Synthetic approaches to break the chemical shift degeneracy of glycans

NMR spectroscopy is the leading technique for determining glycans’ three-dimensional structure and dynamic in solution as well as a fundamental tool to study protein-glycan interactions. To overcome the severe chemical shift degeneracy of these compounds, synthetic probes carrying NMR-active nuclei (e.g., 13 C or 19 F) or lanthanide tags have been proposed. These elegant strategies permitted to simplify the complex NMR analysis of unlabeled analogues, shining light on glycans’ conformational aspects and interaction with proteins. Here, we highlight some key achievements in the synthesis of specifically labeled glycan probes and their contribution towards the fundamental understanding of glycans

The flexibility of oligosaccharides unveiled through residual dipolar coupling analysis

The intrinsic flexibility of glycans complicates the study of their structures and dynamics, which are often important for their biological function. NMR has provided insights into the conformational, dynamic and recognition features of glycans, but suffers from severe chemical shift degeneracy. We employed labelled glycans to explore the conformational behaviour of a β(1-6)-Glc hexasaccharide model through residual dipolar couplings (RDCs). RDC delivered information on the relative orientation of specific residues along the glycan chain and provided experimental clues for the existence of certain geometries. The use of two different aligning media demonstrated the adaptability of flexible oligosaccharide structures to different environments

Automated glycan assembly of ¹⁹F labelled glycan probes enables high‐throughput NMR studies of protein‐glycan interactions

Protein‐glycan interactions mediate important biological processes, including pathogen host invasion and cellular communication. Major challenges to monitoring these low affinity interactions are the required high sensitivity of a biophysical assay and to cover a breath of synthetic well‐defined structures. Here, we showcase an expedite approach that integrates automated glycan assembly (AGA) of 19 F labelled probes and high‐throughput NMR methods, enabling the study of protein‐glycan interactions. Synthetic Lewis type 2 antigens were screened against seven glycan binding proteins (GBPs), including DC‐SIGN and BambL, respectively involved in HIV‐1 and lung infections in immunocompromised patients, confirming the preference for fucosylated glycans (Le x , H type 2, Le y ). Previously unknown glycan‐lectin weak interactions were detected, and thermodynamic data were obtained. Enzymatic reactions were monitored in real‐time, delivering kinetic parameters. These results demonstrate the utility of AGA combined with 19 F NMR for the discovery and characterization of glycan‐protein interactions, opening up new perspectives for 19 F labelled complex glycans

Systematic structural characterization of chitooligosaccharides enabled by Automated Glycan Assembly

Chitin, a polymer composed of β(1-4)-linked N-acetyl-glucosamine monomers, and its partially deacetylated analogue chitosan, are abundant biopolymers with outstanding mechanical as well as elastic properties. Their degradation products, chitooligosaccharides (COS), can trigger the innate immune response in humans and plants. Both material and biological properties are dependent on polymer length, acetylation, as well as the pH. Without well-defined samples, a complete molecular description of these factors is still missing. Automated Glycan Assembly (AGA) enabled rapid access to synthetic well-defined COS. Chitin-cellulose hybrid oligomers were prepared as important tools for a systematic structural analysis. Intramolecular interactions, identified by molecular dynamics simulations and NMR analysis, underscore the importance of the chitosan amino group for the stabilization of specific geometries

Neighboring Group Participation of Benzoyl Protecting Groups in C3- and C6-Fluorinated Glucose

Fluorination is a potent method to modulate chemical properties of glycans. Here, we study how C3- and C6-fluorination of glucosyl building blocks influence the structure of the intermediate of the glycosylation reaction, the glycosyl cation. Using a combination of gas-phase infrared spectroscopy and first-principles theory, glycosyl cations generated from fluorinated and non-fluorinated monosaccharides are structurally characterized. The results indicate that neighboring group participation of the C2-benzoyl protecting group is the dominant structural motif for all building blocks, correlating with the β-selectivity observed in glycosylation reactions. The infrared signatures indicate that participation of the benzoyl group in enhanced by resonance effects. Participation of remote acyl groups such as Fmoc or benzyl on the other hand is unfavored. The introduction of the less bulky fluorine leads to a change in the conformation of the ring pucker, whereas the structure of the active dioxolenium site remains unchanged

Deoxyfluorination tunes the aggregation of cellulose and chitin oligosaccharides and highlights the role of specific hydroxyl groups in the crystallization process

