tSH2 we decided to replace the amino acid residues between the two binding epitopes of ITAM. Replacement with rigid building blocks preserved the high binding affinity. This rigid ITAM mimic was used in a pull-down approach to identify the proteins binding to it. Furthermore, the ITAM mimic was conjugated to different cell penetrating peptides, to evaluate the intracellular effects. The ITAM mimic proved to be a stimulator of mast cells degranulation. A rigid ITAM mimic with a larger linker between the SH2 binding epitopes was found to be an inhibitor of Syk kinase activity, as found by microarray experiments. In addition, a series of photoswitchable azobenzene-containing ITAM mimics was prepared. The interaction of the cis – and trans isomers with Syk tSH2 was assayed with SPR binding studies. For the smallest photoswitchable peptide a large difference in binding affinity between the cis – and trans isomer was found. Furthermore, one ITAM-derived tetrapeptidic SH2 binding epitope was conjugated to dendrimers via click chemistry to create a series of functional phosphopeptide-containing dendrimers ranging from a monovalent to an octavalent dendrimer. The tetra- and octavalent dendrimer had high nanomolar affinity for Syk tSH2, indicating a multivalency effect.

This report describes the design, synthesis, and biochemical evaluation of alkene- and alkane-bridged AB(C)-ring mimics of the lantibiotic nisin. Nisin belongs to a class of natural antimicrobial peptides, and has a unique mode of action: its AB(C)-ring system binds to the pyrophosphate moiety of lipid II. This mode of action was the rationale for the design of smaller nisin-derived peptides to obtain novel potential antibiotics. As a conformational constraint the thioether bridge was mimicked by an alkene- or alkane isostere. The peptides of the linear individual ring precursors were synthesized on solid support or in solution, and cyclized by ring-closing metathesis in solution with overall yields of between 36 and 89 %. The individual alkene-bridged macrocycles were assembled in solution by using carbodiimide-based synthesis protocols for the corresponding AB(C)-ring mimics. These compounds were tested for their binding affinity toward lipid II by evaluation of their potency to inhibit nisin-induced carboxyfluorescein release from large unilamellar vesicles. It was found that these AB(C)-ring mimics were not able to induce membrane leakage; however, they acted by inhibiting nisin-induced carboxy- ACHTUNGTRENUNGfluorescein release; this indicates their affinity toward lipid II. These results imply that an alkene or alkane moiety is a suitable thioether bridge mimic.

Bordetella pertussis, the etiological agent of whooping cough, is re-emerging in several countries with a traditionally high vaccine uptake. In these B. pertussis strains, polymorphisms were found in several proteins, including P.69 pertactin (P.69 Prn). P.69 Prn, an adhesin, contains two variable regions which are composed of repeats, one of which flanks the receptor binding site. Antibody titers against P.69 Prn correlate with protection and P.69 Prn is one of the components of acellular pertussis vaccines. Nevertheless, little is known about the structure and location of P.69 Prn epitopes. We used a three pronged approach to identify discontinuous epitopes that are recognized by mouse monoclonal antibodies, i.e. site-directed mutagenesis, deletion mapping and competition assays. Site-directed mutagenesis was focused on regions of P.69 Prn predicted to form loops according to the crystal structure. In this report we describe the location of several discontinuous epitopes that are also recognized by human antibodies. Our results reveal an important role of the N-terminus in immune recognition. We provide data for an indirect role of loops in immune evasion by masking of epitopes. We propose that the repeat regions have evolved to allow rapid antigenic variation to deflect the immune response from the functional domain of P.69 Prn. The results presented here provide a better understanding of the structure and function of variable loops and their role in the persistence of pathogens in immunologically primed populations.

We have developed a method that allows the rapid improvement of the affinity of phage-displayed antibody fragments by selection on intact eukaryotic cells. A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain variable region and DNA shuffling of both heavy and light chain variable regions. Higher-affinity mutants were selected from small phage display libraries by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displayed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the original antibody. Compared to previously reported affinity maturation schemes, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be expressed as a recombinant molecule or easily purified without disrupting its native configuration. In vitro tumor cell killing assays demonstrated an improved performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated better in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies.