Enzymatic and chemical methods for the generation of pure hyaluronan oligosaccharides with both odd and even numbers of monosaccharide units

Hyaluronan oligosaccharides display physiological activities not associated with the polymer and are widely used to characterize hyaluronan-binding proteins. They can also be used as biocompatible starting blocks for chemical derivatization. Here we present methods for generating milligram quantities of unusual odd- and even-numbered oligosaccharides, greatly increasing the diversity of reagents for use in such studies. These methods are based upon protocols from the 1960s, at which time it was very difficult to assess the stereochemical purity of the products. To address this, products were analyzed with modern high-field nuclear magnetic resonance spectroscopy. Alkaline β-elimination conditions previously used to remove reducing-terminal N-acetylglucosamine residues in fact introduce a significant (∼30%) level of stereoisomerism in the products by alkali-catalyzed keto-enol tautomerizations. Milder alkaline conditions were used to overcome this problem, reducing the contamination to <5%. The elimination by-products from this reaction were isolated and characterized, allowing the mechanism of alkaline degradation of hyaluronan to be investigated for the first time. β-Glucuronidase was used to remove nonreducing-terminal glucuronic acid residues from oligosaccharides. Odd-numbered oligosaccharides with terminal glucuronic acid residues isolated from hyaluronidase digests are shown to originate from acid-catalyzed acetal hydrolysis during boiling denaturation and also have significant levels of stereochemical impurities.