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The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.

We describe the synthesis and coordination behaviour to copper(II) of two close structural triazacyclophane-based mimics of two often encountered aspartic acid and histidine containing metalloenzyme active sites. Coordination of these mimics to copper(I) and their reaction with molecular oxygen leads to the formation of dimeric bis(μ-hydroxo) dicopper(II) complexes.

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP1–19, plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP1–19 in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP1–19 is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP1–19 is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo

Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated monomeric, dimeric, and tetrameric [Tyr 3]octreotide-based analogues as a tool for tumor imaging and/or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar cycloaddition (click reaction) between peptidic azides and dendrimer-derived alkynes and a subsequent metal-free introduction of DOTA via the thio acid/sulfonyl azide amidation (sulfo-click reaction). In a competitive binding assay using rat pancreatic AR42J tumor cells, the monomeric [Tyr 3]octreotide conjugate displayed the highest binding affinity (IC50 = 1.32 nM) followed by dimeric [Tyr3]octreotide (2.45 nM), [DOTA0,Tyr3]octreotide (2.45 nM), and tetrameric [Tyr3]octreotide (14.0 nM). Biodistribution studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the 111In-labeled monomeric [Tyr3]octreotide conjugate had the highest tumor uptake (42.3 ± 2.8 %ID/g) at 2 h p.i., which was better than [111In-DOTA0,Tyr3]octreotide (19.5 ± 4.8 %ID/g). The 111In-labeled dimeric [Tyr 3]octreotide conjugate showed a long tumor retention (25.3 ± 5.9 %ID/g at 2 h p.i. and 12.1 ± 1.3 %ID/g at 24 h p.i.). These promising results can be exploited for therapeutic applications. © 2010 American Chemical Society.

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and b-cell death. Here, we used variants of the hIAPP1–19 fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, 2H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP–membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormone

This study focuses on the motion of passive tracers induced by the joint action of tidal and residual currents in shallow seas with an irregular bottom topography. Interest in this problem has rapidly increased in recent years, because of the detection of large-scale pollution of marine waters by plastics. Early simplified models considered advection of tracers by a two-dimensional depth-averaged velocity field that is solenoidal, thereby resulting in a system that is Hamiltonian and nonintegrable. Here, two new aspects are considered. First, the sensitivity of solutions to three different numerical schemes is investigated. To quantify the behavior of orbits, both the largest Lyapunov exponent and the K-coefficient of the zero-one test for chaos were calculated. It turns out that a new scheme, which extends a known symplectic scheme to systems that also contain non-Hamiltonian terms, performs best. The second aspect concerns the fact that a depth-averaged velocity field is actually divergent, thereby rendering the model of tracer motion to be non-Hamiltonian. It is demonstrated that the divergent velocity components, no matter how small, cause the appearance of attractors in the system and thus they have a strong impact on the fate of tracers. Interpretation of the numerical results is given by deriving and analyzing approximate analytical solutions of the system.

This thesis describes the design and synthesis of a bactericidal poly(ethylene glycol)-based (PEG) hydrogel coating with covalently attached antimicrobial peptides (AMP) stabilized against proteolytic degradation. As such, mimics of the highly active AMP HHC10 (H-KRWWKWIRW-NH2) were designed for optimal stability in human serum while retaining strong antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis, the major causative agents of biomaterial associated infection. In order to investigate the selectivity of the AMPs, their hemolytic activity was determined. A N-terminal cysteine facilitated thiol−ene chemistry for a fast, single-step immobilization/photopolymerization strategy. The antimicrobial activity of the resulting thin layer hydrogel coating on a PET surface was established using the Japanese Industrial Standard (JIS) Z2801 assay, showing complete killing (>99.9%) of inocula of S. aureus, S. epidermidis, and E. coli. The bactericidal hydrogel was molecularly characterized via Coomassie and Lowry assay protein staining agents as well as by X-ray photoelectron spectroscopy. To gain further insight into the biological stability, the hydrogels were incubated with human serum prior to activity testing without loss of activity. These studies revealed a promising bactericidal hydrogel with good stability under physiological conditions. However, the in vivo activity in mice could not be demonstrated and should be further investigated to prove the full potential of this methodology.

