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J. Biol. Chem., Vol. 278, Issue 37, 35199-35203, September 12, 2003

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Rapid Chemoenzymatic Synthesis of Monodisperse Hyaluronan Oligosaccharides with Immobilized Enzyme Reactors^*

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

From the Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (4-glucuronic acid-3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (10–20 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 multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (1–100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting 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 prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.

Received for publication, June 18, 2003 , and in revised form, July 2, 2003.

^* This work was supported in part by National Science Foundation Grant MCB-9876193, Oklahoma Center for Advancement of Science and Technology OARS program Grant AR02.2–019, National Institutes of Health Grant GM56497, and a sponsored research agreement from Hyalose LLC (to P.L.D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104. Tel.: 405-271-2227; Fax: 405-271-3092; E-mail: paul-deangelis{at}ouhsc.edu.

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