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Chemoenzymatic Synthesis of Biologically Important Carbohydrates and Derivatives

Carbohydrates play important roles in a number of pathological processes such as cancer, infectious diseases, and inflammation. Detailed biophysical and biochemical studies of carbohydrates require sufficient quantities of defined oligosaccharides. Due to structure complexity, synthesis of complex carbohydrates with special linkages and configurations by traditional (non-biological) organic synthetic approaches continue to pose difficulties in spite of the significant advances in the field. Enzymatic synthesis by glycosyltransferases is considered an alternative solution. Chemoenzymatic synthesis combining the advantages of both organic synthesis and enzymatic synthesis is a method of choice to obtain complex carbohydrates and glycoconjugates.

Site-specific Glycoconjugation (SSGC)

Carbohydrates are widely distributed as glycoproteins or glycolipids on mammalian cell surface. Most of these carbohydrate moieties have sialic acid residues as the terminal units. Despite their important roles, carbohydrates in glycoproteins usually do not occupy or block the protein critical active sites. Therefore, carbohydrates, especially the terminal sialic acid residues, are ideal sites for modification and conjugation.

Anti-cancer monoclonal antibodies have widely applications in both experimental and clinical studies. Recently developed humanized monoclonal antibodies found great potential in anticancer therapy. Both human and mouse immunoglobulin IgG1 antibodies contains two heavy chains. There is an asparagine-linked (N-linked) oligosaccharide on the CH2 domain of each heavy chain. These N-linked oligosaccharides are generally restricted to biantennary oligosaccharides terminating in 2, 1, and 0 galactose residues. Using a beta-1,4-galactosyltransferase and a sialyltransferase, functional group-tagged sialic acid can be enzymatically conjugated to anticancer monoclonal antibodies. These functional groups-tagged antibodies can be applied in anticancer pretargeting as attractive delivery vehicles for radionuclides, drugs, toxins, and 10B-enriched carboranes to tumors.

Synthetic Polysaccahride Vaccines

Many invasive bacteria produce capsular polysaccharides, which are essential virulence factors for pathogen invasion to human body. Vaccination using capsular polysaccharides conjugated to a protein carrier is a powerful approach for protecting humankind against infectious diseases caused by bacterial pathogens, such as Streptococcus pneumoniae and Haemophilus influenzae type b (Hib). Sufficient amounts of pure and well-defined polysaccharides, which are not easily obtained by purification or purely organic synthesis, are important to reveal their biological roles. Therefore, producing structure-defined synthetic capsular polysaccharides by following their biosynthetic pathway is an alternative and efficient solution. Furthermore, unravel of the biosynthetic pathway of the bacterial polysaccharides will open a door for further modification and improvement of polysaccharide vaccines by combinatorial biosynthesis (producing novel polysaccharides by switching genes in the capsular polysaccharide loci amongst populations of same/related species), unnatural substrate substitution, and/or chemical modification. The relationship of the capsular polysaccharide with the host immunogenicity can be further exploited using the produced novel synthetic polysaccharide products.

Production of homogeneous human-like therapeutic glycoproteins

Majority of proteins for therapeutic use, such as growth hormones, cytokines, and antibodies are glycoproteins (proteins with carbohydrate chains). The pattern of carbohydrate structures on glycoproteins is important in influencing the biological properties of the proteins, such as protein stability, pharmacokinetics, antigenicity, cell adhesion, protein targeting, and many other intercellular recognition processes. Current biological techniques only provide heterogeneous mixtures of glycoproteins with non-human glycoforms, causing inconsistent efficiency and immunogenicity. We are developing a variety of chemical (e.g. novel glycosylation reactions, and glycoconjugation reactions) and biological (e.g. bioengineering expression hosts) methods to create homogenous glycoproteins for both basic research and biomedical applications. One strategy will be using genetic-engineered filamentous fungal system. Trichoderma reesei is the organism of our choice.

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