Novel regio- and stereoselective modifications of heparin in alkaline solution. Nuclear magnetic resonance spectroscopic evidence

Abstract

Nuclear magnetic resonance spectroscopic evidence is presented in characterizing three new structurally modified forms of heparin. One of these, polymer M-I, represents a conversion of about two-thirds of the α-L-iduronic acid 2-sulfate residues (1) into residues of a 2,3-anhydro derivative (3), through the action of sodium hydroxide. The formation of 3 is attributed to a base-catalysed displacement of the sulfate group of 1 by an intramolecular attack of O-3 on C-2. In more concentrated sodium hydroxide solution, heparin is transformed almost quantitatively into polymer M-II, which differs from it in having residues of (non-sulfated) α-L-iduronic acid (4) in place of 1. It is likely that 3 is an intermediate, and that a selective nucleophilic attack of hydroxide ion at C-2 accounts for the ido configuration in 4. The third modification, giving polymer M-III, is induced when a neutral or weakly alkaline solution of M-I is heated at 70 °C or above, which promotes a different stereochemistry in the hydrolysis of the 2,3-oxirane ring of 3. Hence, in contrast to residues of 4 in M-II, most of the uronic acid residues of M-III appear to have the alternate, α-L-galacto, configuration. As shown by a comparison of beef lung and hog mucosal heparin, the rate at which M-I is converted into M-III is facilitated by the higher level of structural heterogeneity in the mucosal heparin. Whereas the formation of M-I, -II, and -III is accompanied by only moderate depolymerization, these novel polymers retain little of the anti-coagulant and anti-XA activities of the unmodified heparin. Keywords: NMR spectroscopy, heparin, desulfation, anhydroaldoside, base-catalysed displacement.