Key Points We present a comprehensive, seamless bed topography across the ice‐ocean margin around GreenlandTwo to 4 times more glaciers have calving fronts grounded below 200 m compared to previous mappingsTotal ice volume of Greenland is 2.99 ± 0.02 times 106 km3, yielding a potential sea level rise of 7.42 m, 7 cm greater than previous estimates, Greenland's bed topography is a primary control on ice flow, grounding line migration, calving dynamics, and subglacial drainage. Moreover, fjord bathymetry regulates the penetration of warm Atlantic water (AW) that rapidly melts and undercuts Greenland's marine‐terminating glaciers. Here we present a new compilation of Greenland bed topography that assimilates seafloor bathymetry and ice thickness data through a mass conservation approach. A new 150 m horizontal resolution bed topography/bathymetric map of Greenland is constructed with seamless transitions at the ice/ocean interface, yielding major improvements over previous data sets, particularly in the marine‐terminating sectors of northwest and southeast Greenland. Our map reveals that the total sea level potential of the Greenland ice sheet is 7.42 ± 0.05 m, which is 7 cm greater than previous estimates. Furthermore, it explains recent calving front response of numerous outlet glaciers and reveals new pathways by which AW can access glaciers with marine‐based basins, thereby highlighting sectors of Greenland that are most vulnerable to future oceanic forcing.

Magnetic inequivalence of nuclear spins is well known to cause additional splittings that complicate spectral analysis. Here, we present an extraordinary case of magnetic inequivalence, manifested in the 13-spin system of a 13C,15N-labeled trimethylated amine. All methyl group protons are chemically equivalent due to the molecular symmetry, but not all are magnetically equivalent as they have different 1JCH and 3JCH couplings. In general, spectra of such a large spin system can be expected to be extremely complicated by the presence of hundreds if not thousands of extra lines, caused by the strong coupling between inequivalent nuclei. Surprisingly, the 1H spectrum presented consists of very few lines, in a pattern of the utmost simplicity. Using sub-spectral analysis we show that this is due to weak coupling between the magnetically inequivalent nuclei, as a consequence of the particular combination of coupling constants. We find that the 4JHH geminal methyl coupling constant is 0.43 Hz and 2JCC is ∼0 Hz. In addition, we demonstrate that homo-decoupling can be used to transform the spin system to a set of fully equivalent spins, resulting in disappearance of 4JHH-splittings. We believe this curious case is a highly instructive example of magnetic inequivalence. The spectra may be considered deceptively simple, as fewer lines are observed than one would anticipate. At the same time, the spectra are deceptively complicated, as they can very well be approximated by intuitive reasoning.

Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manβ(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate and thus this structure can be uniquely extended. Next, the β-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and β-Man terminating antennae can sequentially be de-caged by an appropriate glycosidase to liberate a terminal β-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosylransferases. Asymmetric bi- and tri-antennary glycans could be obtained by removal of a terminal β-GlcNAc moiety by treatment with β-N-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary N-glycan found in human breast carcinoma tissue, which represents the most complex N-glycan ever synthesized. Multistage mass spectrometry of the two isomeric tri-antennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a α2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a α2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with α2-3 or α2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

As more and more bacteria become resistance against the drugs currently used in the clinic we are in dire need of novel antimicrobial compounds. To prevent resistance to occur against novel compounds it is important to carefully select the target against which such novel compounds act. Lipid II is an essential component in the synthesis of the bacterial cell wall and nature itself has proven that it is an effective target for antibacterial purposes. Several naturally occurring antibiotics bind to lipid II and are often not susceptible to resistance. However, these naturally occurring compounds are often not suitable for use in humans. The complex structure of such compounds prevents optimization of the molecule for therapeutic use. Therefore we set out to identify novel peptides that bind to lipid II which can potentially be developed into novel drugs. By using carbohydrate and peptide chemistry we synthesized analogues of lipid II that were suitable for use in phage display experiments. Phage display is an established and potent technique to identify binding peptides from a vast peptide library. As lipid II is a small molecule compared to the proteins that are commonly used in phage display, optimization of the screening conditions was first required. When the optimal conditions were found we analyzed the output of the phage display experiment by high-throughput DNA analysis. The obtained peptide sequences were synthesized and tested for their antibiotic activity. The activity assays led to the identification of three lead peptides that showed a lipid II mediated antibiotic effect against indicator organisms. To further optimize the potency of our lead peptides we modified them either N- or C-terminally with a lipid. The modification led to a significant increase in potency (8-64 fold) and more interestingly one of the compounds was now also effective against clinically relevant resistant enterococci. The lead peptide was further analyzed to prove its mode of action was through sequestration of lipid II. The phage display strategy we designed allowed the identification of novel lipid II binding antibiotic peptides from large libraries. By changing the library or make modifications to the target a large chemical space can quickly be searched for novel antibiotics.

