Glycosaminoglycans (GAGs) such as heparin, heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS) are sulfated polysaccharides that exist on animal cell surfaces and in the extracellular matrix. GAGs are important in providing structural and hydrating support and interaction points for proteins of varied functions, for example growth factors and homeostasis regulatory proteins. Hepatocyte Growth Factor (HGF) is a protein growth factor that regulates cell growth, survival, proliferation, chemotaxis, cell morphology, tissue regeneration and angiogenesis. It is involved in embryogenesis, wound healing and many cancers. In this project, the interactions between the GAG binding N and NK -domains of HGF (HGF-N and HGF-NK) and different types of GAGs are characterised with biophysical techniques. GAG oligosaccharides were produced by enzymatic digestion and purified by preparative gel filtration and ion exchange chromatography. Different constructs of HGF were cloned from human cDNA, expressed with the Pichia pastoris expression system, purified to homogeneity and characterised by mass spectrometry and nuclear magnetic resonance (NMR). The dissociation constants between the different HGF protein constructs, different heparin oligosaccharide lengths and the drug Fondaparinux were shown by isothermal calorimetry (ITC) to vary between 0.35 and 9.26 μM. It was found that the entropy contribution was favourable for short oligosaccharides and disfavourable for long oligosaccharides and that the enthalpy contribution was less important for shorter oligosaccharides than for longer oligosaccharides. NMR titrations of CS, DS, heparin, Fondaparinux and sulfated maltose into 15N labelled protein samples showed that all ligands bind to the same HGF-N binding site, but different binding modes exists. The binding site consists of three regions, with the α2-helix and L2 loops playing key roles (residues 70-84). All GAGs also utilise the N-terminal residues 32-42, whereas long heparin oligosaccharides can also utilise a binding region formed mainly by the β2-strand (residues 59-64, 66, 95, 96). The GAG binding mode changes if HGF-N has an N-terminal truncation and the β2- strand residues become more important, emphasising the role of the N-terminal residues in the HGF-GAG interaction. Spin-labelled fully sulfated heparin-derived hexasaccharide was used to determine its binding direction on the HGF-N surface. Affinity chromatography confirmed the importance of the N-terminal residues and that HGF binds to all investigated GAGs. The oligomeric states of HGF-N and HGF-NK were investigated by AUC, gel filtration and ITC. The results suggest that the proteins oligomerise like beads on a string for long oligosaccharides. An HGF-N self-associating dimer with a slow on/off rate was characterised by affinity chromatography, gel filtration and NMR.

Glycosaminoglycans (GAGs) are a family of linear bio-polysaccharides, which are heterogeneously modified with negatively charged sulfate and carboxylate groups. They are located on every cell surface, extracellular matrix or intracellular space in the body. GAGs are composed of alternating units of an amino sugars (glucosamine or galactosamine) and hexuronic acid/hexose (iduronic acid, glucoronic acid/ or galactose), which are linked by glycosidic bonds with different geometries. In recent years, GAGs have attracted considerable interest. GAGs play vital roles in fundamental biological processes, such as hemostasis, angiogenesis, cell signaling, growth and differentiation. Thus, GAGs contribute to a number of diseases such as thrombosis, cancer, inflammation, osteoarthritis and degenerative diseases. One of the most studied GAGs is heparin, which is widely used as an anticoagulant and is also implicated in other biological processes. Despite its extensive clinical use, heparin continues to suffer from major problems, such as life threatening hemorrhagic complications and heparin-induced thrombocytopenia. These activities originate from the large number of glycan sequences generated during its biosynthesis. Many different enzymes act in a non-template fashion to produce heparin chains with various chain lengths, sulfation and acetylation patterns. Their inherent heterogeneity, complexity and highly anionic nature have seriously limited the development of tools for rapid identification of sequences critical for many biological activities. A RPIP-UPLC MS protocol was developed to separate and characterize structures of heparin oligosaccharides prepared through enzymatic cleavage process and chemoenzymatic synthesis. In designing such protocol, several UPLC and MS parameters were considered. An efficient separation of each oligosaccharide mixture was achieved with different ion-pairing reagents. Yet, the structural elucidation of the multiple chromatographic peaks was hindered by the heterogeneity inherent in these mixtures even with supposedly defined standards. In order to elucidate the structures of these complex molecules, we utilized a strategy, which exploits the ease with which sulfate groups can be fragmented during MS ionization. A systematic increment of the MS cone voltage was employed starting from a minimum dissociation voltage, where the intact molecule is preserved, to a high enough dissociation voltage, where the only core oligosaccharide backbone was retained. This allowed identifying the number of sulfate groups and the core structures. However, positions of substituents were difficult to pinpoint because of the phenomenal number of possibilities. Such analysis would require tandem MS/MS–based approaches.

