This thesis has described novel synthetic methods to produce a variety of (glyco)peptides and their application in the study of various immunological processes. The first part of the thesis... Show moreThis thesis has described novel synthetic methods to produce a variety of (glyco)peptides and their application in the study of various immunological processes. The first part of the thesis describes novel insights into the pathogenesis of multiple sclerosis, in the form of new findings in the areas of lectin-driven immunotolerance and a biochemical comparison between human and animal model antigen. The next part describes novel multivalently glycosylated peptides, that can be used to study lectin binding and lectin mediated antigen uptake. The final part of the thesis describes a novel method to produce trans-cyclooctene protected peptides, an exciting new category of molecular tools within chemical biology that have recently been developed. Show less
Vancomycin is a last-resort antibiotic for the treatment of many Gram-positive bacterial infections, while remaining inactive against Gram-negative strains. Resistance to vancomycin in Gram... Show moreVancomycin is a last-resort antibiotic for the treatment of many Gram-positive bacterial infections, while remaining inactive against Gram-negative strains. Resistance to vancomycin in Gram-positive stains continues to develop. This thesis describes the recent developments in semisynthetically modifying glycopeptide antibiotics to improve their antibacterial activity. Furthermore, the development of several semisynthetic glycopeptide antibiotics are described including the guanidino lipoglycopeptides, the vancomyxins, and the vancomycin-sideromycins. The guanidino lipoglycopeptides are readily synthesized from vancomycin and display potent in vitro and in vivo activity against Gram-positive bacteria, including vancomycin-resistant strains. Assessment of the activity, properties, and mechanism of action of the guanidino lipoglycopeptides shows the potential of these novel glycopeptides to become best-in class. The vancomyxins, which consist of covalently conjugated vancomycin and outer membrane disruptor polymyxin nonapeptide, display enhanced activity against Gram-negative bacterial strains compared to vancomycin monotherapy or co-administration of the two components. The vancomycin-sideromycins are also aimed at conferring antibacterial activity against Gram-negative bacteria by exploiting an iron-uptake system. Overall, a variety of semisynthetic vancomycin derivatives, aimed at overcoming vancomycin resistance or sensitizing Gram-negative strains, are developed and assessed on their activity in this work. Show less
FiveC-glycosyl functionalized lysine building blocks, featuringC-glycosidic derivatives of alpha-rhamnose, alpha-mannose, alpha-galactose, beta-galactose, and beta-N-acetyl glucosamine have been... Show moreFiveC-glycosyl functionalized lysine building blocks, featuringC-glycosidic derivatives of alpha-rhamnose, alpha-mannose, alpha-galactose, beta-galactose, and beta-N-acetyl glucosamine have been designed and synthesized. These derivatives, equipped with acid-labile protecting groups, are eminently suitable for solid-phase synthesis of multivalent glycopeptides. The lysine building blocks were prepared fromC-allyl glycosides that underwent a Grubbs cross-metathesis with an acrylate, followed by a reduction of the C=C double bond in the resulting alpha,beta-unsaturated esters, and liberation of the carboxylate to allow condensation with a lysine side chain. The thus obtainedC-glycosides, five in total, were applied in the solid-phase peptide synthesis (SPPS) of three glycopeptides, showing the potential of the described building blocks in the assembly of well-defined mimics of homo- and heteromultivalent glycopeptides and glycoclusters. Show less
The aim of this thesis is to explore the glycosylation of PSA as well as to study if alterations can be observed between patients with indolent and malignant PCa. For this purpose the powerful... Show moreThe aim of this thesis is to explore the glycosylation of PSA as well as to study if alterations can be observed between patients with indolent and malignant PCa. For this purpose the powerful analytical platform CE-ESI-MS(/MS) was explored with a special focus on the analysis of glycans and glycopeptides (Chapter 1). The first section of the thesis describes the different method developments implemented for the analysis of PSA with CE-ESI-MS. Namely, Chapter 2 describes that CE-ESI-MS enables to separate glycopeptides with differently linked sialic acids without any additional sample treatment, Chapter 3 shows that an introduction of a dopant enriched nitrogen gas improves the limit of detection (sensitivity) of glycopeptides and Chapter 4 introduces a novel labeling procedure of total plasma N-glycome with the hydrazide Girard’s reagent P. Chapter 5 describes the development of a PSA Glycomics Assay which allows the capture of intact PSA from patients’ urine followed by analysis with the optimized CE-ESI-MS platform (Chapters 2 and 3). Finally, Chapter 6 offers a general discussion about future developments, the potential of PSA glycosylation in the clinical setting, showing the relevance of our results and how these may contribute to further clinical applications towards personalized medicine. Show less
Immunoglobulin G (IgG) represents the most abundant antibody class in the human circulation. IgG consists of two heavy chains and two light chains. Parts of the heavy chains, together with the... Show moreImmunoglobulin G (IgG) represents the most abundant antibody class in the human circulation. IgG consists of two heavy chains and two light chains. Parts of the heavy chains, together with the light chains, form two fragment antigen binding (Fab) moieties, whilst the remainders of the two heavy chains form the fragment crystallizable (Fc) moiety. Human IgGs are glycosylated at the highly conserved N-glycosylation site asparagine 297 in the CH2 domain of each heavy polypeptide chain of the Fc part. Fully galactosylated N-glycans are positioned between the Fc polypeptide chains, resulting in an open Fc conformation which is required for high affinity binding to Fc_ receptors. Small changes in the Fc glycosylation can already have a profound influence on the interaction of the Fc portion with receptors modulating the anti and pro-inflammatory properties of IgG. Mass spectrometry provides great opportunities for deta iled structural characterization of protein glycosylation including protein identification, determination of site-specific glycosylation profiles, and structural characterization of glycans at the level of released glycans and glycopeptides. In this thesis novel approaches for fast, miniaturized and high-throughput analysis of IgG Fc N-glycosylation are presented, and the utility of these methods has been demonstrated for clinically relevant research questions. Show less
The application of dedicated mass spectrometry (MS) and nuclear magnetic resonance (NMR) technologies for biomarker discovery and diagnostic purposes has increased substantially in the last decade.... Show moreThe application of dedicated mass spectrometry (MS) and nuclear magnetic resonance (NMR) technologies for biomarker discovery and diagnostic purposes has increased substantially in the last decade. In the studies presented in this thesis, we have used these technologies to identify parasite or host-derived products (biomarkers) related to infection and morbidity associated with schistosomiasis and to better understand the host-parasite interaction. The application of our peptidomics and metabonomics studies on schistosomiasis have provided some novel, valuable information but they are obviously only the first step. In addition to the potential biomarkers identified with the global biomarker discovery approaches, we showed in this thesis that a more targeted approach, looking at glycosylation, also resulted in novel information on S. mansoni infection. We have identified and characterized a set of human Apolipoprotein C-III peptides aberrantly glycosylated at the O-glycosylation site (Thr74), in urine of S. mansoni- infected individuals. The presented study is the first in which MS and NMR were used for the analysis of a cohort of human S. mansoni-infected individuals. This has resulted in the identification of a number of novel markers. Nevertheless, further studies are needed to evaluate the overall applicability of these putative biomarkers Show less
