Chondroitin and Dermatan Sulfate Oligosaccharide Preparation

Chondroitin sulfate A (CS-A) and dermatan sulfate (DS) oligosaccharides were independently prepared by partial enzymatic depolymerization of bovine trachea chondroitin sulfate A (Celsus Laboratories, Cincinnati, OH, USA) and porcine intestinal mucosa dermatan sulfate (Celsus Laboratories). A 20 mg/mL solution of each, in 50 mM Tris-HCl/60 mM sodium acetate buffer, pH 8 was incubated at 37 °C with chondroitin ABC lyase from Proteus vulgaris, EC 4.2.2.4. (Associates of Cape Cod Inc., East Falmouth, MA, USA). After the absorbance at 232 nm indicated the digestion was 50 % completed, the digestion mixture was heated at 100 °C for 3 min. The resulting oligosaccharide mixture was filtered by a 0.22 μm unit (Millipore, Billerica, MA, USA) and fractionated by low pressure GPC on a Bio-Gel P10 (Bio-Rad, Richmond, CA, USA) column. Fractions containing oligosaccharides of interest were desalted by GPC on a Bio-Gel P2 column and freeze-dried [48 (link)]. Further purification of compounds was carried out using strong anion exchange high-pressure liquid chromatography (SAX-HPLC) on a semi-preparative SAX S5 Spherisorb column (Waters Corp, Milford, MA, USA). The SAX-HPLC fractions containing >90 % of oligosaccharides were collected, desalted by GPC, and freeze-dried. The solid was reconstituted in water and purified a second time by SAX-HPLC. Only the top 30 % of the chromatographic peak was collected, desalted, and freeze-dried. Concentration of the oligosaccharide solutions was determined by measuring the absorbance at 232 nm (e=3800M⁻¹ cm⁻¹). The resulting fractions containing individual oligosaccharides were characterized by PAGE, ESI-MS, and high-field nuclear magnetic resonance (NMR) spectroscopy [49 (link)].