Glycosaminoglycans (GAGs), a large family of complex, unbranched polysaccharides, display a variety of essential physiological functions. The structural complexity of GAGs greatly impedes their availability, thus making it difficult to understand the biological roles of GAGs and structure-property relationships. A method that can access GAGs and their analogs with defined structure at relatively large scales will facilitate our understandings of GAG biological roles and biosynthesis modulation. Cellulose is an abundant and renewable natural polymer. Applications of cellulose and cellulose derivatives have drawn increasing attention in recent decades. Chemical modification is an efficient method to append new functionalities to the cellulose backbones. This dissertation describes chemical modification of cellulose and cellulose derivatives to prepare unsulfated and sulfated GAG analogs. Through these studies, we have also discovered novel chemical reactions to modify cellulose. Systematic study of these novel chemistries is also included in this dissertation. We first demonstrated our preparation of two unsulfated GAG analogs by chemical modification of a commercially available cellulose ester. Cellulose acetate was first brominated, followed by azide displacement to introduce azides as the GAG amine precursors. The resulting 6-N3 cellulose acetate was then saponified to liberate 6-OH groups, followed by subsequent (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation of the liberated primary hydroxyl groups to carboxyl groups. Finally, the azides were reduced to amines using a novel reducing reagent, dithiothreitol (DTT). Alternatively, another process utilized thioacetic acid to reduce azides to a mixture of amine and acetamido groups. Through pursuing these GAG analogs, we applied novel azide reductions by DTT and thioacetic acid that are new to polysaccharide chemistry. We systematically investigated the scope of DTT and thioacetic acid azide reduction chemistry under different conditions, substrates, and functional group tolerance. Selective chlorination is another interesting reaction we discovered in functionalization of cellulose esters. We applied this chlorination reaction to hydroxyethyl cellulose (HEC). We then utilized the chlorinated HEC as a substrate for displacement reactions with different types of model nucleophiles to demonstrate the scope of its utility. Overall, we have designed a novel synthetic route to two unsulfated GAG analogs by chemical modification of cellulose acetate. Through exploration of GAG analogs synthesis, we discovered novel methods to modify polysaccharide and polysaccharide derivatives, including azide reduction chemistry and selective chlorination reactions. Successful synthesis of various types of GAG analogs will have great potential biomedical applications and facilitate structure-activity relationship studies. / Doctor of Philosophy / Polysaccharides are long chains of natural sugars. Glycosaminoglycans (GAGs) are an important class of polysaccharides which have complicated chemical structures and play critical roles in many biological processes, including regulation of cell growth, promotion of cell adhesion, anticoagulation, and wound repair. Current methods to obtain these GAGs and GAG analogs are expensive, lengthy, and limited in capability. Novel methods to access these GAGs and their analogs would be promising and would facilitate understanding of biological activities of GAGs. Cellulose is an abundant polymer on earth and provides structural reinforcement in plant cell walls. Cellulose can be further chemically modified to tailor its physiochemical properties. Cellulose and cellulose derivatives have been widely used in many industries for various applications, such as textiles, plastic films, automotive coatings, and drug formulation. This dissertation focuses on modifying inexpensive, abundant cellulose and its derivatives to GAGs and GAG analogs. We start from the simple plant polysaccharide cellulose and obtain structurally complicated analogs of animal-sourced GAGs and GAG analogs. We reached our goal by designing a carefully crafted synthetic route, finally successfully obtaining two types of novel GAG analogs. During this process, we discovered two useful chemical reactions. We systematically investigated these chemical reactions and demonstrated their utility for polysaccharide chemical modification. These successful chemical syntheses of GAGs and their analogs will accelerate our understanding of their natural functions and have potential biomedical applications. The novel chemical methods we discovered will be helpful in chemical modification of polysaccharides.

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.

