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Papers In Press, published online ahead of print July 2, 2003
J. Biol. Chem, 10.1074/jbc.M306431200
Submitted on June 18, 2003
Revised on July 2, 2003
Accepted on July 2, 2003

Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors

Paul L. DeAngelis, Leonard C. Oatman, and Daniel F. Gay

Biochem. & Molec. Biol., OUHSC, Oklahoma City, OK 73104

Corresponding Author: paul-deangelis{at}ouhsc.edu

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan [HA = beta4-glucuronic acid-beta3-N-acetylglucosamine] oligosaccharides. Potential medical applications for HA oligosaccharides (approximately 10 to 20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (~1 to 100 sugars/second), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid-transferase and N-acetylglucosamine-transferase). The resulting two enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes proves both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.


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