The structural complexity of glycans poses a serious challenge in the chemical synthesis of glycosides, oligosaccharides and glycoconjugates. Glycan complexity, determined by composition, ...connectivity, and configuration far exceeds what nature achieves with nucleic acids and proteins. Consequently, glycoside synthesis ranks among the most complex tasks in organic synthesis, despite involving only a simple type of bond-forming reaction. Here, we introduce the fundamental principles of glycoside bond formation and summarize recent advances in glycoside bond formation and oligosaccharide synthesis.
Here readers are introduced to the fundamental principles of glycoside bond formation and recent advances in glycoside and oligosaccharide synthesis.
Cryptococcus neoformans is an opportunistic fungal pathogen, which is a frequent cause of a life-threatening meningitis in immunocompromised individuals. We report the first total synthesis of the ...serotype B heptasaccharide repeating motif. The use of di- and trisaccharide building blocks enabled a concise convergent synthesis of the protected 6-O-acetylated repeating motif in three steps. Glycosylations gave total 1,2-trans selectivity, despite the absence of a neighboring participating group. Using our recently disclosed catalyst pre-tuning strategy global deprotection gave the desired 6-O-acetylated heptasaccharide with no saturation by-products, overall in four steps 31% yield. The serotype B glucuronoxylomannan (GXM) glycans accessed in this study will increase the structurally diversity of our GXM microarray, allowing further steps towards the development of semi-synthetic vaccines against cryptococcal infections.
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•Total synthesis of the Cryptococcus neoformans serotype B repeating motif.•Di- and trisaccharide building blocks enabled a convergent synthesis of the 6-O-acetylated glycan.•Glycosylations gave total 1,2-trans selectivity, despite the absence of a neighboring participating group.•Catalyst pre-tuning strategy gave the desired 6-O-acetylated heptasaccharide with no saturation by-products.•The serotype B glycan accessed in this study will increase the structurally diversity of our GXM microarray.
Fucoidan, a sulfated polysaccharide found in algae, plays a central role in marine carbon sequestration and exhibits a wide array of bioactivities. However, the molecular diversity and structural ...complexity of fucoidan hinder precise structure–function studies. To address this, we present an automated method for generating well-defined linear and branched α-fucan oligosaccharides. Our syntheses include oligosaccharides with up to 20 cis-glycosidic linkages, diverse branching patterns, and 11 sulfate monoesters. In this study, we demonstrate the utility of these oligosaccharides by (i) characterizing two endo-acting fucoidan glycoside hydrolases (GH107), (ii) utilizing them as standards for NMR studies to confirm suggested structures of algal fucoidans, and (iii) developing a fucoidan microarray. This microarray enabled the screening of the molecular specificity of four monoclonal antibodies (mAb) targeting fucoidan. It was found that mAb BAM4 has cross-reactivity to β-glucans, while mAb BAM2 has reactivity to fucoidans with 4-O-sulfate esters. Knowledge of the mAb BAM2 epitope specificity provided evidence that a globally abundant marine diatom, Thalassiosira weissflogii, synthesizes a fucoidan with structural homology to those found in brown algae. Automated glycan assembly provides access to fucoidan oligosaccharides. These oligosaccharides provide the basis for molecular level investigations into fucoidan’s roles in medicine and carbon sequestration.
Palladium-catalyzed hydrogenolysis is often the final step in challenging natural product total syntheses and a key step in industrial processes producing fine chemicals. Here, we demonstrate that ...there is wide variability in the efficiency of commercial sources of palladium on carbon (Pd/C) resulting in significant differences in selectivity, reaction times, and yields. We identified the physicochemical properties of efficient catalysts for hydrogenolysis: (1) small Pd/PdO particle size (2) homogeneous distribution of Pd/PdO on the carbon support, and (3) palladium oxidation state are good predictors of catalytic efficiency. Now chemists can identify and predict a catalyst’s efficiency prior to the use of valuable synthetic material and time.
Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans ’ capsular polysaccharide and its shed exopolysaccharide function both as ...key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J -coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac 3 , calculated from MD simulations. GXM10-Ac 3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by β-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a β-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac 3 adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O -acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac 3 structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.
Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of ...catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of
From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing
cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.
The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to ...either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall–associated polysaccharide aggregates that we call “capsule ghosts”, implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones.
A convergent synthetic strategy to
glucuronoxylomannan (GXM) capsular polysaccharide part structures was developed based on di-, tri-, tetra-, penta- and hexasaccharide thioglycoside building blocks. ...The approach permitted the synthesis of a library of spacer-containing serotype A and D related GXM oligosaccharide structures, ranging from di- to octadecasaccharides. Ten deprotected GXM compounds (mono- to decasaccharide) were printed onto microarray plates and screened with seventeen mouse monoclonal antibodies (mAbs) to GXM. For the first time a GXM oligosaccharide structure (a serotype A decasaccharide), capable of being recognized by neutralizing forms of these GXM-specific mAbs, has been identified, offering insight into the binding epitopes of a range of protective monoclonal antibodies and furthering our efforts to develop semi-synthetic conjugate vaccine candidates against
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Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen ...Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall–membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.
Abstract 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.
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