Introduction: Consumer Quality Index questionnaires are used to assess quality of care from patients’ experiences. Objective: To provide insight into the agreement about quality of pharmaceutical care, measured both by a patient questionnaire and video observations. Methods: Pharmaceutical encounters in four pharmacies were video-recorded. Patients completed a questionnaire based upon the Consumer Quality Index Pharmaceutical Care after the encounter containing questions about patients’ experiences regarding information provision, medication counseling, and pharmacy staff’s communication style. An observation protocol was used to code the recorded encounters. Agreement between video observation and patients’ experiences was calculated. Results: In total, 109 encounters were included for analysis. For the domains “medication counseling” and “communication style”, agreement between patients’ experiences and observations was very high (90%). Less agreement (45%) was found for “information provision”, which was rated more positive by patients compared to the observations, especially for the topic, encouragement of patients’ questioning behavior. Conclusion: A questionnaire is useful to assess the quality of medication counseling and pharmacy staff’s communication style, but might be less suitable to evaluate information provision and pharmacy staff’s encouragement of patients’ questioning behavior. Although patients may believe that they have received all necessary information to use their new medicine, some information on specific instructions was not addressed during the encounter. When using questionnaires to get insight into information provision, observations of encounters are very informative to validate the patient questionnaires and make necessary adjustments.

The majority of the thesis concerns the work conducted on nisin-derived semisynthetic lipopeptides. These compounds were evaluated for their antimicrobial activity and a selection of compounds showed to be as active as the parent compound nisin, while having superior stability, lower charge and lower molecular weight, making for an overall more drug-like entity. Further work was done on PRMT (Protein Arginine Methyl Transferase) peptide substrates comprising unnatural amino acids and their ability to tune PRMT activity. Finally, the synthesis of a fluorescent amino acid suitable for solid phase peptide synthesis is described, as well as a photocleavable linker, that can be of use in activity based protein profiling.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid-liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 0.5-500ng/ml calibration range with r(2)=0.995±0.002 (n=6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n=18) were 2.7-11.7% and between-run (3 runs; n=18) precisions 3.0-14.5%. Accuracies were between 96-109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.

OBJECTIVE To investigate the relationship between ajmaline serum levels (pharmacokinetics, PK) and changes in the ST segment on the electrocardiogram (pharmacodynamics, PD) in patients with suspected Brugada syndrome (BS). DESIGN AND METHODS We determined the PK profile of ajmaline in 34 patients referred to the cardiology clinic for diagnosis of Brugada syndrome. The PD profile was derived from the ST elevations on the electrocardiograms of 26 patients during the diagnostics of BS. The PK and PD data were examined and fitted into non-linear mixed effect modelling (NONMEM 7). The final PK model was evaluated with data from 8 newly included patients. RESULTS Pharmacokinetics of ajmaline was best described with a two compartment model and the PK/PD data as a direct effect with a linear relationship between ajmaline serum levels and ST segment elevation. In BS-positive patients the baseline ST segment was 0.06 mV (rsd 35%) with a slope of 0.03 mV•mL/mg (rsd 94%). In BS-negative patients no association was found between ajmaline serum levels and ST segment elevation. CONCLUSION Pharmacokinetics of ajmaline in patients with suspected BS is best described with a two compartment model with linear elimination. A direct association between ajmaline serum levels and ST segment elevation in BS-positive patients was found. No association was found between ajmaline serum levels and ST segment elevation in BSnegative patients. In future studies this model can be used to examine the effects of co-variates in order to optimize the use of ajmaline in the diagnosis of BS

In recent years (2010-present) there has been an increase in the number of publications reporting the development, validation and use of bioanalytical methods in the rapidly expanding drug class of small molecule protein kinase inhibitors. Most reports describe the technological set-up of the methods that have allowed for drug concentration measurements from various sample types. This includes plasma, dried blood-spot, and tissue-analysis. Also method development, exploration of various techniques, as well as measurement and identification of metabolites were addressed. For the bioanalysis, a variety of sample-pretreatment methods like protein-precipitation, liquid-liquid extraction, and solid-phase extraction have been employed, all varying in complexity, cleanliness and time-consumption. Chromatographic separation, nowadays, is more focused on separating components from ion-suppressive effects, since for MS/MS detection, various components do not have to be baseline separated. For detection multiple types of detectors were used, ranging from state-of-the-art high resolution, and tandem mass spectrometry with low picogram per milliliter detection limits to the classical UV-detector with several nanograms per milliliter limits. As new bioanalytical methods have arisen that do rely on chromatographic separation, for example for high-throughput analysis, these are addressed in this review as well.

