Heung Sik Hahm

Identification of carbohydrate anomers using ion mobility–mass spectrometry

Carbohydrates are ubiquitous biological polymers that are important in a broad range of biological processes. However, owing to their branched structures and the presence of stereogenic centres at each glycosidic linkage between monomers, carbohydrates are harder to characterize than are peptides and oligonucleotides. Methods such as nuclear magnetic resonance spectroscopy can be used to characterize glycosidic linkages, but this technique requires milligram amounts of material and cannot detect small amounts of coexisting isomers. Mass spectrometry, on the other hand, can provide information on carbohydrate composition and connectivity for even small amounts of sample, but it cannot be used to distinguish between stereoisomers. Here, we demonstrate that ion mobility–mass spectrometry—a method that separates molecules according to their mass, charge, size, and shape—can unambiguously identify carbohydrate linkage-isomers and stereoisomers. We analysed six synthetic carbohydrate isomers that differ in composition, connectivity, or configuration. Our data show that coexisting carbohydrate isomers can be identified, and relative concentrations of the minor isomer as low as 0.1 per cent can be detected. In addition, the analysis is rapid, and requires no derivatization and only small amounts of sample. These results indicate that ion mobility–mass spectrometry is an effective tool for the analysis of complex carbohydrates. This method could have an impact on the field of carbohydrate synthesis similar to that of the advent of high-performance liquid chromatography on the field of peptide assembly in the late 1970s.

Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans.

Carbohydrates are the most prevalent class of biopolymers on earth. Bound to proteins and lipids, carbohydrates form four structurally and functionally distinct, biologically significant glycoconjugate classes: glycoproteins, glycolipids, glycosylphosphatidylinositol (GPI) anchors, and glycosaminoglycans (GAGs; Figure 1). These structurally diverse macromolecules, which are usually located in the extracellular matrix, are essential for many fundamental cellular processes. GAGs are acidic, negatively charged polysaccharides that transduce extracellular signals to the interior of the cell. Localization is manifested by connection to a transmembrane core protein, to form a proteoglycan (Figure 1). GAGs are highly variable in size, ranging from 20–200 disaccharide repeating units, backbone composition, and the degree and pattern of sulfation. Chondroitin sulfate contains N-acetylb-d-galactosamine and b-d-glucuronic acid and the sulfation and acetylation of particular hydroxy and amino groups varies. The sulfation patterns of GAGs influence the bioactivity of the molecules but limited access to defined GAG structures has impeded mapping structure– activity patterns. Tailor-made GAG oligosaccharides can be synthesized chemically or enzymatically, and they have become valuable for analyzing GAG–protein interactions and their biological relevance. Introducing sulfate groups to specific positions of an oligosaccharide chain adds an additional level of complexity on top of the already challenging synthesis of oligosaccharides. Therefore, currently available methods for the assembly of GAG oligosaccharides, including modular approaches, are time-consuming and lack generality as the synthesis of each target molecule poses an individual challenge. Herein, we describe a novel approach for automated solid-phase synthesis of GAG oligosaccharides that is based in part on established methods for generating the glycan portion of glycoproteins and glycolipids. Key to the success of this procedure was a stable supply of tailor-made differentially protected building blocks, a robust but easily-cleaved linker, to connect the first monosaccharide of the nascent oligosaccharide to the solid support, and the automated synthesizer. A recently-developed automated solid-phase oligosaccharide synthesizer that allows for fully automated, computer-controlled glycan coupling cycles and the introduction of sulfate groups on solid support was further improved to carry out automated sulfation and modification on solid support. Figure 1. Glycoconjugates of the extracellar matrix. Oand N-glycans are linked to proteins by the side chains of serine, threonine, or asparagine. Glycolipids are composed of glycans that are attached to lipids and play an essential role in cellular recognition processes. Glycophosphatidylinositols (GPI) anchor proteins via two fatty acids to the cell membrane. Glycosaminoglycans occur as the glycan side chain in proteoglycans.

A General Method for Synthesis of GPI Anchors Illustrated by the Total Synthesis of the Low‐Molecular‐Weight Antigen from Toxoplasma gondii

Building blocks: a new, general synthetic strategy, which allows the construction of branched glycosylphosphatidylinositols (GPIs), enables the synthesis of parasitic glycolipid 1 from Toxoplasma gondii. In addition, the structure is further confirmed by recognition of monoclonal antibodies.

