Dissecting the Conformational Stability of a Glycan Hairpin.

Systematic structural studies of model oligopeptides revealed important aspects of protein folding and offered design principles to access non-natural materials. In the same way, the rules that regulate glycan folding could be established by studying synthetic oligosaccharide models. However, their analysis is often limited due to the synthetic and analytical complexity. By utilizing a glycan capable of spontaneously folding into a hairpin conformation as a model system, we investigated the factors that contribute to its conformational stability in aqueous solution. The modular design of the hairpin model featured a trisaccharide turn unit and two ß-1,4-oligoglucoside stacking strands that allowed for systematic chemical modifications of the glycan sequence, including the introduction of NMR labels and staples. Nuclear magnetic resonance assisted by molecular dynamics simulations revealed that stereoelectronic effects and multiple glycan-glycan interactions are the major determinants of folding stabilization. Chemical modifications in the glycan primary sequence (e.g., strand elongation) can be employed to fine-tune the rigidity of structural motifs distant from the modification sites. These results could inspire the design of other glycan architectures, with implications in glycobiology and material sciences.

Scanning Probe Microscopy Characterization of Biomolecules enabled by Mass-Selective, Soft-landing Electrospray Ion Beam Deposition.

Scanning probe microscopy (SPM), in particular at low temperature (LT) under ultra-high vacuum (UHV) conditions, offers the possibility of real-space imaging with resolution reaching the atomic level. However, its potential for the analysis of complex biological molecules has been hampered by requirements imposed by sample preparation. Transferring molecules onto surfaces in UHV is typically accomplished by thermal sublimation in vacuum. This approach however is limited by the thermal stability of the molecules, i.e. not possible for biological molecules with low vapour pressure. Bypassing this limitation, electrospray ionisation offers an alternative method to transfer molecules from solution to the gas-phase as intact molecular ions. In soft-landing electrospray ion beam deposition (ESIBD), these molecular ions are subsequently mass-selected and gently landed on surfaces which permits large and thermally fragile molecules to be analyzed by LT-UHV SPM. In this concept, we discuss how ESIBD+SPM prepares samples of complex biological molecules at a surface, offering controls of the molecular structural integrity, three-dimensional shape, and purity. These achievements unlock the analytical potential of SPM which is showcased by imaging proteins, peptides, DNA, glycans, and conjugates of these molecules, revealing details of their connectivity, conformation, and interaction that could not be accessed by any other technique.

Impact of glycan nature on structure and viscoelastic properties of glycopeptide hydrogels.

Mucus is a complex biological hydrogel that acts as a barrier for almost everything entering or exiting the body. It is therefore of emerging interest for biomedical and pharmaceutical applications. Besides water, the most abundant components are the large and densely glycosylated mucins, glycoproteins of up to 20 MDa and carbohydrate content of up to 80 wt%. Here, we designed and explored a library of glycosylated peptides to deconstruct the complexity of mucus. Using the well-characterized hFF03 coiled-coil system as a hydrogel-forming peptide scaffold, we systematically probed the contribution of single glycans to the secondary structure as well as the formation and viscoelastic properties of the resulting hydrogels. We show that glycan-decoration does not affect α-helix and coiled-coil formation while it alters gel stiffness. By using oscillatory macrorheology, dynamic light scattering microrheology, and fluorescence lifetime-based nanorheology, we characterized the glycopeptide materials over several length scales. Molecular simulations revealed that the glycosylated linker may extend into the solvent, but more frequently interacts with the peptide, thereby likely modifying the stability of the self-assembled fibers. This systematic study highlights the interplay between glycan structure and hydrogel properties and may guide the development of synthetic mucus mimetics.

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.

Nanovesicles displaying functional linear and branched oligomannose self-assembled from sequence-defined Janus glycodendrimers.

