Parametric Analysis of Donor Activation for Glycosylation Reactions.

The chemical synthesis of complex oligosaccharides relies on efficient and highly reproducible glycosylation reactions. The outcome of a glycosylation is contingent upon several environmental factors, such as temperature, acidity, the presence of residual moisture, as well as the steric, electronic, and conformational aspects of the reactants. Each glycosylation proceeds rapidly and with a high yield within a rather narrow temperature range. For better control over glycosylations and to ensure fast and reliable reactions, a systematic analysis of 18 glycosyl donors revealed the effect of reagent concentration, water content, protecting groups, and structure of the glycosyl donors on the activation temperature. With these insights, we parametrize the first step of the glycosylation reaction to be executed reliably and efficiently.

Synthesis of a heparan sulfate tetrasaccharide using automated glycan assembly.

Herein we utilise automated glycan assembly to complete solid-phase synthesis of defined heparan sulfate oligosaccharides, employing challenging D-glucuronate disaccharide donors. Using an orthogonally protected D-GlcN-α-D-GlcA donor, milligram-scale synthesis of a heparan sulfate tetrasaccharide is completed in 18% yield over five steps. Furthermore, orthogonal protecting groups enabled regiospecific on-resin 6-O-sulfation. This methodology provides an important benchmark for the rapid assembly of biologically relevant heparan sulfate sequences.

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.

Enabling Technologies in Carbohydrate Chemistry: Automated Glycan Assembly, Flow Chemistry and Data Science.

The synthesis of defined oligosaccharides is a complex task. Several enabling technologies have been introduced in the last two decades to facilitate synthetic access to these valuable biomolecules. In this concept, we describe the technological solutions that have advanced glycochemistry using automated glycan assembly, flow chemistry and data science as examples. We highlight how the synergies between these different technologies can further advance the field, with progress toward the realization of a self-driving lab for glycan synthesis.

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.

Controlled-Alignment Patterns of Dipeptide Micro- and Nanofibers.

Ordered assemblies of the peptide diphenylalanine (FF) are produced and deposited on planar substrates. The FF aggregate growth is achieved through precipitation from aqueous ammonia solutions induced by solvent evaporation. The applied dip-coating technique confines the FF assembly growth to a narrow zone near the three-phase contact. The growth was observed online by optical microscopy and was investigated systematically as a function of the process parameters. Depending on the external gas flow (to influence solvent evaporation), the withdrawal speed, the initial FF, and the initial ammonia concentrations, FF forms long, straight, and rigid microfibers and/or shorter, curved nanofibers. Under certain process conditions, the FF fibers can also aggregate into stripes. These can be deposited as large arrays of uniform stripes with regular widths and spacings. Scenarios leading to the various types of fibers and the stripe formation are presented and discussed in view of the experimental findings.

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi.

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)6 fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms.

Towards a Systematic Understanding of the Influence of Temperature on Glycosylation Reactions.

Glycosidic bond formation is a continual challenge for practitioners. Aiming to enhance the reproducibility and efficiency of oligosaccharide synthesis, we studied the relationship between glycosyl donor activation and reaction temperature. A novel semi-automated assay revealed diverse responses of members of a panel of thioglycosides to activation at various temperatures. The patterns of protecting groups and the thiol aglycon combine to cause remarkable differences in temperature sensitivity among glycosyl donor building blocks. We introduce the concept of donor activation temperature to capture experimental insights, reasoning that glycosylations performed below this reference temperature evade deleterious side reactions. Activation temperatures enable a simplified temperature treatment and facilitate optimization of glycosyl donor usage. Isothermal glycosylation below the activation temperature halved the equivalents of building block required in comparison to the standard "ramp" regime used in solution- and solid-phase oligosaccharide synthesis to-date.

Expedited synthesis of mannose-6-phosphate containing oligosaccharides.

Currently, the reaction toolbox for the functionalization of glycans assembled on solid-phase is quite limited. Automated (1 h) and manual (overnight) phosphorylation protocols that enable the solid-phase synthesis of oligosaccharides containing up to two mannose-6-phosphates are presented. Automated glycan assembly expedited access to substrates and facilitated the screening of experimental conditions.

