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The sulfated polysaccharide heparin has been used as a life-saving anticoagulant in clinics well before its detailed structure was known. This mini-review is a survey of the evolution in the discovery of the primary and secondary structure of heparin. Highlights in this history include elucidation and synthesis of the specific sequence that binds to antithrombin, the development of low-molecular-weight heparins currently used as antithrombotic drugs, and the most promising start of chemo-enzymatic synthesis. Special emphasis is given to peculiar conformational properties contributing to interaction with proteins that modulate different biological properties., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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 alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) 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 IdoA2SO(3) 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: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). 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.

13C NMR relaxation measurements at three different magnetic field strengths have been used to analyse the motional properties of a low molecular weight K5 polysaccharide (delta UA-[-->4)-beta-D-GlcNAc(1-->4)-beta-D-GlcA(1-->]n-GlcNAcred) from E. coli. Two-dimensional double INEPT spectra with suppression of cross-correlation effects between dipolar and chemical shift anisotropy relaxation mechanisms were collected in order to determine carbon longitudinal and transverse relaxation times. The values of the overall correlation time and the rate of internal motions were obtained using the model free spectral densities. The data indicate that the overall motion of the molecule is non-isotropic and can be approximated with the symmetric top model with an axial ratio of approximately 22. The magnitude of the generalized order parameters (S2 approximately 0.8) and the internal motion correlation time (tau e approximately 30 ps) differ from those found for iduronic acid-containing glycosaminoglycans and suggest that the internal motions in K5 polysaccharide are more limited.

The complete 1H and 13C NMR spectral assignments are described for the most prevalent patterns of sulfation and acetylation which can be found in polymeric heparin or can be obtained by standard chemical modifications. These include a number of novel structures containing unsubstituted or acetylated amino groups and the first complete NMR assignments of many of the other derivatives. Beef lung heparin was chosen as a model system and studies were carried out using conditions to control the influences on the chemical shift positions in heparin samples of divalent cations and variations in pH and temperature.

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

The 1H-n.m.r. 3J values for the L-iduronic acid (IdoA) residues for solutions in D2O of natural and synthetic oligosaccharides that represent the biologically important sequences of dermatan sulfate, heparan sulfate, and heparin have been rationalized by force-field calculations. The relative proportions of the low-energy conformers 1C4, 2S0, and 4C1 vary widely as a function of sequence and of pattern of sulfation. When IdoA or IdoA-2-sulfate units are present inside saccharide sequences, only 1C4 and 2S0 conformations contribute significantly to the equilibrium. This equilibrium is displaced towards the 2S0 form when IdoA-2-sulfate is preceded by a 3-O-sulfated amino sugar residue, and towards the 1C4 form when it is a non-reducing terminal. For terminal non-sulfated IdoA, the 4C1 form also contributes to the equilibrium. N.O.e. data confirm these conclusions. Possible biological implications of the conformational flexibility and the counter-ion induced changes in conformer populations are discussed.