Identifying the origin of local flexibility in a carbohydrate polymer.

Correlating the structures and properties of a polymer to its monomer sequence is key to understanding how its higher hierarchy structures are formed and how its macroscopic material properties emerge. Carbohydrate polymers, such as cellulose and chitin, are the most abundant materials found in nature whose structures and properties have been characterized only at the submicrometer level. Here, by imaging single-cellulose chains at the nanoscale, we determine the structure and local flexibility of cellulose as a function of its sequence (primary structure) and conformation (secondary structure). Changing the primary structure by chemical substitutions and geometrical variations in the secondary structure allow the chain flexibility to be engineered at the single-linkage level. Tuning local flexibility opens opportunities for the bottom-up design of carbohydrate materials.

Visualizing Chiral Interactions in Carbohydrates Adsorbed on Au(111) by High-Resolution STM Imaging.

Carbohydrates are the most abundant organic material on Earth and the structural "material of choice" in many living systems. Nevertheless, design and engineering of synthetic carbohydrate materials presently lag behind that for protein and nucleic acids. Bottom-up engineering of carbohydrate materials demands an atomic-level understanding of their molecular structures and interactions in condensed phases. Here, high-resolution scanning tunneling microscopy (STM) is used to visualize at submolecular resolution the three-dimensional structure of cellulose oligomers assembled on Au(1111) and the interactions that drive their assembly. The STM imaging, supported by ab initio calculations, reveals the orientation of all glycosidic bonds and pyranose rings in the oligomers, as well as details of intermolecular interactions between the oligomers. By comparing the assembly of D- and L-oligomers, these interactions are shown to be enantioselective, capable of driving spontaneous enantioseparation of cellulose chains from its unnatural enantiomer and promoting the formation of engineered carbohydrate assemblies in the condensed phases.

Controlling the Assembly of Cellulose-Based Oligosaccharides through Sequence Modifications.

Peptides and nucleic acids with programmable sequences are widely explored for the production of tunable, self-assembling functional materials. Herein we demonstrate that the primary sequence of oligosaccharides can be designed to access materials with tunable shapes and properties. Synthetic cellulose-based oligomers were assembled into 2D or 3D rod-like crystallites. Sequence modifications within the oligosaccharide core influenced the molecular packing and led to the formation of square-like assemblies based on the rare cellulose IVII allomorph. In contrast, modifications at the termini generated elongated aggregates with tunable surfaces, resulting in self-healing supramolecular hydrogels.

Bottom-Up Approach to Understand Chirality Transfer across Scales in Cellulose Assemblies.

Cellulose is a polysaccharide that displays chirality across different scales, from the molecular to the supramolecular level. This feature has been exploited to generate chiral materials. To date, the mechanism of chirality transfer from the molecular level to higher-order assemblies has remained elusive, partially due to the heterogeneity of cellulose samples obtained via top-down approaches. Here, we present a bottom-up approach that uses well-defined cellulose oligomers as tools to understand the transfer of chirality from the single oligomer to supramolecular assemblies beyond the single cellulose crystal. Synthetic cellulose oligomers with defined sequences self-assembled into thin micrometer-sized platelets with controllable thicknesses. These platelets further assembled into bundles displaying intrinsic chiral features, directly correlated to the monosaccharide chirality. Altering the stereochemistry of the oligomer termini impacted the chirality of the self-assembled bundles and thus allowed for the manipulation of the cellulose assemblies at the molecular level. The molecular description of cellulose assemblies and their chirality will improve our ability to control and tune cellulose materials. The bottom-up approach could be expanded to other polysaccharides whose supramolecular chirality is less understood.

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.

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.

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 ß-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.

Targeted Chemical Modifications Identify Key Features of Carbohydrate Assemblies and Generate Tailored Carbohydrate Materials.

The molecular level description of carbohydrate assemblies is hampered by their structural complexity and the lack of suitable analytical methods. Here, we employed systematic chemical modifications to identify key non-covalent interactions that triggered the supramolecular assembly of a disaccharide model. While some modifications disrupted the supramolecular organization, others were tolerated, delivering important information on the aggregation process. The screening identified new geometries, including nanotubes, and twisted ribbons that were characterized with electron tomography and electron diffraction (ED) methods. This work demonstrates that the combination of synthetic chemistry and ED methods is a powerful tool to draw correlations between the molecular structure and the nanoscale architecture of carbohydrate assemblies.

Systematic Structural Characterization of Chitooligosaccharides Enabled by Automated Glycan Assembly.

Chitin, a polymer composed of ß(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.

Automated Glycan Assembly of 19 F-labeled 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. Herein, we showcase an expedite approach that integrates automated glycan assembly (AGA) of 19 F-labeled 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 (Lex , H type 2, Ley ). 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-labeled complex glycans.

Progress and challenges in the synthesis of sequence controlled polysaccharides.