Microwave-Assisted Automated Glycan Assembly.

Automated synthesis of DNA, RNA, and peptides provides quickly and reliably important tools for biomedical research. Automated glycan assembly (AGA) is significantly more challenging, as highly branched carbohydrates require strict regio- and stereocontrol during synthesis. A new AGA synthesizer enables rapid temperature adjustment from -40 to +100 °C to control glycosylations at low temperature and accelerates capping, protecting group removal, and glycan modifications using elevated temperatures. Thereby, the temporary protecting group portfolio is extended from two to four orthogonal groups that give rise to oligosaccharides with up to four branches. In addition, sulfated glycans and unprotected glycans can be prepared. The new design reduces the typical coupling cycles from 100 to 60 min while expanding the range of accessible glycans. The instrument drastically shortens and generalizes the synthesis of carbohydrates for use in biomedical and material science.

Automated Glycan Assembly in a Variable-Bed Flow Reactor Provides Insights into Oligosaccharide-Resin Interactions.

A pressure-based variable-bed flow reactor built for peptide synthesis and capable of real-time monitoring of resin swelling was adapted for automated glycan assembly. In the context of the solid-phase synthesis of several oligosaccharides, the coupling efficiencies, resin growth patterns, and saccharide solvation during the synthesis were determined. The presented work provides the first estimation of on-resin oligosaccharide solvation and an alternative technique to UV-vis monitoring.

Controlled Deposition of Nanosize and Microsize Particles by Spin-Casting.

The deposition of nanosize and microsize spherical particles on planar solid substrates by hydrodynamic-evaporative spin-casting is studied. The particles are dispersed in a volatile liquid, which evaporates during the process, and the particles are finally deposited on the substrate. Their coverage, Γ, depends on the processing parameters (concentration by weight, particles size, etc.). The behavior of the particles during the spin-casting process and their final Γ values are investigated. It is found that for up to particle diameters of a few micrometers, particle deposition can be described by a theoretical approach developed for the spin-casting of polymer solutions (Karpitschka, S.; Weber, C. M.; Riegler, H. Chem. Eng. Sci. 2015, 129, 243-248. Danglad-Flores, J.; Eickelmann, S.; Riegler, H. Chem. Eng. Sci. 2018, 179, 257-264). For large particles, this basic theory fails. The causes of this failure are analyzed, and a corrected, more general theoretical approach is presented. It takes into account particle size effects as well as particle sedimentation. In summary, we present new insights into the spin-cast process of particle dispersions, analyze the contributions affecting the final particle coverage, and present a theoretical approach which describes and explains the experimental findings.

Meniscus Shape around Nanoparticles Embedded in Molecularly Thin Liquid Films.

Individual nanoparticles embedded in molecularly thin films at planar substrates and the resulting film surface distortion (meniscus) adjacent to the nanoparticles are investigated by conventional optical reflection microscopy. Even for nanoparticles much smaller than the Rayleigh diffraction limit, the meniscus creates such a pronounced optical footprint that the location of the nanoparticles can be identified. This is because the decay length (lateral extension) of the meniscus exceeds the size of the nanoparticle by orders of magnitude. Therefore, for the first time, the exact shape of the meniscus of the liquid adjacent to a nanosize object could be measured and analyzed. The meniscus has a zero curvature shape (cosine hyperbolic). The liquid in the meniscus is in pressure equilibrium with the far-field planar film. The decay length decreases with the decreasing nanoparticle size. However, it is independent of the far-field film thickness. Supposedly, the decay length is determined by van der Waals interactions although it is unknown what determines its (unexpectedly large) absolute value. The presented technical approach may be used to investigate biological systems (for instance, surface distortions in supported membranes caused by proteins or protein aggregates).

Capillary-Enhanced Immobilization of Nanoparticles.

The contact between a spherical nanoparticle and a planar substrate is surrounded by an annular wedge cavity. In adsorption processes, this acts like a small pore. Interfacial/capillarity effects lead to an accumulation of adsorbate in this region ("capillary condensation"). This effectively increases the contact area between the particles and the substrate. Thus, capillary-enhanced adsorbate accumulation increases the adhesion between the nanoparticles and the planar surface, which effectively immobilizes the particles.