The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface. Such simultaneous binding arising from the toxins multivalency is believed to enhance its affinity. Thus, blocking the initial attachment of the toxin to the cell surface using inhibitors with GM1 subunits has the potential to stop the disease. Previously we showed that tetravalent GM1 molecules were sub-nanomolar inhibitors of CTB5. In this study, we synthesized a pentavalent version and compared the binding and potency of penta- and tetravalent cholera toxin inhibitors, based on the same scaffold, for the first time. The pentavalent geometry did not yield major benefits over the tetravalent species, but it was still a strong inhibitor, and no major steric clashes occurred when binding the toxin. Thus, systems which can adopt more geometries, such as those described here, can be equally potent, and this may possibly be due to their ability to form higher-order structures or simply due to more statistical options for binding. Carbohydrates combating cholera! Simultaneous binding of the five B-subunits of the cholera toxin (CTB5) enhances its affinity and facilitates its cellular entry. Thus, blocking the toxins initial attachment to the cell surface could stop the disease. The binding pattern and potency of tetra- and pentavalent CTB5 inhibitors based on the same GM1 ganglioside scaffold are compared for the first time.

Herein the synthesis of two nisin AB dicarba analogs is described, focusing on amino acid modifications at positions 2 and 5. The nisin mimics were synthesized by a combination of solid phase synthesis of the linear peptides, followed by macrocyclization via ring-closing metathesis and fragment assembly by means of solution phase chemistry. The two N-terminal nisin AB-fragment mimics contain either the native dehydrobutyrine (Dhb)/dehydroalanine (Dha) amino acid residues or alanine at position 2 and 5, respectively. The native dehydrobutyrine at position 2 and dehydroalanine at position 5 were introduced as their precursors, namely threonine and serine, respectively, and subsequent dehydration was carried out by EDCI/CuCl as the condensing agent. Both AB-fragment mimics were analyzed in a lipid II binding assay and it was found that the Ala2/Ala5 AB-mimic (2) showed a reduced activity, while the Dhb2/Dha5 AB-mimic (3) was as active as the native AB-fragment (1).

Summary: No effective targeted therapy is currently available for NRAS mutant melanoma. Experimental MEK inhibition is rather toxic and has only limited efficacy in clinical trials. At least in part, this is caused by the emergence of drug resistance, which is commonly seen for single agent treatment and shortens clinical responses. Therefore, there is a dire need to identify effective companion drug targets for NRAS mutant melanoma. Here, we show that at concentrations where single drugs had little effect, ROCK inhibitors GSK269962A or Fasudil, in combination with either MEK inhibitor GSK1120212 (Trametinib) or ERK inhibitor SCH772984 cooperatively caused proliferation inhibition and cell death in vitro. Simultaneous inhibition of MEK and ROCK caused induction of BimEL, PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.

A synthetic approach for the preparation of a new class of highly conjugated unnatural α-amino acids bearing a 5-arylpyrazole side-chain has been developed. Horner-Wadsworth-Emmons reaction of an aspartic acid derived β-keto phosphonate ester with a range of aromatic aldehydes gave β-aryl α,β-unsaturated ketones. Treatment of these with phenyl hydrazine followed by oxidation allowed the regioselective synthesis of pyrazole derived α-amino acids. As well as evaluating the fluorescent properties of the α-amino acids, their synthetic utility was also explored with the preparation of a sulfonyl fluoride derivative, a potential probe for serine proteases.

The lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity. Nisin is the most-prominent representative of the lantibiotics, and it inhibits the bacterial cell-wall biosynthesis by binding to lipid II via its N-terminus. The lipid II - nisin complex is responsible for pore-formation since the C-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. In order to increase the metabolic stability of nisin, the oxidation-sensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin AB(C) analogs containing alkane/alkene bridges.[1] The most interesting fragment of nisin is the C-terminal intertwined DE-ring which has a i->i+3, i+2->i+5 connectivity pattern. To obtain more insight into the importance of this cross-bridged structure on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged DE-ring mimic. This synthesis is based on the cyclization of a linear allylglycine-containing hexapeptide into the correctly knotted 1->4, 3->6 bicyclic hexapeptide using ring-closing metathesis, and all four diastereoisomers were obtained by HPLC and structurally characterized by NMR spectroscopy. An orthogonal protection scheme was used, to enable the independent N- or C-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. Via Cu(I)-catalyzed cycloaddition chemistries, alkyne-functionalized natural ABC-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by HPLC purification, have been conjugated to synthetic DE-ring mimics to obtain novel nisin derivatives and their affinity toward lipid II and pore-forming capacity have been studied. Herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic DE-ring mimics, and their use as synthons in Cu(I)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics.

Purpose: Regorafenib is a novel multikinase inhibitor, currently approved for the treatment of metastasized colorectal cancer and advanced gastrointestinal stromal tumors. We investigated whether regorafenib is a substrate for the multidrug efflux transporters ABCG2 and ABCB1 and whether oral availability, brain and testis accumulation of regorafenib and its active metabolites are influenced by these transporters. Methods: We used in vitro transport assays to assess human (h)ABCB1- or hABCG2- or murine (m)Abcg2-mediated active transport at high and low concentrations of regorafenib. To study the single and combined roles of Abcg2 and Abcb1a/1b in oral regorafenib disposition and the impact of Cyp3a-mediated metabolism, we used appropriate knockout mouse strains. Results: Regorafenib was transported well by mAbcg2 and hABCG2 and modestly by hABCB1 in vitro. Abcg2 and to a lesser extent Abcb1a/1b limited brain and testis accumulation of regorafenib and metabolite M2 (brain only) in mice. Regorafenib oral availability was not increased in Abcg2 -/- ;Abcb1a/1b -/- mice. Up till 2 h, metabolite M5 was undetectable in plasma and organs. Conclusions: Brain and testis accumulation of regorafenib and brain accumulation of metabolite M2 are restricted by Abcg2 and Abcb1a/1b. Inhibition of these transporters may be of clinical relevance for patients with brain (micro)metastases positioned behind an intact blood-brain barrier.

This review surveys developments in the field of ring-closing metathesis and cross-metathesis reactions applied to the synthesis of constrained amino acids, peptides, and peptidomimetics. Examples, in which metathesis is used as one of the synthetic tools to arrive at the desired peptide molecules as well as examples that describe in-depth optimized metathesis protocols, will be discussed. Currently, metathesis reactions are well-accepted synthetic tools within the field of peptide chemistry and provide peptides unprecedented properties like conformational, metabolic, and chemical stability and improved bioactivity.

Peptide and protein self-assembly resulting in the formation of amyloidogenic aggregates is generally thought of as a pathological event associated with severe diseases. However, amyloid formation may also provide a basis for advanced bionanomaterials, since amyloid fibrils combine unique material-like properties that make them very useful for design of new types of conducting nanowires, bioactive ligands, and biodegradable coatings as drug-encapsulating materials. The morphology of the supramolecular aggregates determines the properties and application range of these bionanomaterials. An important parameter to control the supramolecular morphology, is the overall charge of the peptide, which is related to the pH of the environment. Herein, we describe the design, synthesis and morphological analysis of a series of N-terminally functionalized Aβ(16-22) peptides (∼1/4Lys-Leu-Val-Phe-Phe-Ala-Glu-OH), that underwent a pH-induced polymorphism, ranging from lamellar sheets, helical tapes, peptide nanotubes, and amyloid fibrils as was observed by transmission electron microscopy. Infrared spectroscopy and wide angle X-ray scattering studies showed that peptide self-assembly was driven by β-sheet formation, and that the supramolecular morphology was directed by subtle variations in electrostatic interactions. Finally, a structural model and hierarchy of self-assembly of a peptide nanotube, assembled at pH 1, is proposed.

