The proposed research focuses on the development of efficient and widely applicable chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is a ubiquitous posttranslational modification of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. The covalently linked oligosaccharides can profoundly affect proteins'structure, function, and their serum half-life. However, homogeneous glycoproteins with structural defined oligosaccharides are difficult to obtain from natural or recombinant sources, since they are typically produced as a mixture of heterogeneous glycoforms. To obtain homogeneous materials for structural/biological studies and for biomedical applications, we propose to systematically explore the potential of endo-?-N-acetylglucosaminidases (ENGases), a special class of endoglycosidases, for constructing N-glycopeptides and N-glycoproteins. The biggest advantage is that some ENGases are able to transfer an intact oligosaccharide to a GlcNAc-containing peptide or protein in a single step without the need of any protecting groups, thus providing a highly convergent route to large glycopeptides and glycoproteins. But the method has hitherto suffered with low transglycosylation yield, the risk of product hydrolysis, and the limitation to the use of only natural N-glycans as donor substrates that themselves are difficult to prepare. Our preliminary studies have shown that the use of sugar oxazolines (the transition state mimics) as donor substrates not only expanded the substrate availability, but also resulted in a high-yield transglycosylation to form large glycopeptides and homogeneous glycoproteins. This proposal intends to systematically explore the scope and limitation of the novel methodology through pursuing five specific aims: 1) synthesis and examination of a range of oligosaccharide oxazolines as donor substrates for the enzymatic transglycosylation;2) evaluation of novel acceptor substrates for synthesizing complex fucose-containing N-glycopeptides and for detecting O-GlcNAc glycosylation;3) total synthesis of large HIV-1 gp120 fragments for structural and functional studies;4) exploitation of the methodology for synthesizing natural and tailor-made homogeneous glycoproteins;and 5) synthesis and functional studies of homogeneous glycoforms of human antibody IgG-Fc. The knowledge gained from the proposed research will eventually facilitate the development of glycoprotein-based therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080374-04
Application #
7629692
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Marino, Pamela
Project Start
2007-06-01
Project End
2011-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
4
Fiscal Year
2009
Total Cost
$285,000
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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