Cellulose and chitin are abundant structural polysaccharides exploited by nature in a large number of applications thanks to their crystallinity. Chemical modifications are commonly employed to tune polysaccharide physical and mechanical properties, but generate heterogeneous mixtures. Thus, the effect of such modifications is not well understood at the molecular level. In this work, we examined how deoxyfluorination (site and pattern) impact the solubility and aggregation of well-defined cellulose and chitin oligomers. While deoxyfluorination increased solubility in water and lowered the crystallinity of cellulose oligomers, chitin was much less affected by the modification. The OH/F substitution also highlighted the role of specific hydroxyl groups in the crystallization process. This work provides guidelines for the design of cellulose- and chitin-based materials. A similar approach can be imagined to prepare cellulose and chitin analogues capable of withstanding enzymatic degradation

Non-natural oligosaccharides : from structural studies to the design of synthetic carbohydrate materials

Biopolymers self-assemble generating dynamic and complex functional materials that carry out sophisticated tasks. Nature has been a source of inspiration for chemists to create artificial analogs that mimic biopolymer self-assembly. In contrast to peptides and DNAs, carbohydrates are less understood at the molecular level and therefore have rarely been employed as scaffolds to construct self-assembling materials using bottom-up approaches. To date, carbohydrates’ potential in supramolecular chemistry remains mostly untapped. In this thesis, I established structure-property correlations for oligosaccharides and then translated this knowledge into design principles to access self-assembling carbohydrate materials. The use of non-natural monosaccharides and bottom-up approaches were central to deeply understand and design synthetic carbohydrate materials. In Chapter 2, I developed synthetic routes to fluorinated monosaccharide building blocks (BBs) and employed these BBs to assemble a broad collection of complex fluorinated glycans using Automated Glycan Assembly (AGA). Fluorination was exploited as a method to manipulate the hydrogen bonds of cellulose, a polysaccharide with high propensity to crystallize. The subtle role of the substitution pattern and position of the fluorine atom was investigated using X-ray diffraction analysis (XRD). In the second part of Chapter 2, I explored the use of fluorinated glycans as NMR probes. First, 19F-labelled glycans served for structural analysis via NMR revealing the tendency of some oligosaccharides to adopt helical conformations. Last, 19F-labelled Lewis antigens, a class of biologically relevant glycans, were employed to study their binding to proteins and to monitor in real-time enzymatic reactions. In Chapter 3, I developed a bottom-up approach to study the self-assembly and the chirality of supramolecular carbohydrate materials. In the first part, I employed a simple disaccharide that self-assembles into helical fibers to study the transfer of chirality across scales. Site-specific modifications identified key non-covalent interactions stabilizing the assembly. In the second part, I translated the bottom-up approach to a more complex natural polysaccharide and used synthetic oligomers to understand chirality transfer across scales in cellulose. Synthetic D- and L-cellulose oligomers were found to assemble into platelets with controlled dimensions that further aggregate into bundles, displaying chiral features directly connected to their monosaccharide composition. The insertion of L-Glc units in the sequence of D-Glc cellulose oligomers drastically impacted macroscopic properties such as solubility, crystallinity, and chirality of bundles. In Chapter 4, I presented the rational design and synthesis of a glycan adopting a stable secondary structure, challenging the common belief that glycans are not capable of folding due to their flexibility. By combining natural glycan motifs, stabilized by a non-conventional hydrogen bond and hydrophobic interactions, I designed a glycan hairpin, a secondary structure not present in nature. AGA enabled rapid access to synthetic analogs, including site-specific 13C-labelled ones, for NMR conformational analysis. Structural analysis via NMR unequivocally confirmed the folded conformation of the synthetic glycan hairpin. This work demonstrates that it is possible to program glycans to adopt defined conformations in aqueous solution. Overall, the bottom-up approaches used in this doctoral thesis allowed for a deeper