The glycome describes the complete repertoire of glycoconjugates composed of carbohydrate chains, or glycans, that are covalently linked to lipid or protein molecules. Glycoconjugates are formed through a process called glycosylation and can differ in their glycan sequences, the connections between them and their length. Glycoconjugate synthesis is a dynamic process that depends on the local milieu of enzymes, sugar precursors and organelle structures as well as the cell types involved and cellular signals. Studies of rare genetic disorders that affect glycosylation first highlighted the biological importance of the glycome, and technological advances have improved our understanding of its heterogeneity and complexity. Researchers can now routinely assess how the secreted and cell-surface glycomes reflect overall cellular status in health and disease. In fact, changes in glycosylation can modulate inflammatory responses, enable viral immune escape, promote cancer cell metastasis or regulate apoptosis; the composition of the glycome also affects kidney function in health and disease. New insights into the structure and function of the glycome can now be applied to therapy development and could improve our ability to fine-tune immunological responses and inflammation, optimize the performance of therapeutic antibodies and boost immune responses to cancer. These examples illustrate the potential of the emerging field of ‘glycomedicine’. Glycosylation refers to the addition of carbohydrate chains to proteins and lipids. In this Review, the authors discuss the broad role of glycans in immunity, cancer, xenotransplantation and glomerular filtration and the potential of ‘glycomedicine’.

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Glycosylation in health and disease.

Increased understanding of the important roles that oligosaccharides and glycoconjugates play in biological processes has led to a demand for significant amounts of these materials for biological, medicinal, and pharmacological studies. Therefore, tremendous effort has been made to develop new procedures for the synthesis of glycosides, whereby the main focus is often the formation of the glycosidic bonds. Accordingly, quite a few review articles have been published over the past few years on glycoside synthesis; however, most are confined to either a specific type of glycoside or a specific strategy for glycoside synthesis. In this Review, new principles for the formation of glycoside bonds are discussed. Developments, mainly in the last ten years, that have led to significant advances in oligosaccharide and glycoconjugate synthesis have been compiled and are evaluated.

New principles for glycoside-bond formation.

Our understanding of the different glycoconjugates present on cells, proteins and entire organisms is lagging far behind advances in genomics and proteomics. Carbohydrate sequencing and the synthesis of defined oligosaccharides are two key technologies that have contributed to progress in glycomics research. Synthetic tools and high-throughput experiments such as carbohydrate arrays are beginning to affect biological research. These techniques are now being applied to the development of carbohydrate-based diagnostics, vaccines and therapeutics.

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Synthesis and medical applications of oligosaccharides

Recent technological advances in glycobiology and glycochemistry are paving the way for a new era in carbohydrate vaccine design. This is enabling greater efficiency in the identification, synthesis and evaluation of unique glycan epitopes found on a plethora of pathogens and malignant cells. Here, we review the progress being made in addressing challenges posed by targeting the surface carbohydrates of bacteria, protozoa, helminths, viruses, fungi and cancer cells for vaccine purposes.

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Carbohydrate vaccines: developing sweet solutions to sticky situations?

Synthetic oligosaccharides and glycoconjugates are increasingly used as probes for biological research and as lead compounds for drug and vaccine discovery. These endeavors are, however, complicated by a lack of general methods for the routine preparation of this important class of compounds. Recent development such as one-pot multi-step protecting group manipulations, the use of unified monosaccharide building blocks, the introduction of stereoselective glycosylation protocols, and convergent strategies for oligosaccharide assembly, are beginning to address these problems. Furthermore, oligosaccharide synthesis can be facilitated by chemo-enzymatic methods, which employ a range of glycosyl transferases to modify a synthetic oligosaccharide precursor. Glycosynthases, which are mutant glycosidases, that can readily form glycosidic linkages are addressing a lack of a wide range glycosyltransferases. The power of carbohydrate chemistry is highlighted by an ability to synthesize glycoproteins.

