Effect of oversulfation on the chemical and biological properties of chondroitin-6-sulfate

Chondroitin-6-sulfate was oversulfated using chlorosulfonic acid-pyridine complex and isolated as the sodium salt. Infrared analysis of the native and oversulfated compound gave identical results in respect to the O-H stretching, hemiacetal stretching and S=O stretching. Absorption around 825 cm(-1) was also present in both the compounds representing the sulfate group of galactosamine at equatorial C-6 position. The oversulfated compound showed a peak at 855 cm(-1) representing the new sulfate group on axial C-4 position of galactosamine. H-1 NMR studies confirmed the I R results and further showed that the oversulfated compound was a mixture of 6 1 % of chondroitin-4,6-disulfate and 39% of the native compound. The oversulfated compound showed a 2.8-fold increase in sulfate content and a significant anticoagulant activity by doubling the prothrombin time of normal citrated human plasma using 9.5 mu g of the sulfated compound while the native compound was inactive even at 2000 mu g level. During in vitro studies using 0.05 M Tris buffer pH 7.35 containing physiological concentrations of NaCl (0.9%) the oversulfated compound gave a 1.8-fold enhancement of the activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) and a 3.2-fold enhancement of Glu-Plg activation by urokinase (u-PA) in comparison to the control while the native compound or the unfractionated heparin were less active. (c) 2007 Published by Elsevier Ltd.