The sequence, length and substitution of a polysaccharide influence its physical and biological properties. Thus, sequence controlled polysaccharides are important targets to establish structure-properties correlations. Polymerization techniques and enzymatic methods have been optimized to obtain samples with well-defined substitution patterns and narrow molecular weight distribution. Chemical synthesis has granted access to polysaccharides with full control over the length. Here, we review the progress towards the synthesis of well-defined polysaccharides. For each class of polysaccharides, we discuss the available synthetic approaches and their current limitations.

Structural Studies Using Unnatural Oligosaccharides: Toward Sugar Foldamers.

Biopolymers, like DNA and proteins, fold in specific conformations in order to exert complex biological functions. Synthetic modifications are commonly used to alter those conformations and create engineered biomaterials. In stark contrast, the chemical complexity and dynamic nature of polysaccharides have hampered a detailed structural characterization and structure-function correlations are still incomplete. Many synthetic strategies have been developed to access complex unnatural oligosaccharides, capable of mimicking or even improving the properties of the natural counterpart. However, the structural features behind these results are often neglected. This perspective highlights the approaches adopted to develop unnatural glycans, with a particular focus on how the insertion of specific modifications results in more flexible or more constrained structures. Synthetic analogues of natural oligosaccharides could shine light on fundamental structural features. The combination of modern synthetic, computational, and analytical methods will result in novel carbohydrate based foldamers, with defined shape and aggregation behavior. Multiple applications in biology, material science, and nanotechnology can be envisioned.

Supramolecular Assembly and Chirality of Synthetic Carbohydrate Materials.

Hierarchical carbohydrate architectures serve multiple roles in nature. Hardly any correlations between the carbohydrate chemical structures and the material properties are available due to the lack of standards and suitable analytic techniques. Therefore, designer carbohydrate materials remain highly unexplored, as compared to peptides and nucleic acids. A synthetic D-glucose disaccharide, DD, was chosen as a model to explore carbohydrate materials. Microcrystal electron diffraction (MicroED), optimized for oligosaccharides, revealed that DD assembled into highly crystalline left-handed helical fibers. The supramolecular architecture was correlated to the local crystal organization, allowing for the design of the enantiomeric right-handed fibers, based on the L-glucose disaccharide, LL, or flat lamellae, based on the racemic mixture. Tunable morphologies and mechanical properties suggest the potential of carbohydrate materials for nanotechnology applications.

Systematic Hydrogen-Bond Manipulations To Establish Polysaccharide Structure-Property Correlations.

A dense hydrogen-bond network is responsible for the mechanical and structural properties of polysaccharides. Random derivatization alters the properties of the bulk material by disrupting the hydrogen bonds, but obstructs detailed structure-function correlations. We have prepared well-defined unnatural oligosaccharides including methylated, deoxygenated, deoxyfluorinated, as well as carboxymethylated cellulose and chitin analogues with full control over the degree and pattern of substitution. Molecular dynamics simulations and crystallographic analysis show how distinct hydrogen-bond modifications drastically affect the solubility, aggregation behavior, and crystallinity of carbohydrate materials. This systematic approach to establishing detailed structure-property correlations will guide the synthesis of novel, tailor-made carbohydrate materials.

Phosphates as Assisting Groups in Glycan Synthesis.

In nature, phosphates are added to and cleaved from molecules to direct biological pathways. The concept was adapted to overcome limitations in the chemical synthesis of complex oligosaccharides. Phosphates were chemically placed on synthetic glycans to ensure site-specific enzymatic elongation by sialylation. In addition, the deliberate placement of phosphates helped to solubilize and isolate aggregating glycans. Upon traceless removal of the phosphates by enzymatic treatment with alkaline phosphatase, the native glycan structure was revealed, and the assembly of glycan nanostructures was triggered.

Synthesis of a glycan hairpin.

The primary sequence of a biopolymer encodes the essential information for folding, permitting to carry out sophisticated functions. Inspired by natural biopolymers, peptide and nucleic acid sequences have been designed to adopt particular three-dimensional (3D) shapes and programmed to exert specific functions. In contrast, synthetic glycans capable of autonomously folding into defined 3D conformations have so far not been explored owing to their structural complexity and lack of design rules. Here we generate a glycan that adopts a stable secondary structure not present in nature, a glycan hairpin, by combining natural glycan motifs, stabilized by a non-conventional hydrogen bond and hydrophobic interactions. Automated glycan assembly enabled rapid access to synthetic analogues, including site-specific 13C-labelled ones, for nuclear magnetic resonance conformational analysis. Long-range inter-residue nuclear Overhauser effects unequivocally confirmed the folded conformation of the synthetic glycan hairpin. The capacity to control the 3D shape across the pool of available monosaccharides has the potential to afford more foldamer scaffolds with programmable properties and functions.

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 ß(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.