Les Héparanes sulfates (HS) appartiennent à la famille des glycosaminoglycanes (GAGs) qui sont des polysaccharides existants sur la surface cellulaire ou dans la matrice extracellulaire des cellules animales. Les GAGs jouent des rôles essentiels dans plusieurs processus biologiques via leurs interactions avec certaines protéines (chemokines, cytokines, facteurs de croissance, enzymes…) dont ils modulent les activités biologiques. Ils sont constitués par la répétition d’un motif disaccharidique de base comportant un acide uronique lié à un 2-amino-sucre. Une diversité moléculaire considérable provient de l’existence de divers motifs de O-et/ou N-sulfatation ainsi que de motifs d’épimérisation au niveau de l’acide uronique. Cette diversité qui est responsable d’interactions spécifiques de haute affinité avec différentes protéines serait due à l’action des différentes enzymes de biosynthèse. Ce travail de thèse vise à développer de nouvelles méthodologies nécessaires à la préparation d’une chimiothèque octasaccharidique de fragments d’HS, dans le but d’étudier la spécificité des enzymes de biosynthèse des HS et principalement la N-déacétylase N-sulfotransférase (NDST) et la C-5 épimérase. La chimiothèque octasaccharidique peut être obtenue à partir d’un seul octasaccharide dont les quatre atomes d’azote seraient protégés par des groupements protecteurs différents (octasaccharide « N-différencié »). La synthèse de cet octasaccharide constitue notre objectif principal.Nous nous sommes intéressés dans un premier temps à l’optimisation de la synthèse d’une brique disaccharidique impliquée dans la synthèse des fragments d’héparane sulfate. Dans cet objectif nous avons mis au point une méthode d’acétylation sélective de l’hydroxyle primaire, une méthode de benzylation compatible avec la présence à l’acétate et une procédure « one-pot » comportant quatre étapes de synthèse du disaccharide et facilitant l’accès au disaccharide avec 45 % de rendement global.Par la suite, nous avons optimisé la réaction de glycosylation entre les briques disaccharidiques en utilisant le donneur N-phényltrifluoroacétimidate, dans le but d’avoir une stéréosélectivité α maximale et d’améliorer le rendement de glycosylation. Les conditions optimisées ont permis l’accès au tétrasaccharide et à l’octasaccharide ayant les fonctions amines sous forme de groupements azido, avec un excellent rendement (95 %) et une bonne stéréosélectivité (96/4) en faveur de l’anomère α et sont reproductibles à grande échelle. La troisième partie était consacrée à une étude méthodologique afin de permettre la synthèse de l’octasaccharide « N-différencié » précurseur de la chimiothèque octasaccharidique. Dans ce but, nous avons choisi les groupements Fmoc, Alloc, pNZ et N3 comme groupements protecteurs des fonctions amines de l’octasaccharide. Nous avons préparé différents accepteurs ayant les fonctions amines protégées avec ces groupements afin d’étudier l’influence des différents groupements N-protecteurs de la fonction amine de l’accepteur sur la réaction de glycosylation. Les différents tétrasaccharides ont été obtenus avec d’excellents rendements (87–97 %) et une bonne stéréosélectivité (autour de 95/5 en faveur de l’anomère α). Enfin, nous avons effectué une étude de l’orthogonalité de la déprotection de ces groupements protecteurs (N3, Alloc, Fmoc et pNZ). Cette étude est essentielle avant la préparation de l’octasaccharide pour prouver la stratégie de la synthèse. En plus cette étude facilitera la préparation de la chimiothèque octasaccharidique une fois l’octasaccharide « N-différencié » préparé. / Heparan sulfate (HS), a highly sulfated glycosaminoglycan present in the extracellular matrix and at the cell surface, is known to play vital functional roles in various biological processes due to its interactions with proteins (chemokines, cytokines, growth factors, enzymes ...). HS consist of a repeating disaccharide unit, composed of a glucosamine and a hexuronic acid (glucuronic acid or its C5 epimer, iduronic acid).The HS chains are further modified by some epimerases and sulfotransferases during their biosynthesis. These sulfation/epimerization patterns provide considerable complexity. These modifications are required for interaction with many protein ligands.This thesis aims to develop new methodologies for the preparation of a library of octasaccharides, HS fragments, in order to study the specificity of HS biosynthesis enzymes, mainly N-deacetylase N-sulfotransferase (NDST) and the C-5 epimerase. The library can be obtained starting from a single octasaccharide whose four nitrogen atoms are protected by various protecting groups (octasaccharide N-differentiated). The synthesis of this octasaccharide is our main goal.We are interested in first to optimize the synthesis of a fully protected disaccharide which is used as building block in our heparan sulfate fragments synthesis. For this purpose we have developed a regioselective acetylation method of a disaccharide bearing three hydroxyl groups using a temporary protection, a benzylation method compatible with the presence of one acetate group using silver oxide and a one-pot procedure incorporating up to four steps, reducing work-up and time-consuming purifications. In the second chapter, we optimized the glycosylation reaction between two disaccharides using the N- phényltrifluoroacétimidate donor in order to have good α stereoselectivity and yield. We are now able to obtain the tetrasaccharide and the octasaccharide with 95 % yield and (α/β : 96/4) stereoselectivity.The third part was devoted to the methodological study to obtain the N-differentiated octasaccharide, precursor of a octasaccharides library. For this purpose, we chose Fmoc, Alloc, pNZ and N3 as protecting groups of the amine groups. We have prepared various acceptors with these different protecting groups in order to study them in glycosylation reactions. The tetrasaccharides were obtained with excellent yields (87-97%) and good stereoselectivity (around 95/5 for the α anomer) with the four protecting groups. Finally, we conducted a study to deprotect these protecting groups (N3, Alloc, Fmoc and pNZ) on the tetrasaccharides. This study is essential before preparing the octasaccharide as a proof of the synthesis strategy. In addition this study will facilitate the preparation of the octasaccharides library once the N-differentiated octasaccharide prepared.

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.