understanding of the principles dictating carbohydrate self-assembly. The work presented here opens the way to future explorations of glycans as scaffolds for self-assembly or to perform complex functions as catalysis.Biopolymere sind selbstanordnende, dynamisch-komplexe Funktionsmaterialien, die bedeutende Aufgaben erfüllen. Für die Synthese künstlicher Analoga dienen der Wissenschaft die von der Natur genutzten Mechanismen der Selbstorganisation noch immer als Vorbild und Inspirationsquelle. Im Gegensatz zu Peptiden und DNA sind Kohlenhydrate auf molekularer Ebene bis jetzt nur wenig erforscht. Bisher wurden sie nur selten als Basis für selbstorganisierende Materialien mit Bottom-up-Ansätzen verwendet. Bis heute bleibt das Potenzial von Kohlenhydraten in der supramolekularen Chemie weitgehend ungenutzt. In dieser Arbeit habe ich Struktur-Eigenschafts-Korrelationen für Oligosaccharide erforscht und mit Hilfe dieser Designprinzipien gestaltet, die den Zugang zu selbstorganisierenden Kohlenhydratmaterialien ebnen. Dabei war die Verwendung nicht-natürlicher Monosaccharide und Bottom-up-Ansätze von zentraler Bedeutung, um ein tieferes Verständnis für die Entwicklung synthetischer Kohlenhydratmaterialien zu gewinnen. In Kapitel 2 entwickelte ich synthetische Wege zu fluorierten Monosaccharid-Bausteinen (BBs) und erschloss, unter zur Hilfenahme von Automated Glycan Assembly (AGA), eine breite Sammlung komplexer fluorierter Glykane. Die Fluorierung wurde als Methode zur Manipulation der Wasserstoffbrückenbindungen von Cellulose, einem Polysaccharid mit hoher Kristallisationsneigung, genutzt. Die Rolle des Substitutionsmusters und der Position des Fluoratoms wurde mit Hilfe der Röntgenbeugungsanalyse (XRD) untersucht. Im zweiten Teil von Kapitel 2 testete ich das Potential der fluorierten Glykane als NMR-Sonden. Zunächst dienten 19F-markierte Glykane zur Strukturanalyse mittels NMR, wobei die Tendenz einiger Oligosaccharide zur Annahme helikaler Konformationen deutlich wurde. Schließlich wurden 19F-markierte Lewis-Antigene, eine Klasse biologisch relevanter Glykane, zur Untersuchung ihrer Bindung an Proteine und zur Überwachung enzymatischer Reaktionen in Echtzeit eingesetzt. In Kapitel 3 entwickelte ich einen Bottom-up-Ansatz zur Untersuchung der Selbstorganisation und der Chiralität von supramolekularen Kohlenhydratmaterialien. Im ersten Teil konnte ich mit Hilfe eines einfachen Disaccharids, dass sich selbst zu spiralförmigen Fasern zusammensetzt, die Übertragung von Chiralität über mehrere Maßstäbe hinweg verfolgen. Durch ortsspezifische Modifikationen wurden wichtige nicht-kovalente Wechselwirkungen identifiziert, die den spezifischen Aufbau stabilisieren. Im zweiten Teil übertrug ich den Bottom-up-Ansatz auf das natürlich vorkommende Polysaccharid Cellulose. Die Studien der synthetischen Oligomere erlaubten es den Chiralitätstransfer über mehrere Maßstäbe hinweg zu verstehen. Ich konnte feststellen, dass sich synthetische D- und L-Cellulose-Oligomere zu Plättchen mit kontrollierten Abmessungen zusammensetzen, die sich wiederum zu chiralen Bündeln anordnen. Die Chiralität dieser Aggregate ist dabei direkt von der Monosaccharidzusammensetzung abhängig. Die Einfügung von L-Glc-Einheiten in die Sequenz von D-Glc-Cellulose-Oligomeren wirkte sich drastisch auf die makroskopischen Eigenschaften wie Löslichkeit, Kristallinität und Chiralität der Bündel aus. In Kapitel 4 habe ich das rationale Design und die Synthese eines Glykans mit einer stabilen Sekundärstruktur vorgestellt und damit die gängige Meinung widerlegt, dass Glykane aufgrund ihrer Flexibilität nicht faltbar sind. Durch die Kombination natürlicher Glykanmotive, die durch eine unkonventionelle Wasserstoffbrückenbindung und hydrophobe Wechselwirkungen stabilisiert werden, habe ich eine Glykan-Haarnadel entworfen, eine Sekundärstruktur, die in der Natur nicht vorkommt. AGA ermöglichte einen schnellen Zugang zu synthetischen Analoga, einschließlich ortsspezifischer 13C-markierter Spezies, die für NMR-Konformationsanalysen genutzt werden können. Die Strukturanalyse mittels NMR bestätigte eindeutig die gefaltete Konformation der synthetischen Glykan-Haarnadel. Diese Arbeit zeigt, dass es möglich ist, Glykane so zu programmieren, dass sie in wässriger Lösung definierte Konformationen annehmen. Insgesamt ermöglichten die in dieser Dissertation verwendeten Bottom-up-Ansätze ein tieferes Verständnis der Prinzipien, die die Selbstorganisation von Kohlenhydraten bestimmen. Die hier vorgestellte Arbeit öffnet den Weg für künftige Untersuchungen von Glykanen als Gerüste für die Selbstorganisation oder zur Ausführung komplexer Funktionen wie der Katalyse