Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins Original Research Article

In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the α(1→4)-linked 2-O-sulfated l-iduronic acid (IdoA2SO3)→N,6-O-sulfated d-glucosamine (GlcNSO36SO3) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO3 residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO–MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one d-glucuronic acid was: GlcNSO3>GlcN6SO3>IdoA3SO3≅GlcA2SO3≅GlcN3SO3>IdoA2SO3≅GlcA3SO3. Starting from a ‘supersulfated’ low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.