Cell surfaces are often decorated with glycoconjugates that contain linear and more complex symmetrically and asymmetrically branched carbohydrates essential for cellular recognition and communication processes. Mannose is one of the fundamental building blocks of glycans in many biological membranes. Moreover, oligomannoses are commonly found on the surface of pathogens such as bacteria and viruses as both glycolipids and glycoproteins. However, their mechanism of action is not well understood, even though this is of great potential interest for translational medicine. Sequence-defined amphiphilic Janus glycodendrimers containing simple mono- and disaccharides that mimic glycolipids are known to self-assemble into glycodendrimersomes, which in turn resemble the surface of a cell by encoding carbohydrate activity via supramolecular multivalency. The synthetic challenge of preparing Janus glycodendrimers containing more complex linear and branched glycans has so far prevented access to more realistic cell mimics. However, the present work reports the use of an isothiocyanate-amine "click"-like reaction between isothiocyanate-containing sequence-defined amphiphilic Janus dendrimers and either linear or branched oligosaccharides containing up to six monosaccharide units attached to a hydrophobic amino-pentyl linker, a construct not expected to assemble into glycodendrimersomes. Unexpectedly, these oligoMan-containing dendrimers, which have their hydrophobic linker connected via a thiourea group to the amphiphilic part of Janus glycodendrimers, self-organize into nanoscale glycodendrimersomes. Specifically, the mannose-binding lectins that best agglutinate glycodendrimersomes are those displaying hexamannose. Lamellar "raft-like" nanomorphologies on the surface of glycodendrimersomes, self-organized from these sequence-defined glycans, endow these membrane mimics with high biological activity.

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.

Linker, loading, and reaction scale influence automated glycan assembly.

Automated glycan assembly (AGA) affords collections of well-defined glycans in a short amount of time. We systematically analyzed how parameters connected to the solid support affect the AGA outcome for three different glycan sequences. We showed that, while loading and reaction scale did not significantly influence the AGA outcome, the chemical nature of the linker dramatically altered the isolated yields. We identified that the major determinants of AGA yields are cleavage from the solid support and post-AGA purification steps.

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.

Design, Synthesis, and Characterization of Stapled Oligosaccharides.

Stapling short peptides to lock specific conformations and thereby obtain superior pharmacological properties is well established. However, similar concepts have not been applied to oligosaccharides. Here, we describe the design, synthesis, and characterization of the first stapled oligosaccharides. Automated assembly of ß-(1,6)-glucans equipped with two alkenyl side chains was followed by on-resin Grubbs metathesis for efficient ring closure with a variety of cross-linkers of different sizes. Oligosaccharide stapling increases enzymatic stability and cell penetration, therefore opening new opportunities for the use of glycans in medicinal chemistry.

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.

Comparative analysis of lanthanide excited state quenching by electronic energy and electron transfer processes.

The relative sensitivities of structurally related Eu(III) complexes to quenching by electron and energy transfer processes have been compared. In two sets of 9-coordinate complexes based on 1,4,7-triazacyclononane, the Eu emission lifetime decreased as the number of conjugated sensitising groups and the number of unbound ligand N atoms increased, consistent with photoinduced electron transfer to the excited Eu(III) ion that is suppressed by N-protonation. Quenching of the Eu 5D0 excited state may also occur by electronic energy transfer, and the quenching of a variety of 9-coordinate complexes by a cyanine dye with optimal spectral overlap occurs by an efficient FRET process, defined by a Förster radius (R0) value of 68 Å and characterised by second rate constants in the order of 109 M-1 s-1; these values were insensitive to changes in the ligand structure and to the overall complex hydrophilicity. Quenching of the Eu and Tb excited states by energy transfer to Mn(II) and Cu(II) aqua ions occurred over much shorter distances, with rate constants of around 106 M-1 s-1, owing to the much lower spectral overlap integral. The calculated R0 values were estimated to be between 2.5 to 4 Å in the former case, suggesting the presence of a Dexter energy transfer mechanism that requires much closer contact, consistent with the enhanced sensitivity of the rate of quenching to the degree of steric shielding of the lanthanide ion provided by the ligand.

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.

Automated Assembly of Starch and Glycogen Polysaccharides.

Polysaccharides are Nature's most abundant biomaterials essential for plant cell wall construction and energy storage. Seemingly minor structural differences result in entirely different functions: cellulose, a ß (1-4) linked glucose polymer, forms fibrils that can support large trees, while amylose, an α (1-4) linked glucose polymer forms soft hollow fibers used for energy storage. A detailed understanding of polysaccharide structures requires pure materials that cannot be isolated from natural sources. Automated Glycan Assembly provides quick access to trans-linked glycans analogues of cellulose, but the stereoselective installation of multiple cis-glycosidic linkages present in amylose has not been possible to date. Here, we identify thioglycoside building blocks with different protecting group patterns that, in concert with temperature and solvent control, achieve excellent stereoselectivity during the synthesis of linear and branched α-glucan polymers with up to 20 cis-glycosidic linkages. The molecules prepared with the new method will serve as probes to understand the biosynthesis and the structure of α-glucans.

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.