Conformational changes and anticoagulant activity of chondroitin sulfate following its O-sulfonation

Abstract

Chondroitin sulfate from bovine tracheal cartilage, with the basic structure (4- O-sulfo- d-Gal pNAc β1→4- d-Glc pA) n , was chemically modified by O-sulfonation. Depending on the reaction conditions, the products showed a different degree of O-sulfonation. A fully O-sulfonated chondroitin sulfate, having no free hydroxyl groups, and a sulfo ester group:disaccharide unit ratio of 4.0 was prepared. This chondroitin sulfate derivative was shown by 1 H NMR spectroscopy to have a uronate residue with an altered conformation. Usually, the uronate residue in chondroitin sulfate resides in the 4C 1 form. Fully O-sulfonated chondroitin sulfate had an uronate residue in the 1C 4 form at 30 °C, similar to the preferred conformation of the 2- O-sulfo-iduronate residue most commonly found in heparin. The 2S 0 form of the uronate residue was also found in fully O-sulfonated chondroitin sulfate at 60 °C. The anti-factor IIa activity of fully O-sulfonated chondroitin sulfate was 40 units/mg. This value is similar to the activities reported for various low-molecular-weight heparins, and substantially higher than those previously reported for partially O-sulfonated chondroitin sulfates having an average sulfate group/disaccharide unit of 2.5 to 3.3. The anti-factor Xa activity of the fully O-sulfonated chondroitin sulfate was 12 units/mg. This value is considerably lower than the activities reported for various low-molecular-weight heparins, consistent with the critical importance of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. These findings suggest that the conformational change of glucuronic acid residue in chondroitin sulfate resulting from its full O-sulfonation can result in enhanced anticoagulant activity, particularly as measured by anti-factor IIa assay.