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.

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.

A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

Flagellin Glycoproteomics of the Periodontitis Associated Pathogen Selenomonas sputigena Reveals Previously Not Described O-glycans and Rhamnose Fragment Rearrangement Occurring on the Glycopeptides.

Flagellated, Gram-negative, anaerobic, crescent-shaped Selenomonas species are colonizers of the digestive system, where they act at the interface between health and disease. Selenomonas sputigena is also considered a potential human periodontal pathogen, but information on its virulence factors and underlying pathogenicity mechanisms is scarce. Here we provide the first report of a Selenomonas glycoprotein, showing that S. sputigena produces a diversely and heavily O-glycosylated flagellin C9LY14 as a major cellular protein, which carries various hitherto undescribed rhamnose- and N-acetylglucosamine linked O-glycans in the range from mono- to hexasaccharides. A comprehensive glycomic and glycoproteomic assessment revealed extensive glycan macro- and microheterogeneity identified from 22 unique glycopeptide species. From the multiple sites of glycosylation, five were unambiguously identified on the 437-amino acid C9LY14 protein (Thr149, Ser182, Thr199, Thr259, and Ser334), the only flagellin protein identified. The O-glycans additionally showed modifications by methylation and putative acetylation. Some O-glycans carried hitherto undescribed residues/modifications as determined by their respective m/z values, reflecting the high diversity of native S. sputigena flagellin. We also found that monosaccharide rearrangement occurred during collision-induced dissociation (CID) of protonated glycopeptide ions. This effect resulted in pseudo Y1-glycopeptide fragment ions that indicated the presence of additional glycosylation sites on a single glycopeptide. CID oxonium ions and electron transfer dissociation, however, confirmed that just a single site was glycosylated, showing that glycan-to-peptide rearrangement can occur on glycopeptides and that this effect is influenced by the molecular nature of the glycan moiety. This effect was most pronounced with disaccharides. This study is the first report on O-linked flagellin glycosylation in a Selenomonas species, revealing that C9LY14 is one of the most heavily glycosylated flagellins described to date. This study contributes to our understanding of the largely under-investigated surface properties of oral bacteria. The data have been deposited to the ProteomeXchange with identifier PXD005859.

Using carbohydrate-based biomaterials as scaffolds to control human stem cell fate.

Given the important roles of carbohydrates in numerous signalling pathways, cell-cell interactions and cell-matrix interactions, a variety of scaffolds based on natural polysaccharides and glycoproteins that are mostly found in the native extracellular matrix (ECM) have been established and investigated as stem cell culture systems. Stem cells have been extensively used in cell therapy and tissue engineering, and the ability to create a suitable environment to direct stem cell behavior is critical for such endeavors. It is a great challenge to construct scaffolds that mimic the native "niche" of stem cells. The present review describes the current statuses and applications of several natural polysaccharide and glycoprotein scaffolds for use in tissue engineering. In the future, carbohydrate-based biomaterials may be used to create powerful scaffolds that can regulate the stem cell fate.

Continuous flow photochemistry.

Due to the narrow width of tubing/reactors used, photochemistry performed in micro- and mesoflow systems is significantly more efficient than when performed in batch due to the Beer-Lambert Law. Owing to the constant removal of product and facility of flow chemical scalability, the degree of degradation observed is generally decreased and the productivity of photochemical processes is increased. In this Personal Account, we describe a wide range of photochemical transformations we have examined using both visible and UV light, covering cyclizations, intermolecular couplings, radical polymerizations, as well as singlet oxygen oxygenations.

Synthesis of carbohydrate-functionalised sequence-defined oligo(amidoamine)s by photochemical thiol-ene coupling in a continuous flow reactor.

