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 couple with hyaluronan and cross-linked derivatives (hylan) has been studied using a rapid-flaw electron paramagnetic resonance spectroscopy (EPR) system. Radicals were detected as a result of hydrogen atom abstraction from the carbohydrate at pH 3.6; these gave rise to both relatively broad and sharp isotropic features. The broad signals are assigned to high-molecular-weight hyaluronan-derived radicals, whereas the isotropic features are due to rapidly tumbling radicals present either at the ends of the polymer or on low-molecular-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 parameters identical to those observed for the radical generated as a result of hydrogen abstraction from the C-5 position of the model compound glucuronic acid, and is therefore assigned to this species on the polymer. The second 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 hydrogen 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 the linear polymer. These EPR data fully support the chain scission processes previously proposed for aqueous hyaluronan and hylan systems, where each hydroxyl radical results in a single chain scission. (C) 1999 Elsevier Science Ltd. All rights reserved.