Identification of radicals from hyaluronan (hyaluronic acid) and crosslinked derivatives using electron paramagnetic resonance spectroscopy

The reaction of hydroxyl radicals generated using a Ti(III)-H2O2 redox coup le with hyaluronan and cross-linked derivatives (hylan) has been studied us ing a rapid-flaw electron paramagnetic resonance spectroscopy (EPR) system. Radicals were detected as a result of hydrogen atom abstraction from the c arbohydrate at pH 3.6; these gave rise to both relatively broad and sharp i sotropic features. The broad signals are assigned to high-molecular-weight hyaluronan-derived radicals, whereas the isotropic features are due to rapi dly tumbling radicals present either at the ends of the polymer or on low-m olecular-weight fragments. These isotropic signals have been interpreted in terms of the presence of two major radicals; one of these gives rise to a doublet signal (a(H) 1.36 mT, g 2.0049), the other a doublet of doublets (a (alpha-H) 1.86 mT, a(beta-H) 0.81 mT, g 2.0035). The former signal has para meters identical to those observed for the radical generated as a result of hydrogen abstraction from the C-5 position of the model compound glucuroni c acid, and is therefore assigned to this species on the polymer. The secon d signal, which has parameters characteristic of a radical with both alpha- H and beta-H splittings, is believed to be generated as a result of hydroge n abstraction from C-6 on the N-acetyl-D-glucosamine monomer. Less intense signals were observed with the cross-linked material hylan, in accord with previous data which show that this material is less readily degraded than t he linear polymer. These EPR data fully support the chain scission processe s previously proposed for aqueous hyaluronan and hylan systems, where each hydroxyl radical results in a single chain scission. (C) 1999 Elsevier Scie nce Ltd. All rights reserved.