Streptococcus pneumoniae, the pneumococcus, is an important human pathogen causing considerable morbidity and mortality worldwide. This thesis addresses several interactions between pneumococcus and man. The first part of the thesis deals with the host immune response against pneumococci. We studied the anticapsular antibody response during pneumonia and found that only 45% of adult pneumococcal pneumonia patients mount a significant serotype-specific antibody response. We showed that corticosteroid use during pneumonia does not affect anticapsular pneumococcal antibody formation. In the second part of the thesis diagnostic options for pneumococcal pneumonia are discussed. We investigated the measurement of antibodies as an alternative method to detect pneumococcal pneumonia as well as two additional newly developed techniques: a quantitative PCR followed by capsular sequence typing, and a serotype-specific urinary antigen assay. Using all three techniques we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we investigated whether a urinary antigen test (UAT) could serve as a method to diagnose invasive pneumococcal pneumonia, classically defined as pneumonia in which S. pneumoniae is cultured from a normally sterile body site, by comparing the outcome of bacteraemic and non-bacteraemic/UAT-positive patients. A non-significant association with mortality was found for bacteraemia. In the third part of the thesis various techniques for distinguishing and typing cultured pneumococci are described. Two molecular serotyping techniques, a real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the cpsB gene within the pneumococcal capsulation locus, were compared. The rmPCR assay was shown to perform well for the 21 serotypes/-groups included in the assay and for serotype mixtures. The sequetyping assay performs well, with specific exceptions, and may be more useful for regions where vaccine serotypes are less common. To gain more insight in the genetic background of pneumococci, we developed a mixed genome microarray containing 470 DNA fragments and evaluated genetic differences between 445 invasive and non-invasive pneumococcal isolates. The microarray was able to genetically distinguish pneumococci at a deeper level than the typing techniques generally used. The fourth and last part of the thesis focuses on pneumococcal vaccination. We have studied the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) introduction in the Netherlands on the incidence and presentation of invasive pneumococcal disease (IPD). Four years after introduction of the vaccine, significant declines in overall IPD incidence were observed in children

OBJECTIVE: To increase the potential of membrane-acting peptides as possible novel drug-like compounds by increasing lipophilicity and thereby enhancing membrane affinity. DESIGN: An Fmoc-protected enantiomerically pure lipophilic amino acid (Fmoc-Lad-OH), which contains a nine carbon atom hydrophobic side chain, was designed. Fmoc-Lad-OH can be introduced into any peptide sequence using standard solid phase peptide synthesis to increase the lipophilicity of a peptide without sacrificing important polar segments of a peptide like for instance the N and C-termini. The antimicrobial decapeptide anoplin was chosen as a model peptide to test the hypothesis. METHODS: Fmoc-Lad-OH was prepared via organic synthesis and incorporated into the anoplin peptide sequence using solid-phase peptide synthesis followed by reversed-phase HPLC purification. Biological activity was evaluated using microtiter dilution bacterial growth assays, haemolytic assays and membrane vesicle leakage experiments. RESULTS: All three lipophilic analogues show a dramatic increase in antimicrobial activity: up to 4-8 times better for Escherichia coli (Gram-negative] and over one order of magnitude for Staphylococcus aureus (Gram-positive) compared to anoplin. Although the haemolytic activity was increased for the lipophilic analogues, the concentration at which 50% lysis will occur (EC50) was still one order of magnitude higher than the determined MICs. In the membrane vesicle leakage experiments the lipophilic analogues showed a higher lytic activity than anoplin, in agreement with the observed MIC values. CONCLUSION: Introduction of Lad into anoplin clearly showed a positive effect, which suggests that Fmoc-Lad-OH could be used as a general approach to increase membrane affinity of membrane-acting peptides.