Poly/oligo(amidoamine)s (PAAs) have recently been recognised for their potential as well-defined scaffolds for multiple carbohydrate presentation and as multivalent ligands. Herein, we report two complimentary strategies for the preparation of such sequence-defined carbohydrate-functionalised PAAs that use photochemical thiol-ene coupling (TEC) as an alternative to the established azide-alkyne cycloaddition ("click") reaction. In the first approach, PAAs that contained multiple olefins were synthesised on a solid support from a new building block and subsequent conjugation with unprotected thio-carbohydrates. Alternatively, a pre-functionalised building block was prepared by using TEC and assembled on a solid support to provide a carbohydrate-functionalised PAA. Both methods rely on the use of a continuous flow photoreactor for the TEC reactions. This system is highly efficient, owing to its short path length, and requires no additional radical initiator. Performing the reactions at 254 nm in Teflon AF-2400 tubing provides a highly efficient TEC procedure for carbohydrate conjugation, as demonstrated in the reactions of O-allyl glycosides with thiols. This method allowed the complete functionalisation of all of the reactive sites on the PAA backbone in a single step, thereby obtaining a defined homogeneous sequence. Furthermore, reaction at 366 nm in FEP tubing in the flow reactor enabled the large-scale synthesis of an fluorenylmethyloxycarbonyl (Fmoc)-protected glycosylated building block, which was shown to be suitable for solid-phase synthesis and will also allow heterogeneous sequence control of different carbohydrates along the oligomeric backbone. These developments enable the synthesis of sequence-defined carbohydrate-functionalised PAAs with potential biological applications.

Magnetic porous sugar-functionalized PEG microgels for efficient isolation and removal of bacteria from solution.

Here, we present a new microparticle system for the selective detection and magnetic removal of bacteria from contaminated solutions. The novelty of this system lies in the combination of a biocompatible scaffold reducing unspecific interactions with high capacity for bacteria binding. We apply highly porous poly(ethylene glycol) (PEG) microparticles and functionalize them, introducing both sugar ligands for specific bacteria targeting and cationic moieties for electrostatic loading of superparamagnetic iron oxide nanoparticles. The resulting magnetic, porous, sugar-functionalized (MaPoS) PEG microgels are able to selectively bind and discriminate between different strains of bacteria Escherichia coli . Furthermore, they allow for a highly efficient removal of bacteria from solution as their increased surface area can bind three times more bacteria than nonporous particles. All in all, MaPoS particles represent a novel generation of magnetic beads introducing for the first time a porous, biocompatible and easy to functionalize scaffold and show great potential for various biotechnological applications.

Snowballing radical generation leads to ultrahigh molecular weight polymers.

Styrene is the classical monomer obeying zero-one kinetics in radical emulsion polymerization. Accordingly, particles that are less than 100 nm in diameter contain either one or no growing radical(s). We describe a unique photoinitiated polymerization reaction accelerated by snowballing radical generation in a continuous flow reactor. Even in comparison to classical emulsion polymerization, these unprecedented snowballing reactions are rapid and high-yielding, with each particle simultaneously containing more than one growing radical. This is a consequence of photoinitiator incorporation into the nascent polymer backbone and repeated radical generation upon photo-irradiation.

Synthetic phosphoethanolamine-modified oligosaccharides reveal the importance of glycan length and substitution in biofilm-inspired assemblies.

Bacterial biofilm matrices are nanocomposites of proteins and polysaccharides with remarkable mechanical properties. Efforts understanding and tuning the protein component have been extensive, whereas the polysaccharide part remained mostly overlooked. The discovery of phosphoethanolamine (pEtN) modified cellulose in E. coli biofilms revealed that polysaccharide functionalization alters the biofilm properties. To date, the pattern of pEtN cellulose and its mode of interactions with proteins remains elusive. Herein, we report a model system based on synthetic epitomes to explore the role of pEtN in biofilm-inspired assemblies. Nine pEtN-modified oligosaccharides were synthesized with full control over the length, degree and pattern of pEtN substitution. The oligomers were co-assembled with a representative peptide, triggering the formation of fibers in a length dependent manner. We discovered that the pEtN pattern modulates the adhesion of biofilm-inspired matrices, while the peptide component controls its stiffness. Unnatural oligosaccharides tune or disrupt the assembly morphology, revealing interesting targets for polysaccharide engineering to develop tunable bio-inspired materials.

