Enzymatic preparation of heparin oligosaccharides containing antithrombin III binding sites

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact-antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional H-1 NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are Delta UAp2S(1-->4)-alpha-D-GlcNpS 6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp (1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpS6S (where Delta UAp is 4-deoxy-alpha-L-threo-hexenopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and Delta UAp2S(1-->4)-alpha-D-Glc-NpS6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S (1-->4)-beta-D-GlcAp( 1-->4)-alpha-D-GlcNp3S6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpS6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated, The structure of the hexasaccharide is Delta UAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp (1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpS6S. The octasaccharide and decasaccharide correspond to the predominant structural moth found in porcine intestinal mucosal heparin, Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry, This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay, Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.