Steffen Eller

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.

Glycan arrays containing synthetic Clostridium difficile lipoteichoic acid oligomers as tools toward a carbohydrate vaccine.

Clostridium difficile is a leading cause of severe nosocomial infections. Cell-surface carbohydrate antigens are promising vaccine candidates. Here we report the first total synthesis of oligomers of the lipoteichoic acid antigen repeating unit. Synthetic glycan microarrays revealed anti-glycan antibodies in the blood of patients that help to define epitopes for vaccine development.

SOLID PHASE SYNTHESIS OF OLIGOSACCHARIDES

Oligosaccharides are involved in many fundamental biological processes. However, relatively little is known about the precise molecular mechanism of action of these macromolecules, because the complexity of these structures impeded their synthesis by chemical methods analogous to those employed to create oligonucleotides and peptides. Herein, we describe recently developed techniques for solid-supported oligosaccharide synthesis. Several key aspects of solid phase synthesis are highlighted and examined, including the choice of resin and the challenge of real-time reaction monitoring. Recent examples of manual and automated solid-supported syntheses of complex oligosaccharides are given and the automated solid phase synthesis of the tumor-associated carbohydrate antigen Globo-H is highlighted.

Synthesis of Multiantennary Complex Type N‐Glycans by Use of Modular Building Blocks

A modular set of oligosaccharide building blocks was developed for the synthesis of multiantennary N-glycans of the complex type, which are commonly found on glycoproteins. The donor building blocks were laid out for the elongation of a core trisaccharide acceptor (beta-mannosyl chitobiose) conveniently protected with a single benzylidene moiety at the beta-mannoside. Through two consecutive regio- and stereoselective couplings the donors gave N-glycans with three to five antennae in high yields. Due to the consistent protection group pattern of the donors the deprotection of the final products can be performed by using a general reaction sequence.

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.

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.

Insights into the Dynamics and Molecular Recognition Features of Glycopeptides by Protein Receptors: The 3D Solution Structure of Hevein Bound to the Trisaccharide Core of N‐Glycoproteins

Protein-carbohydrate interactions are at the heart of a variety of essential molecular recognition events. Hevein, a model lectin related to the superantigen family, recognizes the trisaccharide core of N-glycoproteins (1). A combined approach of NMR spectroscopy and molecular modeling has permitted us to demonstrate that an Asn-linked Man(GlcNAc)(2) (2) is bound with even higher affinity than (GlcNAc)(3). The molecular recognition process entails conformational selection of only one of the possibilities existing for chitooligosaccharides. The deduced 3D structure of the hevein/2 complex permits the extension of polypeptide chains from the Asn moiety of 2, as well as glycosylation at Man O-3 and Man O-6 of the terminal sugar. Given the ubiquity of the Man(GlcNAc)(2) core in all mammalian N-glycoproteins, the basic recognition mode presented herein might be extended to a variety of systems with biomedical importance.

Highly Efficient Synthesis of Multiantennary Bisected N-glycans Based on Imidates

The occurrence of N-glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N-glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N-glycans of the complex type by employing modular TFAc-protected donors for all antennae. The TFAc-protected N-glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N-glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one-pot double glycosylation. With this new method, rare N-glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.

Glycosylation efficiencies on different solid supports using a hydrogenolysis-labile linker.

Summary Automated oligosaccharide assembly requires suitable linkers to connect the first monosaccharide to a solid support. A new hydrogenolysis-labile linker that is stable under both acidic and basic conditions was designed, synthesized and coupled to different resins. Glycosylation and cleavage efficiencies on these functionalized solid supports were investigated, and restrictions for the choice of solid support for oligosaccharide synthesis were found.

Acylsulfonamide safety-catch linker: promise and limitations for solid–phase oligosaccharide synthesis

Summary Safety-catch linkers are useful for solid-phase oligosaccharide synthesis as they are orthogonal to many common protective groups. A new acylsulfonamide safety-catch linker was designed, synthesized and employed during glycosylations using an automated carbohydrate synthesizer. The analysis of the cleavage products revealed shortcomings for oligosaccharide synthesis.