DIRECT-DETECTION AND IDENTIFICATION OF RADICALS GENERATED DURING THE HYDROXYL RADICAL-INDUCED DEGRADATION OF HYALURONIC-ACID AND RELATED MATERIALS

HO. attack on hyaluronic acid, related polymers and monomers has been studied by both direct, rapid-flow, EPR (ESR) and EPR spin trapping us ing a variety of traps. Evidence has been obtained, with the monomers, for essentially random hydrogen-atom abstraction at all the ring C-H bonds with glucuronic acid, and at all sites except the N-acetyl side chain and C(2) with N-acetylglucosamine. The initial radicals do not u ndergo rapid rearrangement reactions at pH 4; however at both lower an d higher pH values, acid- and base-catalysed rearrangement processes, respectively, result in the loss of these species. The rate of loss of these species is dependent on the substrate, with those derived from N-acetylglucosamine undergoing slower acid-catalysed rearrangement tha n the glucuronic acid-derived species. This is rationalised in terms o f a rearrangement reaction of 1,2-dihydroxyalkyl (1,2-diol) radicals i nvolving an electron-deficient radical-cation intermediate; the format ion of this species would be disfavoured by the electron-withdrawing N -acetyl substituent. The base-catalysed process, which is believed to involve a radical-anion intermediate, occurs rapidly at pH 7.4, and ap pears to be less substrate dependent. In the case of glucuronic acid- (but not N-acetylglucosamine-) derived species this latter process res ults in the detection of ring-opened semidione species. With equimolar mixtures of the two monomers essentially random attack occurs on the two rings. However with chondroitin sulphate A, attack appears to be m uch more selective, with a radical generated at C(5) on the glucuronic acid ring present at highest concentration. The initial radicals obta ined with this polysaccharide also undergo base- and acid-catalysed re arrangements; this leads to strand-breakage and the formation of low-m olecular-weight material. Spin-trapping experiments carried out with h yaluronic acid, and a number of other polysaccharides, resulted in the detection of a number of novel spin adducts, the formation of which a re consistent with attack on both the sugar rings in the polymer. The pH dependence of the observed spectra, and the detection of additional species at some pH values, suggest that at least some of the initial radicals undergo base-catalysed rearrangement reactions which result i n strand-breakage and the formation of low-molecular-weight fragments. The extent of fragmentation at a particular pH, is also affected by t he radical flux, with high radical yields giving more low-molecular-we ight material. These observations suggest that pH-independent processe s also contribute to strand-cleavage; this may be due to beta-cleavage of the radicals formed at C(1) on either ring, C(3) on N-acetylglucos amine or C(4) on the glucuronic acid ring.