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Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research

The technical field of this invention is automated oligosaccharide synthesizers. There is a need in this field for more efficient oligosaccharide synthesizers. For example, the present invention is an apparatus for solid phase oligosaccharide synthesis, which includes a reaction vessel for holding a reaction mixture, such that the reaction vessel is equipped with a temperature control system, a donor vessel for holding a saccharide donor; an activation vessel for holding activator, a pump operably connected to a fluidic valve; an additional fluidic valve connected to the activation vessel, to the first fluidic valve via a first fluid line, and to the reaction vessel via a second fluid line, such that the activator or saccharide donor can be delivered via the second fluidic valve into the first fluid line and then through the second fluid line into the reaction vessel.

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Automated oligosaccharide synthesizer

Understanding the structure–function relationship of carbohydrates in biological systems is a major challenge. Such investigations require fast and reliable access to structurally-defined oligosaccharides. To date, oligosaccharide synthesis has been considered technically difficult and only parts of the synthetic process have been automated using solution and solid-phase techniques. Here, we describe a versatile platform integrating a new synthesis strategy and a fully-automated oligosaccharide synthesizer. Structurally diverse conjugation-ready oligosaccharides can be generated for the creation of glycoconjugates and microarrays. Biologically-significant oligosaccharides of increasing length, structural complexity, and chemical diversity were produced, including glycans found on the surfaces of pathogenic bacteria, and those with integral roles in inflammatory and immune responses.

Streamlined access to conjugation-ready glycans by automated synthesis

The Globo series of tumor-associated antigens are glycosphingolipids implicated in several cancer types. These oligosaccharides contain an α-linked galactose. Reported are methods to efficiently install cis-galactosidic linkages by automated solid-phase oligosaccharide synthesis. The complex tumor-associated oligosaccharides Globo-H and Gb-3 were assembled in about 1 day using the new approach that extends the scope of automated oligosaccharide synthesis to structurally more challenging carbohydrates.

Automated Synthesis of the Tumor-Associated Carbohydrate Antigens Gb-3 and Globo-H: Incorporation of α-Galactosidic Linkages

Carbohydrates are the basis for many therapeutic and diagnostic strategies, yet the full potential of glycans in medicine has not been realized. The study of the precise role of different carbohydrates, bound to either proteins or lipids, is hampered by difficulties in accessing pure, well-defined glycoconjugates. This Review highlights recent advances in glycobiology with a particular emphasis on oligosaccharide synthesis and conjugation techniques for the construction of homogeneous glycoconjugates. New methods for the study of protein-glycan interactions such as carbohydrate arrays and in vivo visualization of glycosylation pattern changes will also be addressed. The development of glycotherapeutics is just beginning, and much remains to be understood about the relationship between glycoconjugate structure and function. The emergence of novel tools will certainly facilitate and expand the use of carbohydrates in therapeutics and diagnostics.

Combined Approaches to the Synthesis and Study of Glycoproteins

For automated oligosaccharide synthesis to impact glycobiology, synthetic access to most carbohydrates has to become efficient and routine. Methods to install "difficult" glycosidic linkages have to be established and incorporated into the overall synthetic concept. Described here is the first automated solid-phase synthesis of oligosaccharides containing the challenging beta-mannosidic linkage. Carboxybenzyl mannoside building blocks proved effective beta-mannosylation agents and resulted in excellent conversion and good to moderate selectivities. [(Triisopropylsilyl)oxy]-methyl ether (Tom), served as an orthogonal, minimally intrusive, and readily cleavable protecting group for the elongation of the C3 position of mannose. The desired oligosaccharide products were readily separated from by-products containing unwanted stereoisomers using reverse-phase HPLC. The methods described here expand the scope of carbohydrates currently accessible by automation as many oligosaccharides of biological interest contain beta-mannosidic linkages.

Bastien Castagner

Papers

Automated oligosaccharide synthesizer

Streamlined access to conjugation-ready glycans by automated synthesis

Automated Synthesis of the Tumor-Associated Carbohydrate Antigens Gb-3 and Globo-H: Incorporation of α-Galactosidic Linkages

Combined Approaches to the Synthesis and Study of Glycoproteins

Automated Solid-Phase Synthesis of Protected Oligosaccharides Containing β-Mannosidic Linkages