Evaluation of Multivalent Sialylated Polyglycerols for Resistance Induction in and Broad Antiviral Activity against Influenza A Viruses.

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.

Design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting.

Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialoglycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized ß-cyclodextrins (ßCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded ßCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids containing a terminal D-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that ßCDs and liposomes displaying terminal D-Gal/D-GalNAc residues were preferentially endocytosed. In contrast, uptake of ßCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized ßCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized ßCDs are efficient molecular carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.

In vitro imaging and in vivo liver targeting with carbohydrate capped quantum dots.

PEGylated quantum dots (QDs) capped with d-mannose, d-galactose, and d-galactosamine have been synthesized. The stable, high quantum yield fluorescence of QDs was exploited to study specific carbohydrate-protein interactions in vitro and in vivo.

Synthesis, Liposomal Formulation, and Immunological Evaluation of a Minimalistic Carbohydrate-α-GalCer Vaccine Candidate.

Fully synthetic glycan-based vaccines hold great potential as preventive and therapeutic vaccines against infectious diseases as well as cancer. Here, we present a two-component platform based on the facile conjugation of carbohydrate antigens to α-galactosylceramide (α-GalCer) to yield fully synthetic vaccine candidates. Formulation of the cancer-associated Tn antigen glycolipid model vaccine candidate into liposomes of different sizes and subsequent immunization of mice generated specific, high-affinity antibodies against the carbohydrate antigen with characteristics of T cell-dependent immunity. Liposome formulation elicited more reproducible glycan immunity than a conventional glycoconjugate vaccine bearing the same glycan antigen did. Further evaluation of the immune response revealed that the size of the liposomes influenced the glycan antibody responses toward either a cellular (Th1) or a humoral (Th2) immune phenotype. The glycolipid vaccine platform affords strong and robust antiglycan antibody responses in vivo without the need for an external adjuvant.

Microarrays of heparin oligosaccharides obtained by nitrous acid depolymerization of isolated heparin.

Heparin oligosaccharides derived by nitrous acid depolymerization of heparin have been immobilized on amine-coated glass slides. The formation of a Schiff base creates heparin chips that are a suitable platform for the high-throughput analysis of carbohydrate-protein interactions.

Merging organic and polymer chemistries to create glycomaterials for glycomics applications.

[Image: see text] Oligosaccharides at cell surfaces are known to play a critical role in many biological processes such as biorecognition, interactions between cells and with artificial surfaces, immune response, infection and inflammation. In order to facilitate studies of the role of sugars, an increasing number of novel tools are becoming available. New synthetic strategies now provide much more efficient access to complex carbohydrates or glycoconjugates. Branched carbohydrates and hybrids of carbohydrates conjugated to polymers have been prepared using solution and/or solid-phase synthesis and advanced methods of polymerization. These materials are essential for the development of methodologies to study and map the molecular structure-function relationship at interfaces. This article highlights recent advances in the synthesis of carbohydrates and polymer hybrids mimicking the properties and functionalities of the natural oligosaccharides, as well as selected applications in biology, biotechnology and diagnostics.

RAFT-derived polymer-drug conjugates: poly(hydroxypropyl methacrylamide) (HPMA)-7-ethyl-10-hydroxycamptothecin (SN-38) conjugates.

A series of well-defined polymer-drug conjugates were prepared in order to modify the physical properties of a known cytotoxic drug, 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11). Reversible addition-fragmentation chain transfer (RAFT) polymerisation was used to covalently and site-specifically append a defined N-(2-hydroxypropyl)methacrylamide (HPMA) polymer to SN-38 using a graft-from process. These poly-HPMA-SN-38 conjugates displayed excellent aqueous solubility and stability, whilst retaining the cytotoxic activity of the parent SN-38. In vitro co-culture assays containing both cancer and noncancer cell lines demonstrated the specificity of RAFT-derived poly-HPMA-SN-38 conjugates for cancerous cells. The concept of post-optimisation modification of small-molecule drugs through a graft-from polymer conjugation method is introduced.