Peptide-based materials constitute a class of molecules that play an important role in many biological processes and are utilized by many organisms to interact with their environment. One of the most well-known examples is spider silk, a material produced by web-spinning spiders composed of repeating stretches of amino acids that has a very high tensile strength. Many peptide-based materials are composed of repeating stretches of certain amino acids that confer function and (macroscopic) structure to these materials. In the first three research chapters a peptide material based on naturally occurring antifreeze molecules from arctic fish is described. In nature this material is composed of repeating stretches of a glycosylated tripeptide. Our mimic is synthesized by a copper-catalyzed polymerization reaction from a modified glycotripeptide with polymerization handles. This polymerization reaction introduces a non-natural element in the peptide backbone that was known to mimic the natural amide bond in previously published examples to a certain extent. A one-pot reduction and polymerization yielded a mixture of oligomers that could be separated by size. Upon analysis of the antifreeze activity of the resulting antifreeze mimics by an ice recrystallization experiment, the polymeric material was shown to have a strongly reduced antifreeze activity compared to the natural compound and a synthetic derivative synthesized previously. Structural analysis by circular dichroism showed a similar structure to the previously described and potent synthetic antifreeze glycopeptide, indicating that a small structural change can abolish the potent antifreeze properties of such molecules. The chemistry for introducing a urea moiety that can be incorporated into the antifreeze glycopeptides to enhance the interaction with the ice-lattice is described, using a 4-chlorophenyl glycoside synthon that can react with amines under mild conditions to form a ureido glycoside linkage. As a case study, this chemistry was used to synthesize a urea-containing glycopolymer based on the previously synthesized mimic. In chapter five a modification of the naturally occurring antibiotic nisin is described, wherein commercially available nisin was conjugated on the C-terminus with propargylamine, a handle for copper catalyzed click chemistry. In a second step this modified nisin could be coupled to fluorescent reporter molecules, and as a proof-of-concept, a small molecule with two azide handles. The successful synthesis of the dimeric nisin structure and retention of antimicrobial activity of all conjugates indicated the possibility to easily incorporate nisin in a functionalized (peptide) material or surface to obtain antimicrobial peptide based materials. In chapter six a dicysteine motif is described, which easily oxidizes to a cyclocystine. Initially found in a peptide derived from Tamm-Horsfall protein (F991) that was indicated to bind free-light chains, it became apparent that in serum this peptide rapidly formed the cyclocystine moiety. Attempts to use this structural motif in the beta-sheet forming human islet amyloid polypeptide as an oxidative switch are described. Preliminary results show a distorted structure by circular dichroism, but the resulting peptide still formed similar structures in aqueous buffer. Further attempts at increasing the change upon oxidation are described.

The interaction between proteins is important in all biological functions. In practically every cellular process protein complexes have been identified as essential components. Defects or disturbance in the regulation of protein-protein interactions are responsible for many diseases. Therefore, the development of modulators of protein-protein interactions has become an important topic in drug design. Peptide-based mimics of protein binding sites are believed to be promising candidates for modulating protein-protein interactions. The binding sites of proteins consist of discontinuous epitopes. Mimicry of discontinuous epitopes is a challenging task because of the complex nature of these epitopes. In order to achieve good mimics, the multiple binding segments of the native protein should be combined into one single molecule by employing a molecular scaffold approach. Moreover, discontinuous epitopes are often present as loop-like structures. Therefore, it is believed that it is necessary to use cyclic peptides in discontinuous epitope mimicry. The aim of this thesis was the development of a general applicable method for the mimicry of discontinuous epitopes employing scaffolded cyclic peptides. Although the mimicry of discontinuous protein binding sites is gaining more and more interest, the design and synthesis of molecules, which are functionally capable of mimicking the epitopes of natural proteins, is still a great challenge. In addition, most examples of discontinuous epitope mimicry by scaffolded cyclic peptides concern bicyclic systems, only few approaches allow a regioselective attachment of more than two pre-organized ligands to a molecular scaffold. With respect to design and synthesis of scaffolded cyclic peptides mainly three issues are involved: 1) molecular scaffold, 2) cyclic peptides containing a handle for conjugation, 3) an efficient conjugation method for attaching the cyclic peptides to the scaffold. These topics were studied in this thesis.

The accessibility to collections, libraries and arrays of cyclic peptides is increasingly important since cyclic peptides may provide better mimics of the loop-like structures ubiquitously present in and - especially - on the surface of proteins. The next important step is the preparation of libraries of ensembles of scaffolded cyclic peptides, which upon screening may lead to promising protein mimics. Here we describe the synthesis of a tri-cysteine containing scaffold as well as the simultaneous native chemical ligation of three cyclic peptides thereby affording a clean library of multiple cyclic peptides on this scaffold, representing potential mimics of gp120. Members of this collection of protein mimics showed a decent inhibition of the gp120-CD4 interaction.

Synthetic mimics of protein surfaces have the potential to become inhibitors of protein-protein interactions or even synthetic vaccines. However, the synthesis of these complicated molecular constructs is still difficult. Here we describe an efficient and versatile synthesis of protein mimics containing up to three different cyclic peptides. Using a sequential native chemical ligation strategy, peptide loops containing a thioester handle were introduced onto a triazacyclophane scaffold bearing orthogonal protected cysteine residues.

Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein-protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides. Using an orthogonal alkyne protection strategy, peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)-catalyzed azide alkyne cycloaddition. This method provides rapid access to protein mimics requiring different peptide segments for their interaction and activity.

SummaryNew lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.