Glycan Fingerprinting via Cold-Ion Infrared Spectroscopy

The diversity of stereochemical isomers present in glycans and glycoconjugates poses a formidable challenge for comprehensive structural analysis. Typically, sophisticated mass spectrometry (MS)-based techniques are used in combination with chromatography or ion-mobility separation. However, coexisting structurally similar isomers often render an unambiguous identification impossible. Other powerful techniques such as gas-phase infrared (IR) spectroscopy have been limited to smaller glycans, since conformational flexibility and thermal activation during the measurement result in poor spectral resolution. This limitation can be overcome by using cold-ion spectroscopy. The vibrational fingerprints of cold oligosaccharide ions exhibit a wealth of well-resolved absorption features that are diagnostic for minute structural variations. The unprecedented resolution of cold-ion spectroscopy coupled with tandem MS may render this the key technology to unravel complex glycomes.

Automated glycan assembly using the Glyconeer 2.1 synthesizer

Significance Rapid access to defined, pure biomacromolecules is key to biochemical and biophysical investigations as evidenced by the impact automated solid-phase synthesis of oligonucleotides and oligopeptides had on basic research. Here, a strategy as well as a new instrument to routinely access oligosaccharides by automated synthesis is reported. The method produces glycans quickly and reliably to accelerate fundamental advances in glycobiology. Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined glycans is key to establishing structure–activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydrate-based vaccines.

Modular automated solid phase synthesis of dermatan sulfate oligosaccharides

Dermatan sulfates are glycosaminoglycan polysaccharides that serve a multitude of biological roles as part of the extracellular matrix. Orthogonally protected D-galactosamine and L-iduronic acid building blocks and a photo-cleavable linker are instrumental for the automated synthesis of dermatan sulfate oligosaccharides. Conjugation-ready oligosaccharides were obtained in good yield.

Total Synthesis of the Escherichia coli O111 O‐Specific Polysaccharide Repeating Unit

The first total synthesis of the O-antigen pentasaccharide repeating unit from Gram-negative bacteria Escherichia coli O111 was achieved starting from four monosaccharide building blocks. Key to the synthetic approach was a bis-glycosylation reaction to combine trisaccharide 10 and colitose 5. The colitose building block (5) was obtained de novo from non-carbohydrate precursors. The pentasaccharide was equipped at the reducing end with an amino spacer to provide a handle for subsequent conjugation to a carrier protein in anticipation of immunological studies.

Automated Glycan Assembly of Oligosaccharides Related to Arabinogalactan Proteins

Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the arabinogalactan protein function. Synthetic chemistry is key to the procurement of molecular probes for plant biologists. Described is the automated glycan assembly of 14 oligosaccharides from four monosaccharide building blocks. These linear and branched glycans represent key structural features of natural type-II arabinogalactans and will serve as tools for arabinogalactan biology.

Combination of automated solid-phase and enzymatic oligosaccharide synthesis provides access to α(2,3)-sialylated glycans

A synthetic strategy combining automated solid-phase chemical synthesis and enzymatic sialylation was developed to access α(2,3)-sialylated glycans.

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine

Automated glycan assembly enabled antibody reverse engineering to develop a semisynthetic carbohydrate–based vaccine against the highly virulent Streptococcus pneumoniae serotype 8. Pruning out nonprotective glycotopes Pediatric vaccines targeting bacterial capsular polysaccharides are more effective for certain types of bugs than others, and the manufacturing process as well as immunodominance of different glycan epitopes (glycotopes) can lead to a mixed immune response that does not protect against disease. To directly identify glycotopes that induce a protective response, Schumann et al. combined antibody reverse engineering with automated glycan assembly using Streptococcus pneumoniae serotype 8 as a proof of concept. Promising glycotopes conjugated to a carrier protein induced protective antibodies in mice and were also immunogenic in rabbits. When combined with a commercially available pneumococcal vaccine, these glycoconjugates were able to boost the opsonophagocytic bacterial killing ability of sera from immunized rabbits. This approach leveraging semisynthetic glycoconjugates could lead to the design of more effective bacterial vaccines. Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as Streptococcus pneumoniae successfully protect from disease but suffer from incomplete coverage, are troublesome to manufacture from isolated CPSs, and lack efficacy against certain serotypes. Defined, synthetic oligosaccharides are an attractive alternative to isolated CPSs but require the identification of immunogenic and protective oligosaccharide antigens. We describe a medicinal chemistry strategy based on a combination of automated glycan assembly (AGA), glycan microarray–based monoclonal antibody (mAb) reverse engineering, and immunological evaluation in vivo to uncover a protective glycan epitope (glycotope) for S. pneumoniae serotype 8 (ST8). All four tetrasaccharide frameshifts of ST8 CPS were prepared by AGA and used in glycan microarray experiments to identify the glycotopes recognized by antibodies against ST8. One tetrasaccharide frameshift that was preferentially recognized by a protective, CPS-directed mAb was conjugated to the carrier protein CRM197. Immunization of mice with this semisynthetic glycoconjugate followed by generation and characterization of a protective mAb identified protective and nonprotective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Coformulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent S. pneumoniae vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines.