Structural Characterization of the Low-Molecular-Weight Heparin Dalteparin by Combining Different Analytical Strategies

The sulfation at the 3-OH position of glucosamine is an important modification in forming structural domains for heparan sulfate to enable its biological functions. Seven 3- O -sulfotransferase isoforms in the human genome are involved in the biosynthesis of 3- O -sulfated heparan sulfate. As a rare modification present in heparan sulfate, the availability of 3- O -sulfated oligosaccharides is very limited. Here, we report the use of a chemoenzymatic synthetic approach to synthesize six 3- O -sulfated oligosaccharides, including three hexasaccharides and three octasaccharides. The synthesis was achieved by rearranging the enzymatic modification sequence to accommodate the substrate specificity of 3- O -sulfotransferase 3. We studied the impact of 3- O -sulfation on the conformation of the pyranose ring of 2- O -sulfated iduronic acid using NMR, and on the correlation between ring conformation and anticoagulant activity. We identified a novel octasaccharide that interacts with antithrombin and displays anti factor Xa activity. Interestingly, the octasaccharide displays a faster clearance rate than fondaparinux, an FDA approved pentasaccharide drug, in a rat model, making this octasaccharide a potential short acting anticoagulant drug candidate that could reduce bleeding risk. Having access to a set of critically important 3- O -sulfated oligosaccharides offers the potential to develop new heparan sulfate-based therapeutics.

As part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin, Tinzaparin and Dalteparin were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT). The HA fractions were further subfractionated and characterised. For the parent LMWHs and selected fractions, molecular weight parameters were measured using a SE chromatographic system with a triple detector (TDA) to obtain absolute molecular weights. The SE chromatograms clearly indicate that Enoxaparin is consistently richer in shorter oligosaccharides than Tinzaparin and Dalteparin. Besides providing the content of terminal groups and individual glucosamine and uronic acid residues with different sulfate substituents, the HSQC-NMR spectra permitted us to evaluate and correlate the content of the pentasaccharide, AT-binding sequence A-G-A*-I-A (AT-bs) through quantification of signals of the disaccharide sequence G-A*. Whereas the percent content of HA species is approximately the same for the three LMWHs, substantial differences were observed for the chain distribution of AT-bs as a function of length, with the AT-bs being preferentially contained in the longest chains of each LMWH. The above information will be useful in establishing structure-activity relationships currently under way. This study is therefore critical for establishing correlations between structural features of LMWHs and their AT-mediated anticoagulant activity.