Controlled γ-ray irradiation of heparin generates oligosaccharides enriched in highly sulfated sequences

Controlled physical depolymerization of heparin was performed in aqueous solution in the presence of isopropanol by gamma-irradiation. Isopropanol both makes selective the radical-induced scission of the glycosidic linkage involved in the depolymerization and acts as hydrogen donor scavenger of heparin intermediate radicals that usually result in UV-absorbing by-products. Several preparations of heparin-derived oligosaccharides (HDO) were reproducibly obtained from different unfractionated heparins (UFH). From each preparation, an intermediate fraction with an average molecular weight of about 2200 Da (gamma-HDO) was isolated by gel permeation chromatography and its sulfation pattern was characterized by nuclear magnetic resonance spectroscopy. The comparison of the sulfation pattern of HDO with that of parent UFHs revealed a significant decrease in the relative content of D-glucuronic acid with respect to the total amount of uronic acid. Further gamma-ray treatments of heparin chains that survived the first depolymerization procedure led to a further decrease of D-glucuronic acid and also slightly reduced the content of N-acetyl-glucosamine residues, thus enriching oligosaccharide chains in the highly sulfated sequences. MALDI mass spectrometric analysis of oligomeric components indicated that gamma-HDO prevalently contains highly sulfated chains (tetra to decasaccharides, including both odd and even number of monosaccharidic units) chiefly constituted of repeating sequences of the trisulfated disaccharide L-iduronic acid 2-sulfate (1 to 4) D-glucosamine-N,6 disulphate. gamma-Irradiation in the presence of isopropanol thus generated heparin fragments largely preserving the prevalent structure of the parent heparin and involved glucuronic acid as the major site of cleavage.